Note: Descriptions are shown in the official language in which they were submitted.
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ELECTRODES LINKED VIA CONDUCTIVE OLIGOMERS
TO NUCLEIC ACIDS
FIELD OF THE INVENTION
The invention relates to nucleic acids covalently coupled to electrodes via
conductive oligomers. More
particularly, the invention is directed to the site-selective modification of
nucleic acids with electron
transfer moieties and electrodes to produce a new class of biomaterials, and
to methods of making
and using them.
BACKGROUND OF THE INVENTION
The detection of specific nucleic acids is an important tool for diagnostic
medicine and molecular
biology research. Gene probe assays currently play roles in identifying
infectious organisms such as
bacteria and viruses, in probing the expression of normal genes and
identifying mutant genes such as
oncogenes, in typing tissue for compatibility preceding tissue
transplantation, in matching tissue or
blood samples for forensic medicine, and for exploring homology among genes
from different species.
Ideally, a gene probe assay should be sensitive, specific and easily
automatable (for a review, see
Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)). The requirement
for sensitivity (i.e. low
detection limits) has been greatly alleviated by the development of the
polymerase chain reaction
(PCR) and other amplification technologies which allow researchers to amplify
exponentially a specific
nucleic acid sequence before analysis (for a review, see Abramson et al.,
Current Opinion in
Biotechnology, 4:41-47 (1993)).
Specificity, in contrast, remains a problem in many currently available gene
probe assays. The extent
of molecular complementarity between probe and target defines the specificity
of the interaction.
Variations in the concentrations of probes, of targets and of salts in the
hybridization medium, in the
reaction temperature, and in the length of the probe may alter or influence
the specificity of the
probe/target interaction.
It may be possible under some limited circumstances to distinguish targets
with perfect
complementarity from targets with mismatches, although this is generally very
difficult using traditional
technology, since small variations in the reaction conditions will alter the
hybridization. New
experimental techniques for mismatch detection with standard probes include
DNA ligation assays
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where single point mismatches prevent ligation and probe digestion assays in
which mismatches
create sites for probe cleavage.
Finally, the automation of gene probe assays remains an area in which current
technologies are
lacking. Such assays generally rely on the hybridization of a labelled probe
to a target sequence
followed by the separation of the unhybridized free probe. This separation is
generally achieved by gel
electrophoresis or solid phase capture and washing of the target DNA, and is
generally quite difficult to
automate easily.
The time consuming nature of these separation steps has led to two distinct
avenues of development.
One involves the development of high-speed, high-throughput automatable
electrophoretic and other
separation techniques. The other involves the development of non-separation
homogeneous gene
probe assays.
PCT applications WO 95/15971, PCT/US96/09769 and PCT/US97/09739 describe novel
compositions
comprising nucleic acids containing electron transfer moieties, including
electrodes, which allow for
novel detection methods of nucleic acid hybridization.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the invention to provide for improved
compositions and methods for the
detection of nucleic acids.
In one aspect, the invention provides compositions comprising (a) a first
electron transfer moiety
comprising an electrode; (b) a first single stranded nucleic acid; (c) a
second electron transfer moiety
covalently attached to the first nucleic acid; and (d) a conductive oligomer
covalently attached to both
the electrode and the first nucleic acid.
In an additional aspect) the invention provides compositions comprising (a) a
first electron transfer
moiety comprising an electrode; (b) a first single stranded nucleic acid; (c)
a conductive oligomer
covalently attached to both the electrode and the first nucleic acid; and (d)
a second electron transfer
moiety covalently attached to a second single stranded nucleic acid.
In one aspect, the conductive oligomer has the formula:
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~Y~B~---D Y
g /n ~ m
wherein
Y is an aromatic group;
- 5 n is an integer from 1 to 50;
g is either 1 or zero;
a is an integer from zero to 10;and
m is zero or 1;
wherein when g is 1, B-D is a conjugated bond; and
wherein when g is zero, a is 1 and D is preferably carbonyl, or a heteroatom
moiety, wherein the
heteroatom is selected from oxygen, sulfur, nitrogen, silicon or phosphorus.
In an additional aspect, the conductive oligomer has the formula:
~C-G-C J
n ~ m
wherein
n is an integer from 1 to 50;
mis0or1;
C is carbon;
J is carbonyl or a heteroatom moeity, wherein the heteroatom is selected from
the group consisting of
oxygen, nitrogen, silicon, phosphorus, sulfur; and
G is a bond selected from alkane, alkene or acetylene.
In a further aspect, the invention provides methods of detecting a target
sequence in a nucleic acid
sample. The method comprises applying a first input signal to a hybridization
complex and detecting
electron transfer. The hybridization complex comprises the target sequence, if
present, and at least a
first probe nucleic acid. The probe nucleic acid comprises a a covalently
attached conductive
oligomer. The conductive oligomer is also covalently attached to a first
electron transfer moiety
comprising an electrode. In addition, the hybridization complex has a
covalently attached second
electron transfer moiety.
b In one aspect, the conductive oligomer has the formula:
~Y~B~---D Y
8 a/n ~ m
or
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-fi-C-G-C J
n ' m
In one aspect, the first input signal comprises an AC component and a non-zero
DC component.
In an additional aspect, the first input signal comprises an AC component at a
first frequency and a
non-zero DC component, and the method further comprises applying a second
input signal comprising
an AC component at at least a second frequency and a non-zero DC component.
In a further aspect, the first input signal comprises an AC component and a
first non-zero DC
component, and the method further comprises applying a second input signal
comprising an AC
component and a second non-zero DC component.
In an additional aspect, the first input signal comprises an AC component at a
fist voltage amplitude
and the method further comprises applying a second input signal comprising an
AC component at a
second voltage amplitude.
In an additional aspect, the invention provides methods of making the
compositions of the invention.
The methodscomprise attaching a conductive oligomer to a nucleic acid, and
attaching the conductive
oligomer to said electrode. These steps may be done in any order.
In a further aspect, the invention provides compositions comprising a
conductive oligomer covalently
attached to a nucleoside, wherein said conductive oligomer has the formula: is
selected from the
group consisting of:
~Y~B~-D Y
B a Jn ~ m
or
-t-C-G-C J
n ' m
wherein
n is an integer from 1 to 50;
mis0or1;
C is carbon;
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J is carbonyl or a heteroatom moeity, wherein the heteroatom is selected from
the group consisting of
oxygen, nitrogen, silicon, phosphorus, sulfur; and
G is a bond selected from alkane, alkene or acetylene, wherein if m = 0, at
least one G is not alkane.
In an additional aspect, the invention provides compositions comprising (a) a
solid support comprising
a monolayer of passivation agent; (b) a nucleic acid comprising at least one
nucleoside) wherein said
nucleic acid is covalently attached to said solid support with a linker
selected from the group selected
from:
Y~B~-D Y
~ ~ ~ g a
n ' m
or ~
~C-G-C J
m
wherein
n is an integer from 1 to 50;
mis0orl;
C is carbon;
J is carbonyl or a heteroatom moeity, wherein the heteroatom is selected from
the group consisting of
oxygen, nitrogen, silicon, phosphorus, sulfur; and
G is a bond selected from alkane, alkene or acetylene, wherein if m = 0, at
least one G is not alkane.
In an additional aspect, the invention provides compositions comprising (a) an
electrode; (b) at least
one metallocene; and (c) a conductive oligomer covalently attached to both
said electrode and said
metallocene, wherein said conductive oligomer is selected from the group
consisting of:
i) ~
--/-C-G-C J
n ' m
or
~Y~B~D Y
B e/n ~ m
In a further aspect, the invention provides peptide nucleic acids with at
least one chemical substituent
covalently attached to the a-carbon of a subunit of the peptide nucleic acid.
In an additional aspect, the invention provides peptide nucleic acids with at
least one chemical
substituent covalently attached to an internal subunit of the peptide nucleic
acid.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the synthetic scheme for a conductive oligomer covalently
attached to a uridine
nucleoside via an amide bond.
Figure 2 depicts the synthetic scheme for covalently attaching a conductive
oligomer covalently
attached to a uridine nucleoside via an amine bond.
Figure 3 depicts the synthetic scheme for a conductive oligomer covalently
attached to a uridine
nucleoside via the base.
Figure 4 depicts the synthetic scheme for a conductive oligomer covalently
attached to a nucleoside
via a phosphate of the ribose-phosphate backbone. The conductive oligomer is a
phenyl-acetylene
Structure 5 oligomer, although other oligomers may be used, and terminates in
an ethyl pyridine
protecting group, as described herein, for attachment to gold electrodes.
Figure 5 depicts the synthetic scheme for a conductive oligomer covalently
attached to a nucleoside
via a phosphate of the ribose-phosphate backbone, using an amide linkage and
an ethylene linker,
although other linkers may be used. The conductive oligomer is a phenyl-
acetylene Structure 5
oligomer, although other oligomers may be used, and terminates in an ethyl
pyridine protecting group,
as described herein) for attachment to gold electrodes.
Figure 6 depicts the synthetic scheme for a conductive polymer containing an
aromatic group with a
substitution group. The conductive oligomer is a phenyl-acetylene Structure 5
oligomer with a single
methyl R group on each phenyl ring, although other oligomers may be used, and
terminates in an ethyl
pyridine protecting group) as described herein, for attachment to gold
electrodes.
Figure 7 depicts the synthetic scheme for the synthesis of a metallocene, in
this case ferrocene, linked
via a conductive oligomer to an electrode. The conductive oligomer is a phenyl-
acetylene Structure 5
oligomer, although other oligomers may be used, and terminates in an ethyl
pyridine protecting group,
as described herein) for attachment to gold electrodes.
Figure 8 depicts a model compound, ferrocene attached to a C,6 alkane molecule
(insulator-1 ), at 200
mV AC amplitude and frequencies of 1, 5 and 100 Hz. The sample responds at all
three frequencies,
with higher currents resulting from higher frequencies.
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Figures 9A and 98 depict the response with varying frequency. Figure 9A shows
overlaid
voltammograms of an electrode coated with a ferrocene-conductive oligomer
model complex (wire-2).
Four excitation frequencies were applied, 10 Hz, 100 Hz, 1 kHz and 10 kHz, all
at 25 mV
overpotential. Again, current increases with frequency. Figure 9B shows
overlaid voltammograms of
electrodes coated with either ssDNA or dsDNA. ssDNA was run at 1 Hz and 10 Hz
at 100 mV
overpotential (bottom two lines). dsDNA was run at 1, 10, 50 and 100 Hz at 10
mV overpotential (top
four lines). Note that the scales between Figure 8 and Figures 9A and 9B are
different.
Figure 10 depicts the frequency response of these systems. The peak currents
at a number of
frequencies are determined and plotted. Sample 3 (filled triangles) responds
to increasing frequencies
through 10 kHz (system limit), while samples 1 (open circles) and 2 (filled
circles) lose their responses
at between 20 and 200 Hz. This data was not normalized to the increase in
current associated with
increasing frequency.
3 5 Figure 11 depicts the frequency responses of ssDNA (open circles; sample
5) and dsDNA (filled
circles; sample 6) at 25 mV overpotential. The current has been normalized.
The curves are not a fit
to the data; rather, these are models of RC circuits, illustrating that the
data can be fit to such curves,
and that the system is in fact mimic standard RC circuits. The top curve was
modeled using a 500
ohm resistor and a 0.001 farad capacitor. The bottom curve was modeled using a
20 ohm resistor and
a 0.002 farad capacitor.
Figure 12 shows that increasing the overpotential will increase the output
current.
Figures 13A and 13B illustrate that the overpotential and frequency can be
tuned to increase the
selectivity and sensitivity, using Sample 1.
Figure 14 shows that ferrocene added to the solution (Sample 7; open circles)
has a frequency
response related to diffusion that is easily distinguishable from attached
ferrocene (Sample 3; filled
circles).
Figures 15A and 15B shows the phase shift that results with different samples.
Figure 15A uses two
experiments of Sample 1, Sample 3 and Sample 4. Figure 15B uses Sample 5 and
Sample 6.
Figure 76 depicts the synthetic scheme for a conductive oiigomer covalently
attached to a uridine
nucleoside via an amine bond) with a CH2 group as a Z linker. Compound C4 can
be extended as
outlined herein and in Figure 1.
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Figures 17A, 17B, 17C, 17D, 17E, 17F and 17G depict other conductive
oligomers, attached either
through the base (A-D) or through the ribose of the backbone (E-G), which have
been synthesized
using the techniques outlined herein. Figure 17H depicts a conductive oligomer
attached to a
ferrocene. As will be appreciated by those in the art, the compounds are shown
as containing CPG
groups, phosphoramidite groups, or neither; however, they may all be made as
any of these.
Figure 18 depicts a synthetic scheme for a four unit conductive oligomer
attached to the base.
Figure 19 depicts a synthetic scheme for a four unit conductive oligomer
attached to the base.
Figure 20 depicts the use of a trimethylsilylethyl protecting group in
synthesizing a five unit wire
attached via the base.
Figure 21 depicts the use of a trimethylsilylethyl protecting group in
synthesizing a five unit wire
attached via the ribose.
Figures 22A and 22B depict simulations based on traditional electrochemical
theory (Figure 22B) and
the simulation model developed herein (Figure 22A).
Figures 23A and 23B depict experimental data plotted with theoretical model,
showing good
correlation. Fc-wire of Example 7 was used as 10 Hz (Figure 23A) and 100 Hz
(Figure 23B).
Figure 24 depicts the synthetic scheme for protecting and derivatizing adenine
for incorporation into
PNA.
Figure 25 depicts the synthetic scheme for protecting and derivatizing
cytosine for incorporation into
PNA.
Figure 26 depicts the synthetic scheme for protecting and derivatizing guanine
for incorporation into
PNA.
Figure 27 depicts the synthetic scheme for protecting and derivatizing thymine
for incorporation into
PNA.
Figures 28A, 28B, 28C, 28D and 28E. Figure 28A depicts the synthetic scheme
for making PNA
monomeric subunits. Figures 28B-28E depict the PNA monomers.
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Figure 29 depicts the synthetic scheme for a PNA monomeric subunit with a
ferrocene covalently
attached to a uracil base, for incorporation into a growing PNA.
Figure 30 depicts the synthetic scheme for a three unit conductive oligomer
covalently attached to a
base of a PNA monomeric subunit.
Figure 31 depicts the synthetic scheme for a three unit conductive oligomer
covalently attached to the
backbone of a PNA monomeric subunit.
Figure 32 depicts the synthetic scheme for a ferrocene covalently attached to
the backbone of a PNA
monomeric subunit.
DETAILED DESCRIPTION OF THE INVENTION
The present invention capitalizes on the previous discovery that electron
transfer apparently proceeds
through the stacked n-orbitals of the heterocyclic bases of double stranded
(hybridized) nucleic acid
("the n-way"). This finding allows the use of nucleic acids containing
electron transfer moieties to be
used as nucleic acid probes. See PCT publication WO 95/15971, hereby
incorporated by reference in
its entirety, and cited references. This publication describes the site-
selective modification of nucleic
acids with redox active moieties, i.e. electron donor and acceptor moieties,
which allow the long-
distance electron transfer through a double stranded nucleic acid. In general,
electron transfer
between electron donors and acceptors does not occur at an appreciable rate
when the nucleic acid is
single stranded, nor does it occur appreciably unless nucleotide base pairing
exists in the double
stranded sequence between the electron donor and acceptor in the double
helical structure. Thus,
PCT publicationWO 95I15971 and the present invention are directed to the use
of nucleic acids with
electron transfer moieties, including electrodes, as probes for the detection
of target sequences within
a sample.
In one embodiment, the present invention provides for novel gene probes, which
are useful in
molecular biology and diagnostic medicine. In this embodiment, single stranded
nucleic acids having
a predetermined sequence and covalently attached electron transfer moieties,
including an electrode,
are synthesized. The sequence is selected based upon a known target sequence,
such that if
hybridization to a complementary target sequence occurs in the region between
the electron donor
and the electron acceptor, electron transfer proceeds at an appreciable and
detectable rate. Thus, the
invention has broad general use, as a new form of labelled gene probe. In
addition, the probes of the
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present invention allow detection of target sequences without the removal of
unhybridized probe.
Thus, the invention is uniquely suited to automated gene probe assays or field
testing.
The present invention provides improved compositions comprising nucleic acids
covalently attached
via conductive oligomers to an electrode, of a general structure depicted
below in Structure 1:
Structure 1
F,-X-F2-nuGeic acid
I n Structure 1, the hatched marks on the left represent an electrode. X is a
conductive oligomer as
defined herein. F, is a linkage that allows the covalent attachment of the
electrode and the conductive
oligomer) including bonds, atoms or linkers such as is described herein, for
example as "A", defined
below. Fz is a linkage that allows the covalent attachment of the conductive
oligomer to the nucleic
acid, and may be a bond, an atom or a linkage as is herein described. Fz may
be part of the
conductive oligomer, part of the nucleic acid, or exogeneous to both, for
example, as defined herein
for "Z".
By "nucleic acid" or "oligonucleotide" or grammatical equivalents herein means
at least two
nucleotides covaiently linked together. A nucleic acid of the present
invention will generally contain
phosphodiester bonds, although in some cases, as outlined below, nucleic acid
analogs are included
that may have alternate backbones, comprising, for example, phosphoramide
(Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and references therein;,Letsinger, J. Org.
Chem. 35:3800 (1970);
Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids
Res. 14:3487 (1986); Sawai
et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470
(1988); and Pauwels et al.,
Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids
Res. 19:1437 (1991);
and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem.
Soc. 111:2321 (1989),
O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and
Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid backbones and
linkages (see Egholm, J.
Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008
(1992); Nielsen, Nature,
365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are
incorporated by reference).
Other analog nucleic acids include those with positive backbones (Denpcy et
al., Proc. Natl. Acad. Sci.
USA 92:6097 {1995); non-ionic backbones (U.S. Patent Nos. 5,386,023,
5,637,684, 5,602,240,
5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English
30:423 (1991); Letsinger
et al.; J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside &
Nucleotide 13:1597 (1994);
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Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in
Antisense Research",
Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et a(., Bioorganic & Medicinal
Chem. Lett. 4:395
(1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett.
37:743 (1996)) and non-
ribose backbones) including those described in U.S. Patent Nos. 5,235,033 and
5,034,506, and
Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in
Antisense Research")
Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also
included within the definition of nucleic acids (see Jenkins et al., Chem.
Soc. Rev. (1995) pp169-
176). Several nucleic acid analogs are described in Rawls, C & E News June 2,
1997 page 35. All of
these references are hereby expressly incorporated by reference. These
modifications of the ribose-
phosphate backbone may be done to facilitate the addition of electron transfer
moieties, or to increase
the stability and half-life of such molecules in physiological environments.
As will be appreciated by those in the art, all of these nucleic acid analogs
may find use in the present
invention. In addition, mixtures of naturally occurring nucleic acids and
analogs can be made; for
example, at the site of conductive oligomer or electron transfer moiety
attachment, an analog structure
may be used. Alternatively, mixtures of different nucleic acid analogs, and
mixtures of naturally
occuring nucleic acids and analogs may be made.
Particularly preferred are peptide nucleic acids (PNA) which includes peptide
nucleic acid analogs.
These backbones are substantially non-ionic under neutral conditions, in
contrast to the highly
charged phosphodiester backbone of naturally occurring nucleic acids. This
results in two
advantages. First, the PNA backbone exhibits improved hybridization kinetics.
PNAs have larger
changes in the melting temperature (Tm) for mismatched versus perfectly
matched basepairs. DNA
and RNA typically exhibit a 2-4~C drop in Tm for an internal mismatch. With
the non-ionic PNA
backbone, the drop is closer to 7-9~C. This allows for better detection of
mismatches. Similarly, due
to their non-ionic nature, hybridization of the bases attached to these
backbones is relatively
insensitive to salt concentration. This is particularly advantageous in the
systems of the present
invention, as a reduced salt hybridization solution has a lower Faradaic
current than a physiological
salt solution (in the range of 150 mM).
The nucleic acids may be single stranded or double stranded, as specified, or
contain portions of both
double stranded or single stranded sequence. The nucleic acid may be DNA, both
genomic and
cDNA, RNA or a hybrid, where the nucleic acid contains any combination of
deoxyribo- and ribo-
nucleotides, and any combination of bases, including uracil, adenine, thymine,
cytosine, guanine,
inosine, xathanine hypoxathanine) isocytosine, isoguanine, etc. As used
herein, the term "nucleoside"
includes nucleotides and nucleoside and nucleotide analogs, and modified
nucleosides such as amino
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modified nucleosides. In addition, "nucleoside" includes non-naturally
occuring analog structures.
Thus for example the individual units of a peptide nucleic acid, each
containing a base, are referred to
herein as a nucleoside.
The nucleosides and nucleic acids are covalently attached to a conductive
oligomer. By "conductive
oligomer" herein is meant a substantially conducting oligomer, preferably
linear, some embodiments of
which are referred to in the literature as "molecular wires". By
"substantially conducting" herein is
meant that the rate of electron transfer through the conductive oligomer is
faster than the rate of
electron transfer through single stranded nucleic acid, such that the
conductive oligomer is not the rate
limiting step in the detection of hybridization) although as noted below,
systems which use spacers
that are the rate limiting step are also acceptable. Stated differently, the
resistance of the conductive
oligomer is less than that of the nucleic acid. Preferably, the rate of
electron transfer through the
conductive oligomer is faster than the rate of electron transfer through
double stranded nucleic acid,
i.e. through the stacked n-orbitals of the double helix. Generally, the
conductive oligomer has
substantially overlapping n-orbitals, i.e. conjugated n-orbitals, as between
the monomeric units of the
conductive oligomer, although the conductive oligomer may also contain one or
more sigma (o) bonds.
Additionally, a conductive oligomer may be defined functionally by its ability
to inject or receive
electrons into or from an attached nucleic acid. Furthermore, the conductive
oligomer is more
conductive than the insulators as defined herein.
In a preferred embodiment, the conductive oligomers have a conductivity, S, of
from between about
10~ to about 10~ f2-'cm-') with from about 10-5 to about 103 W'cm-' being
preferred, with these S values
being calculated for molecules ranging from about 20A to about 200A. As
described below, insulators
have a conductivity S of about 10-' f2-'cm' or lower, with less than about 10-
a W'cm~' being preferred.
See generally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,
incorporated herein by
reference.
Desired characteristics of a conductive oligomer include high conductivity,
sufficient solubility in
organic solvents and/or water for synthesis and use of the compositions of the
invention, and
preferably chemical resistance to reactions that occur i) during nucleic acid
synthesis (such that
nucleosides containing the conductive oligomers may be added to a nucleic acid
synthesizer during
the synthesis of the compositions of the invention), ii) during the attachment
of the conductive oligomer
to an electrode, or iii) during hybridization assays.
The oligomers of the invention comprise at least two monomeric subunits) as
described herein. As is
described more fully below, oligomers include homo- and hetero-oligomers, and
include polymers.
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In a preferred embodiment, the conductive oligomer has the structure depicted
in Structure 2:
Structure 2
~Y~Bj--D Y
B a/n ~ m
As will be understood by those in the art, all of the structures depicted
herein may have additional
atoms or structures; i.e. the conductive oligomer of Structure 2 may be
attached to electron transfer
moieties, such as electrodes, transition metal complexes, organic electron
transfer moieties, and
metallocenes, and to nucleic acids, or to several of these. Unless otherwise
noted, the conductive
oligomers depicted herein will be attached at the left side to an electrode;
that is, as depicted in
Structure 2, the left "Y" is connected to the electrode as described herein
and the right "Y", if present,
is attached to the nucleic acid, either directly or through the use of a
linker, as is described herein.
In this embodiment, Y is an aromatic group, n is an integer from 1 to 50, g is
either 1 or zero, a is an
integer from zero to 10, and m is zero or 1. When g is 1, B-D is a conjugated
bond, preferably
selected from acetylene, alkene, substituted alkene, amide, azo, -C=N-
(including -N=C-, -CR=N- and
-N=CR-), -Si=Si-, and -Si=C- (including -C=Si-, -Si=CR- and -CR=Si-). When g
is zero, a is preferably
1, D is preferably carbonyl, or a heteroatom moiety, wherein the heteroatom is
selected from oxygen,
sulfur, nitrogen, silicon or phosphorus. Thus, suitable heteroatom moieties
include, but are not limited
to, -NH and -NR, wherein R is as defined herein; substituted sulfur; sulfonyl
(-S02-) sulfoxide (-SO-);
phosphine oxide (-PO- and -RPO-); and thiophosphine (-PS- and -RPS-). However,
when the
conductive oligomer is to be attached to a gold electrode, as outlined below)
sulfur derivatives are not
preferred.
By "aromatic group" or grammatical equivalents herein is meant an aromatic
monocyclic or polycyclic
hydrocarbon moiety generally containing 5 to 14 carbon atoms (although larger
polycyclic rings
structures may be made) and any carbocylic ketone or thioketone derivative
thereof, wherein the
carbon atom with the free valence is a member of an aromatic ring. Aromatic
groups include arylene
groups and aromatic groups with more than two atoms removed. For the purposes
of this application
aromatic includes heterocycle. "Heterocycle" or "heteroaryl" means an aromatic
group wherein 1 to 5
of the indicated carbon atoms are replaced by a heteroatom chosen from
nitrogen, oxygen, sulfur)
phosphorus, boron and silicon wherein the atom with the free valence is a
member of an aromatic ring,
and any heterocyclic ketone and thioketone derivative thereof. Thus,
heterocycle includes thienyl,
furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl) quinolyl, isoquinolyl,
thiazolyl, imidozyl, etc.
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Importantly, the Y aromatic groups of the conductive oligomer may be
different, i.e. the conductive
oligomer may be a heterooligomer. That is, a conductive oligomer may comprise
a oligomer of a
single type of Y groups, or of multiple types of Y groups. Thus, in a
preferred embodiment, when a
barrier monolayer is used as is described below, one or more types of Y groups
are used in the
conductive oligomer within the monolayer with a second types) of Y group used
above the monolayer
level. Thus, as is described herein, the conductive oligomer may comprise Y
groups that have good
packing efficiency within the monolayer at the electrode surface, and a second
types) of Y groups
with greater flexibility and hydrophilicity above the monolayer level to
facilitate nucleic acid
hybridization. For example, unsubstituted benzyl rings may comprise the Y
rings for monolayer
packing, and substituted benzyl rings may be used above the monolayer.
Alternatively, heterocylic
rings, either substituted or unsubstituted, may be used above the monolayer.
Additionally, in one
embodiment, heterooligomers are used even when the conductive oligomer does
not extend out of the
monolayer.
The aromatic group may be substituted with a substitution group, generally
depicted herein as R. R
groups may be added as necessary to affect the packing of the conductive
oligomers, i.e. when the
nucleic acids attached to the conductive oligomers form a monolayer on the
electrode, R groups may
be used to alter the association of the oligomers in the monolayer. R groups
may also be added to 1 )
alter the solubility of the oligomer or of compositions containing the
oligomers; 2) after the conjugation
or electrochemical potential of the system; and 3) alter the charge or
characteristics at the surface of
the monolayer.
In a preferred embodiment, when the conductive oligomer is greater than three
subunits, R groups are
preferred to increase solubility when solution synthesis is done. However, the
R groups, and their
positions) are chosen to minimally effect the packing of the conductive
oligomers on a surface,
particularly within a monolayer, as described below. In general, only small R
groups are used within
the monolayer, with larger R groups generally above the surface of the
monolayer. Thus for example
the attachment of methyl groups to the portion of the conductive oligomer
within the monolayer to
increase solubility is preferred, with attachment of longer alkoxy groups, for
example, C3 to C10, is
preferably done above the monolayer surtace. In general, for the systems
described herein, this
generally means that attachment of sterically significant R groups is not done
on any of the first two or
three oligomer subunits, depending on the length of the insulator molecules.
Suitable R groups include) but are not limited to, hydrogen, alkyl, alcohol,
aromatic) amino, amido,
vitro, ethers, esters, aldehydes, sulfonyl, silicon moieties, halogens, sulfur
containing moieties,
phosphorus containing moieties, and ethylene glycols. In the structures
depicted herein, R is
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hydrogen when the position is unsubstituted. It should be noted that some
positions may allow two
substitution groups, R and R', in which case the R and R' groups may be either
the same or different.
By "alkyl group" or grammatical equivalents herein is meant a straight or
branched chain alkyl group,
with straight chain alkyl groups being preferred. If branched, it may be
branched at one or more
positions, and unless specified, at any position. The alkyl group may range
from about 1 to about 30
carbon atoms (C1 -C30), with a preferred embodiment utilizing from about 1 to
about 20 carbon atoms
(C1 -C20), with about C1 through about C12 to about C15 being preferred, and
C1 to C5 being
particularly preferred, although in some embodiments the alkyl group may be
much larger. Also
included within the definition of an alkyl group are cycloalkyl groups such as
C5 and C6 rings, and
heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus. Alkyl also
includes heteroalkyl, with
heteroatoms of sulfur, oxygen, nitrogen, and silicone being preferred. Alkyl
includes substituted alkyl
groups. By "substituted alkyl group" herein is meant an alkyl group further
comprising one or more
substitution moieties "R", as defined above.
By "amino groups" or grammatical equivalents herein is meant -NH2, -NHR and -
NRZ groups) with R
being as defined herein.
By "nitro group" herein is meant an -NOZ group.
By "sulfur containing moieties" herein is meant compounds containing sulfur
atoms, including but not
limited to, thia-, thio- and sulfo- compounds, thiols (-SH and -SR), and
sulfides (-RSR-). By
"phosphorus containing moieties" herein is meant compounds containing
phosphorus, including, but
not limited to, phosphines and phosphates. By "silicon containing moieties"
herein is meant
compounds containing silicon.
By "ether" herein is meant an -O-R group. Preferred ethers include alkoxy
groups, with -O-(CHZ)ZCH3
and -O-(CHZ)4CH3 being preferred.
By "ester" herein is meant a -COOR group.
By "halogen" herein is meant bromine, iodine, chlorine, or fluorine. Preferred
substituted alkyls are
partially or fully halogenated alkyls such as CF3, etc.
By "aldehyde" herein is meant -RCOH groups.
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By "alcohol" herein is meant -OH groups, and alkyl alcohols -ROH.
By "amido" herein is meant -RCONH- or RCONR- groups.
By "ethylene glycol" or "(poly)ethylene glycol" herein is meant a -(O-CHz-
CHZ)~ group, although each
carbon atom of the ethylene group may also be singly or doubly substituted,
i.e. -(O-CRz-CRZ)~ , with
R as described above. Ethylene glycol derivatives with other heteroatoms in
place of oxygen (i.e. -(N-
CHZ CHZ)~ or -(S-CH2-CH2)~ , or with substitution groups) are also preferred.
Preferred substitution groups include, but are not limited to, methyl, ethyl,
propyl, alkoxy groups such
as -O-(CH2)ZCH3 and -O-(CHZ)aCH3 and ethylene glycol and derivatives thereof.
Preferred aromatic groups include, but are not limited to, phenyl, naphthyl,
naphthalene, anthracene,
phenanthroline, pyrole, pyridine, thiophene, porphyrins, and substituted
derivatives of each of these,
included fused ring derivatives.
In the conductive oligomers depicted herein, when g is 1, B-D is a bond
linking two atoms or chemical
moieties. In a preferred embodiment, B-D is a conjugated bond, containing
overlapping or conjugated
rr-orbitals.
Preferred B-D bonds are selected from acetylene (-C--__C-, also called alkyne
or ethyne), alkene (-
CH=CH-, also called ethylene), substituted alkene (-CR=CR-, -CH=CR- and -CR=CH-
), amide (-NH-
CO- and -NR-CO- or -CO-NH- and -CO-NR-), azo (-N=N-), esters and thioesters (-
CO-O-, -O-CO-, -
CS-O- and -O-CS-) and other conjugated bonds such as (-CH=N-, -CR=N-, -N=CH-
and -N=CR-), (-
SiH=SiH-, -SiR=SiH-, -SiR=SiH-, and -SiR=SiR-}, (-SiH=CH-, -SiR=CH-, -SiH=CR-,
-SiR=CR-,
CH=SiH-, -CR=SiH-, -CH=SiR-, and -CR=SiR-). Particularly preferred B-D bonds
are acetylene,
alkene, amide, and substituted derivatives of these three, and azo. Especially
preferred B-D bonds
are acetylene, alkene and amide. The oligomer components attached to double
bonds may be in the
traps or cis conformation, or mixtures. Thus) either B or D may include
carbon, nitrogen or silicon.
The substitution groups are as defined as above for R.
When g=0 in the Structure 2 conductive oligomer, a is preferably 1 and the D
moiety may be carbonyl
or a heteroatom moiety as defined above.
As above for the Y rings) within any single conductive oligomer, the B-D bonds
(or D moieties, when
g=0) may be all the same, or at least one may be different. For example, when
m is zero) the terminal
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B-D bond may be an amide bond, and the rest of the B-D bonds may be acetylene
bonds. Generally,
when amide bonds are present, as few amide bonds as possible are preferable)
but in some
embodiments all the B-D bonds are amide bonds. Thus, as outlined above for the
Y rings, one type of
B-D bond may be present in the conductive oligomer within a monolayer as
described below, and
another type above the monolayer level, to give greater flexibility for
nucleic acid hybridization.
In the structures depicted herein, n is an integer from 1 to 50, although
longer oligomers may also be
used (see for example Schumm et al., Angew. Chem. Int. Ed. Engl. 1994
33(13):1360). Without
being bound by theory, it appears that for efficient hybridization of nucleic
acids on a surface, the
hybridization should occur at a distance from the surface, i.e. the kinetics
of hybridization increase as
a function of the distance from the surface, particularly for long
oligonucleotides of 200 to 300
basepairs. Accordingly, the length of the conductive oligomer is such that the
closest nucleotide of the
nucleic acid is positioned from about 6A to about 100A (although distances of
up to 500A may be
used) from the electrode surface, with from about 15A to about 60h being
preferred and from about
25A to about 60A also being preferred. Accordingly, n will depend on the size
of the aromatic group,
but generally will be from about 1 to about 20, with from about 2 to about 15
being preferred and from
about 3 to about 10 being especially preferred.
In the structures depicted herein, m is either 0 or 1. That is, when m is 0,
the conductive oligomer may
terminate in the B-D bond or D moiety, i.e. the D atom is attached to the
nucleic acid either directly or
via a linker. In some embodiments, for example when the conductive oligomer is
attached to a
phosphate of the ribose-phosphate backbone of a nucleic acid, there may be
additional atoms, such
as a linker, attached between the conductive oligomer and the nucleic acid.
Additionally, as outlined
below, the D atom may be the nitrogen atom of the amino-modified ribose.
Alternatively, when m is 1,
the conductive oligomer may terminate in Y, an aromatic group, i.e. the
aromatic group is attached to
the nucleic acid or linker.
As will be appreciated by those in the art, a large number of possible
conductive oligomers may be
utilized. These include conductive oligomers falling within the Structure 2
and Structure 9 formulas, as
well as other conductive oligomers, as are generally known in the art,
including for example,
compounds comprising fused aromatic rings or Teflon-like oligomers, such as -
(CF2)~ , -(CHF)~ and
-(CFR)~ . See for example, Schumm et al., angew. Chem. Intl. Ed. Engl. 33:1361
(1994);Grosshenny
et al., Platinum Metals Rev. 40(1 ):26-35 (1996); Tour, Chem. Rev. 96:537-553
{1996); Hsung et al.)
Organometallics 14:4808-4815 (1995; and references cited therein, all of which
are expressly
incorporated by reference.
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Particularly preferred conductive oligomers of this embodiment are depicted
below:
Structure 3
-f-Yi---B-0 Y
m
Structure 3 is Structure 2 when g is 1. Preferred embodiments of Structure 3
include: a is zero, Y is
pyrole or substituted pyrole; a is zero) Y is thiophene or substituted
thiophene; a is zero, Y is furan or
substituted furan; a is zero, Y is phenyl or substituted phenyl; a is zero, Y
is pyridine or substituted
pyridine; a is 1, B-D is acetylene and Y is phenyl or substituted phenyl (see
Structure 5 below). A
preferred embodiment of Structure 3 is also when a is one, depicted as
Structure 4 below:
Structure 4
--r-Y-8-D Y
m
Preferred embodiments of Structure 4 are: Y is phenyl or substituted phenyl
and B-D is azo; Y is
phenyl or substituted phenyl and B-D is acetylene; Y is phenyl or substituted
phenyl and B-D is alkene;
Y is pyridine or substituted pyridine and B-D is acetylene; Y is thiophene or
substituted thiophene and
B-D is acetylene; Y is furan or substituted furan and B-D is acetylene; Y is
thiophene or furan (or
substituted thiophene or furan) and B-D are alternating alkene and acetylene
bonds.
Most of the structures depicted herein utilize a Structure 4 conductive
oligomer. However, any
Structure 4 oligomers may be substituted with a Structure 2, 3 or 9 oligomer,
or other conducting
oligomer, and the use of such Structure 4 depiction is not meant to limit the
scope of the invention.
Particularly preferred embodiments of Structure 4 include Structures 5, 6, 7
and 8, depicted below:
Structure 5
R
R R ~ R R m
Particularly preferred embodiments of Structure 5 include: n is two, m is one,
and R is hydrogen; n is
three, m is zero, and R is hydrogen; and the use of R groups to increase
solubility.
Structure 6
R
O
R R R R m
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When the B-D bond is an amide bond, as in Structure 6, the conductive
oligomers are pseudopeptide
oligomers. Although the amide bond in Structure 6 is depicted with the
carbonyl to the left, i.e. -
CONH-, the reverse may also be used, i.e. -NHCO-. Particularly preferred
embodiments of Structure
6 include: n is two, m is one, and R is hydrogen; n is three, m is zero, and R
is hydrogen (in this
embodiment, the terminal nitrogen (the D atom) may be the nitrogen of the
amino-modified ribose);
and the use of R groups to increase solubility.
Structure 7
R R R R R R
O
N
R"R n ~ R~R R ~ R~R /m
Preferred embodiments of Structure 7 include the first n is two, second n is
one, m is zero, and all R
groups are hydrogen, or the use of R groups to increase solubility.
Structure 8
R R R R
/ \ -
R R / n ~ p
Preferred embodiments of Structure 8 include: the first n is three, the second
n is from 1-3, with m
being either 0 or 1, and the use of R groups to increase solubility.
In a preferred embodiment, the conductive oligomer has the structure depicted
in Structure 9:
Structure 9
-f-C-G-C J
m
In this embodiment, C are carbon atoms, n is an integer from 1 to 50, m is 0
or 1, J is a heteroatom
selected from the group consisting of oxygen, nitrogen, silicon, phosphorus,
sulfur, carbonyl or
sulfoxide, and G is a bond selected from alkane, alkene or acetylene, such
that together with the two
carbon atoms the C-G-C group is an alkene (-CH=CH-}, substituted alkene (-
CR=CR-) or mixtures
thereof (-CH=CR- or -CR=CH-), acetylene (-C--_C-), or alkane (-CRZ-CR2-, with
R being either
hydrogen or a substitution group as described herein). The G bond of each
subunit may be the same
or different than the G bonds of other subunits; that is, alternating
oligomers of alkene and acetylene
bonds could be used, etc. However, when G is an alkane bond, the number of
alkane bonds in the
oligomer should be kept to a minimum, with about six or less sigma bonds per
conductive oligomer
being preferred. Alkene bonds are preferred, and are generally depicted
herein, although alkane and
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acetylene bonds may be substituted in any structure or embodiment described
herein as will be
appreciated by those in the art.
In some embodiments, for example when second electron transfer moieties are
not present, if m=0
then at least one of the G bonds is not an alkane bond.
In a preferred embodiment, the m of Structure 9 is zero. In a particularly
preferred embodiment, m is
zero and G is an alkene bond, as is depicted in Structure 10:
Structure 10
R
Y-f-
n ~m
R
The alkene oligomer of structure 10, and others depicted herein, are generally
depicted in the
preferred traps configuration, although ofigomers of cis or mixtures of traps
and cis may also be used.
As above, R groups may be added to alter the packing of the compositions on an
electrode, the
hydrophilicity or hydrophobicity of the oligomer, and the flexibility, i.e.
the rotational, torsional or
longitudinal flexibility of the oligomer. n is as defined above.
In a preferred embodiment, R is hydrogen, although R may be also alkyl groups
and polyethylene
glycols or derivatives.
In an alternative embodiment, the conductive oligomer may be a mixture of
different types of
oligomers, for example of structures 2 and 9.
The conductive oligomers are covalently attached to the nucleic acids. By
"covalently attached" herein
is meant that two moieties are attached by at least one bond, including sigma
bonds, pi bonds and
coordination bonds.
The nucleic acid is covalently attached to the conductive oligomer, and the
conductive oligomer is also
covalently attached to the electrode. In general, the covalent attachments are
done in such a manner
as to minimize the amount of unconjugated sigma bonds an electron must travel
from the electron
donor to the electron acceptor. Thus, linkers are generally short, or contain
conjugated bonds with few
sigma bonds.
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The covalent attachment of the nucleic acid and the conductive oligomer may be
accomplished in
several ways. In a preferred embodiment, the attachment is via attachment to
the base of the
nucleoside, via attachment to the backbone of the nucleic acid (either the
ribose, the phosphate, or to
an analogous group of a nucleic acid analog backbone), or via a transition
metal ligand) as described
below. The techniques outlined below are generally described for naturally
occuring nucleic acids,
although as will be appreciated by those in the art, similar techniques may be
used with nucleic acid
analogs.
In a preferred embodiment, the conductive ofigomer is attached to the base of
a nucleoside of the
nucleic acid. This may be done in several ways, depending on the oligomer, as
is described below. In
one embodiment, the oligomer is attached to a terminal nucleoside, i.e. either
the 3' or 5' nucleoside of
the nucleic acid. Alternatively, the conductive oligomer is attached to an
internal nucleoside.
The point of attachment to the base will vary with the base. While attachment
at any position is
possible, it is preferred to attach at positions not involved in hydrogen
bonding to the complementary
base. Thus, for example, generally attachment is to the 5 or 6 position of
pyrimidines such as uridine,
cytosine and thymine. For purines such as adenine and guanine, the linkage is
preferably via the 8
position. Attachment to non-standard bases is preferably done at the
comparable positions.
In one embodiment, the attachment is direct; that is, there are no intervening
atoms between the
conductive oligomer and the base. in this embodiment, for example, conductive
oligomers with
terminal acetylene bonds are attached directly to the base. Structure 11 is an
example of this linkage,
using a Structure 4 conductive oligomer and uridine as the base, although
other bases and conductive
oligomers can be used as will be appreciated by those in the art:
Structure 11
0
~Y-B-D~ Y
\ NH
N ~0
0
It should be noted that the pentose structures depicted herein may have
hydrogen) hydroxy,
phosphates or other groups such as amino groups attached. In addition, the
pentose and nucleoside
structures depicted herein are depicted non-conventionally, as mirror images
of the normal rendering.
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In addition, the pentose and nucleoside structures may also contain additional
groups, such as
protecting groups, at any position, for example as needed during synthesis.
In addition, the base may contain additional modifications as needed, i.e. the
carbonyl or amine groups
may be altered or protected, for example as is depicted in Figure 3 or 18.
In an alternative embodiment, the attachment is through an amide bond using a
linker as needed, as is
generally depicted in Structure 12 using uridine as the base and a Structure 4
oligomer:
Structure 12:
'O HN ,
~V-B--D~ V~ ~1
[\ ~ \H NH
0 0
Preferred embodiments of Structure 12 include Z is a methylene or ethylene.
The amide attachment
can also be done using an amino group of the base, either a naturally
occurring amino group such as
in cytidine or adenidine, or from an amino-modified base as are known in the
art.
In this embodiment, Z is a linker. Preferably, Z is a short linker of about 1
to about 5 atoms) that may
or may not contain alkene bonds. Linkers are known in the art; for example,
homo-or hetero-
bifunctional Tinkers as are well known (see 1994 Pierce Chemical Company
catalog, technical section
on cross-linkers, pages 155-200, incorporated herein by reference). Preferred
Z linkers include, but
are not limited to, alkyl groups and alkyl groups containing heteroatom
moieties, with short alkyl
groups, esters, epoxy groups and ethylene glycol and derivatives being
preferred, with propyl,
acetylene, and CZ alkene being especially preferred. Z may also be a sulfone
group, forming
sulfonamide linkages as discussed below.
In a preferred embodiment, the attachment of the nucleic acid and the
conductive oligomer is done via
attachment to the backbone of the nucleic acid. This may be done in a number
of ways, including
attachment to a ribose of the ribose-phosphate backbone, or to the phosphate
of the backbone, or
other groups of analogous backbones.
As a preliminary matter, it should be understood that the site of attachment
in this embodiment may be
to a 3' or 5' terminal nucleotide, or to an internal nucleotide, as is more
fully described below.
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In a preferred embodiment, the conductive oligomer is attached to the ribose
of the ribose-phosphate
backbone. This may be done in several ways. As is known in the art,
nucleosides that are modified at
either the 2' or 3' position of the ribose with amino groups, sulfur groups,
silicone groups, phosphorus
groups, or oxo groups can be made (Imazawa et al., J. Org. Chem., 44:2039
(1979); Hobbs et al., J.
Org. Chem. 42(4):714 (1977); Verheyden et al., J. Orrg. Chem. 36(2):250 (1971
); McGee et al., J.
Org. Chem. 61:781-785 (1996); Mikhailopulo et al., Liebigs. Ann. Chem. 513-519
(1993); McGee et al.,
Nucleosides & Nucleotides 14(6):1329 (1995), all of which are incorporated by
reference). These
modified nucleosides are then used to add the conductive oligomers.
A preferred embodiment utilizes amino-modified nucleosides. These amino-
modified riboses can then
be used to form either amide or amine linkages to the conductive oligomers. In
a preferred
embodiment, the amino group is attached directly to the ribose, although as
will be appreciated by
those in the art, short linkers such as those described herein for "Z" may be
present between the
amino group and the ribose.
in a preferred embodiment, an amide linkage is used for attachment to the
ribose. Preferably, if the
conductive oligomer of Structures 2-4 is used, m is zero and thus the
conductive oligomer terminates
in the amide bond. In this embodiment, the nitrogen of the amino group of the
amino-modified ribose
is the "D" atom of the conductive oligomer. Thus, a preferred attachment of
this embodiment is
depicted in Structure 13 (using the Structure 4 conductive oligomer):
Structure 13
~ ~ II
-t-Y-B-~Y-C-H-bas
~n
As will be appreciated by those in the art, Structure 13 has the terminal bond
fixed as an amide bond.
In a preferred embodiment, a heteroatom linkage is used, i.e. oxo, amine)
sulfur, etc. A preferred
embodiment utilizes an amine linkage. Again, as outlined above for the amide
linkages, for amine
linkages, the nitrogen of the amino-modified ribose may be the "D" atom of the
conductive oligomer
when the Structure 4 conductive oligomer is used. Thus, for example,
Structures 14 and 15 depict
nucleosides with the Structures 4 and 10 conductive oligomers, respectively,
using the nitrogen as the
heteroatom, athough other heteroatoms can be used:
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Structure 14
~ ~ \~ O
Y-B- ~Y~ Z
~n ~ ~ t~ t H bas
In Structure 14, preferably both m and t are not zero. A preferred Z here is a
methylene group, or
other aliphatic alkyl linkers. One, two or three carbons in this position are
particularly useful for
synthetic reasons; see Figure 16.
Structure 15
R
\o
Y Z~N
t H base
R
In Structure 15, Z is as defined above. Suitable linkers include methylene and
ethylene.
In an alternative embodiment) the conductive oligomer is covalently attached
to the nucleic acid via the
phosphate of the ribose-phosphate backbone (or analog) of a nucleic acid. In
this embodiment, the
attachment is direct, utilizes a linker or via an amide bond. Structure 16
depicts a direct linkage, and
Structure 17 depicts linkage via an amide bond (both utilize the Structure 4
conductive oligomer,
although Structure 9 conductive oligomers are also possible). Structures 16
and 17 depict the
conductive oligomer in the 3' position, although the 5' position is also
possible. Furthermore, both
Structures 16 and 17 depict naturally occurring phosphodiester bonds, although
as those in the art will
appreciate, non-standard analogs of phosphodiester bonds may also be used.
Structure 16
base
0
0
Y-B-(~Y~ Z-h- ~ =O or S
~ ~ m~ ~t
In Structure 16, if the terminal Y is present (i.e. m=1 ), then preferably Z
is not present (i.e. t=0). If the
terminal Y is not present, then Z is preferably present.
Structure 17 depicts a preferred embodiment, wherein the terminal B-D bond is
an amide bond, the
terminal Y is not present, and Z is a linker, as defined herein.
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Structure 17
base
O
O O
~ ~ II I
-/-V-B-D-f--V-C-p-2-P-O a 5
~n
O
In a preferred embodiment, the conductive oligomer is covalently attached to
the nucleic acid via a
transition metal ligand. In this embodiment, the conductive oligomer is
covalently attached to a ligand
which provides one or more of the coordination atoms for a transition metal.
In one embodiment, the
ligand to which the conductive oligomer is attached also has the nucleic acid
attached) as is generally
depicted below in Structure 18. Alternatively, the conductive oligomer is
attached to one ligand, and
the nucleic acid is attached to another figand, as is generally depicted below
in Structure 19. Thus, in
the presence of the transition metal, the conductive oligomer is covalently
attached to the nucleic acid.
Both of these structures depict Structure 4 conductive oligomers, although
other oligomers may be
utilized. Structures 18 and 19 depict two representative structures:
Structure 18
nuclefcadd
--/-Y-B-D~Y~Z~L
L,
Structure 19
nuGeicacid
--rY-B-D Y Z~L.~ L'
',
Lr
In the structures depicted herein, M is a metal atom, with transition metals
being preferred. Suitable
transition metals for use in the invention include, but are not limited to,
cadmium (Cd), copper (Cu),
cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium
(Rh), osmium (Os)) rhenium
(Re), platinium (Pt), scandium (Sc), titanium (Ti), Vanadium (V), chromium
(Cr), manganese (Mn),
nickel {Ni), Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir).
That is, the first series
of transition metals, the platinum metals (Ru, Rh, Pd, Os, fr and Pt), along
with Fe, Re, W, Mo and Tc,
are preferred. Particularly preferred are ruthenium, rhenium, osmium,
platinium, cobalt and iron.
L are the co-ligands, that provide the coordination atoms for the binding of
the metal ion. As will be
appreciated by those in the art, the number and nature of the co-ligands will
depend on the
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coordination number of the metal ion. Mono-, di- or polydentate co-ligands may
be used at any
position. Thus, for example, when the metal has a coordination number of six,
the L from the terminus
of the conductive oligomer, the L contributed from the nucleic acid, and r,
add up to six. Thus, when
the metal has a coordination number of six, r may range from zero (when all
coordination atoms are
provided by the other two ligands) to four, when all the co-ligands are
monodentate. Thus generally, r
will be from 0 to 8) depending on the coordination number of the metal ion and
the choice of the other
ligands.
In one embodiment, the metal ion has a coordination number of six and both the
ligand attached to the
conductive oligomer and the ligand attached to the nucleic acid are at least
bidentate; that is, r is
preferably zero, one (i.e. the remaining co-ligand is bidentate) or two (two
monodentate co-ligands are
used).
As will be appreciated in the art, the co-ligands can be the same or
different. Suitable ligands fall into
two categories: ligands which use nitrogen, oxygen, sulfur, carbon or
phosphorus atoms (depending
on the metal ion) as the coordination atoms (generally referred to in the
literature as sigma (a) donors)
and organometallic ligands such as metallocene ligands (generally referred to
in the literature as pi (n)
donors, and depicted herein as Lm). Suitable nitrogen donating ligands are
well known in the art and
include, but are not limited to, NH2; NHR; NRR'; pyridine; pyrazine;
isonicotinamide; imidazole;
bipyridine and substituted derivatives of bipyridine; terpyridine and
substituted derivatives;
phenanthroiines, particularly 1,10-phenanthroline (abbreviated phen) and
substituted derivatives of
phenanthrolines such as 4,7-dimethylphenanthroline and dipyridol(3,2-a:2',3'-
c]phenazine (abbreviated
dppz); dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);
9,10-
phenanthrenequinone diimine (abbreviated phi); 1,4,5,8-tetraazaphenanthrene
(abbreviated tap);
1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and isocyanide.
Substituted derivatives)
including fused derivatives, may also be used. In some embodiments, porphyries
and substituted
derivatives of the porphyrin family may be used. See for example,
Comprehensive Coordination
Chemistry, Ed. Wilkinson et al., Pergammon Press, 1987, Chapters 13.2 (pp73-
98), 21.1 (pp. 813-
898) and 21.3 (pp 915-957), all of which are hereby expressly incorporated by
reference.
Suitable sigma donating ligands using carbon, oxygen, sulfur and phosphorus
are known in the art.
For example, suitable sigma carbon donors are found in Cotton and Wilkenson,
Advanced Organic
Chemistry, 5th Edition, John Wiley & Sons, 1988, hereby incorporated by
reference; see page 38, for
example. Similarly, suitable oxygen ligands include crown ethers, water and
others known in the art.
Phosphines and substituted phosphines are also suitable; see page 38 of Cotton
and Wilkenson.
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The oxygen, sulfur, phosphorus and nitrogen-donating ligands are attached in
such a manner as to
allow the heteroatoms to serve as coordination atoms.
In a preferred embodiment, organometallic ligands are used. In addition to
purely organic compounds
for use as redox moieties, and various transition metal coordination complexes
with a-bonded organic
ligand with donor atoms as heterocyclic or exocyclic substituents, there is
available a wide variety of
transition metal organometallic compounds with rr-bonded organic ligands {see
Advanced Inorganic
Chemistry, 5th Ed., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;
Organometaliics, A
Concise Introduction, Elschenbroich et al., 2nd Ed., 1992, VCH; and
Comprehensive Organometallic
Chemistry l l, A Review of the Literature 1982-1994, Abel et al. Ed., Vol. 7,
chapters 7, 8, 10 & 11,
Pergamon Press, hereby expressly incorporated by reference). Such
organometallic ligands include
cyclic aromatic compounds such as the cyclopentadienide ion [C5H5(-1 )] and
various ring substituted
and ring fused derivatives, such as the indenylide (-1 ) ion, that yield a
class of
bis(cyclopentadieyl)metal compounds, {i.e. the metallocenes); see for example
Robins et al., J. Am.
Chem. Soc. 104:1882-1893 (1982); and Gassman et al., J. Am. Chem. Soc.
108:4228-4229
{1986), incorporated by reference. Of these, ferrocene [(C5H5)ZFe] and its
derivatives are prototypical
examples which have been used in a wide variety of chemical (Connelly et al.,
Chem. Rev. 96:877-
910 (1996), incorporated by reference) and electrochemical (Geiger et al.,
Advances in Organometallic
Chemistry 23:1-93; and Geiger et al., Advances in Organometallic Chemistry
24:87, incorporated by
reference) electron transfer or "redox" reactions. Metallocene derivatives of
a variety of the first,
second and third row transition metals are potential candidates as redox
moieties that are covalently
attached to either the ribose ring or the nucleoside base of nucleic acid.
Other potentially suitable
organometallic ligands include cyclic arenes such as benzene, to yield
bis(arene)metal compounds
and their ring substituted and ring fused derivatives, of which
bis(benzene)chromium is a prototypical
example, Other acyclic n-bonded ligands such as the allyl(-1 ) ion, or
butadiene yield potentially
suitable organometallic compounds, and all such ligands, in conjuction with
other n-bonded and a-
bonded ligands constitute the general class of organometallic compounds in
which there is a metal to
carbon bond. Electrochemical studies of various dimers and oligomers of such
compounds with
bridging organic ligands, and additional non-bridging ligands, as well as with
and without metal-metal
bonds are potential candidate redox moieties in nucleic acid analysis.
When one or more of the co-ligands is an organometallic ligand, the ligand is
generally attached via
one of the carbon atoms of the organometallic ligand, although attachment may
be via other atoms for
heterocyclic ligands. Preferred organometallic ligands include metallocene
ligands, including
substituted derivatives and the metalloceneophanes (see page 1174 of Cotton
and Wilkenson, supra).
For example, derivatives of metallocene ligands such as
methylcyclopentadienyl, with multiple methyl
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groups being preferred, such as pentamethylcyclopentadienyl, can be used to
increase the stability of
the metaliocene. In a preferred embodiment, only one of the two metallocene
ligands of a metallocene
are derivatized.
As described herein, any combination of ligands may be used. Preferred
combinations include: a) all
ligands are nitrogen donating ligands; b) all ligands are organometaffic
ligands; and c) the ligand at the
terminus of the conductive oiigomer is a metallocene ligand and the ligand
provided by the nucleic acid
is a nitrogen donating ligand, with the other ligands, if needed, are either
nitrogen donating ligands or
metaliocene ligands, or a mixture. These combinations are depicted in
representative structures using
the conductive oligomer of Structure 4 are depicted in Structures 20 (using
phenanthroline and amino
as representative ligands), 21 (using ferrocene as the metal-ligand
combination) and 22 (using
cyclopentadienyl and amino as representative ligands).
Structure 20
y-B-p~ y~Z
n /m
-N N
......M.",. . ~ T
4'', N~~base
Structure 21
-/-Y-B-D~Y~Z
4...,~w/((L
base
Structure 22
~Y-B-D~Y~Z t
\ /O
~~bas
In a preferred embodiment, the ligands used in the invention show altered
fluoroscent properties
depending on the redox state of the chelated metal ion. As described below,
this thus serves as an
additional mode of detection of electron transfer through nucleic acid.
In a preferred embodiment, as is described more fully below, the ligand
attached to the nucleic acid is
an amino group attached to the 2' or 3' position of a ribose of the ribose-
phosphate backbone. This
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ligand may contain a multiplicity of amino groups so as to form a polydentate
ligand which binds the
metal ion. Other preferred ligands include cyclopentadiene and phenanthroline.
As described herein, the compositions described herein of nucleosides
covalently attached to
conductive oligomers may be incorporated into a longer nucleic acid at any
number of positions)
including either the 5' or 3' terminus of the nucleic acid or any internal
position. As is outlined below,
this is generally done by adding a nucleotide with a covalently attached
conductive oligomer to an
oligonucleotide synthetic reaction at any position. After synthesis is
complete, the nucleic acid with the
covalently attached conductive oligomer is attached to an electrode. Thus) any
number of additional
nucleotides, modified or not, may be included at any position. Alternatively,
the compositions are
made via post-nucleic acid synthesis modifications.
The total length of the nucleic acid will depend on its use. Generally, the
nucleic acid compositions of
the invention are useful as oligonucleotide probes. As is appreciated by those
in the art, the length of
the probe will vary with the length of the target sequence and the
hybridization and wash conditions.
Generally, oligonucleotide probes range from about 8 to about 50 nucleotides,
with from about 10 to
about 30 being preferred and from about 12 to about 25 being especially
preferred. In some cases,
very long probes may be used, e.g. 50 to 200-300 nucleotides in length.
Also of consideration is the distance between the nucleoside containing the
electrode, i.e. a first
electron transfer moiety, and the nucleoside containing a second electron
transfer moiety. Electron
transfer proceeds between the two electron transfer moieties. Since the rate
of electron transfer is
distance dependent, the distance between the two electron transfer moieties
preferably ranges from
about 1 to about 30 basepairs, with from about 1 to about 20 basepairs being
preferred and from about
2 to about 10 basepairs being particularly preferred and from about 2 to 6
being especially preferred.
However, probe specificity can be increased by adding oligonucleotides on
either side of the electron
transfer moieties, thus increasing probe specificity without increasing the
distance an electron must
travel.
Thus, in the structures depicted herein, nucleosides may be replaced with
nucleic acids.
In a preferred embodiment, the conductive oiigomers with covalently attached
nucleosides or nucleic
acids as depicted herein are covalently attached to an electrode. Thus, one
end or terminus of the
conductive oligomer is attached to the nucleoside or nucleic acid, and the
other is attached to an
electrode. In some embodiments it may be desirable to have the conductive
oligomer attached at a
position other than a terminus, or even to have a branched conductive oligomer
that is attached to an
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electrode at one terminus and to two or more nucleosides at other termini,
although this is not
preferred. Similarly, the conductive oligomer may be attached at two sites to
the electrode.
By "electrode" herein is meant a composition, which, when connected to an
electronic device, is able
to sense a current or charge and convert it to a signal. Thus, an electrode is
an electron transfer
moiety as described herein. Preferred electodes are known in the art and
include, but are not limited
to, certain metals and their oxides, including gold; platinum; palladium;
silicon; aluminum; metal oxide
electrodes including platinum oxide) titanium oxide, tin oxide, indium tin
oxide, palladium oxide, silicon
oxide, aluminum oxide, molybdenum oxide (Mo206), tungsten oxide (W03) and
ruthenium oxides; and
carbon (including glassy carbon electrodes, graphite and carbon paste).
Preferred electrodes include
gold, silicon, carbon and metal oxide electrodes.
The electrodes described herein are depicted as a flat surface, which is only
one of the possible
conformations of the electrode and is for schematic purposes only. The
conformation of the electrode
will vary with the detection method used. For example, flat planar electrodes
may be preferred for
optical detection methods, or when arrays of nucleic acids are made, thus
requiring addressable
locations for both synthesis and detection. Alternatively, for single probe
analysis) the electrode may
be in the form of a tube, with the conductive oligomers and nucleic acids
bound to the inner surface.
This allows a maximum of surface area containing the nucleic acids to be
exposed to a small volume
of sample.
The covalent attachment of the conductive oligomer containing the nucleoside
may be accomplished
in a variety of ways, depending on the electrode and the conductive oligomer
used. Generally, some
type of linker is used, as depicted below as "A" in Structure 23) where X is
the conductive oligomer,
and the hatched surface is the electrode:
Structure 23
A -X nuGeosiCe
In this embodiment, A is a linker or atom. The choice of "A" will depend in
part on the characteristics
of the electrode. Thus, for example, A may be a sulfur moiety when a gold
electrode is used.
Alternatively, when metal oxide electrodes are used, A may be a silicon
(silane) moiety attached to the
oxygen of the oxide (see for example Chen et al., Langmuir 10:3332-3337
(1994); Lenhard et al., J.
Electroanal. Chem. 78:195-201 (1977), both of which are expressly incorporated
by reference). When
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carbon based electrodes are used, A may be an amino moiety (preferably a
primary amine; see for
example Deinhammer et al., Langmuir 10:1306-1313 (1994)). Thus, preferred A
moieties include, but
are not limited to, silane moieties, sulfur moieties (including alkyl sulfur
moieties), and amino moieties.
In a preferred embodiment, epoxide type linkages with redox polymers such as
are known in the art
are not used.
Although depicted herein as a single moiety, the conductive oligomer may be
attached to the electrode
with more than one "A" moiety; the "A" moieties may be the same or different.
Thus, for example,
when the electrode is a gold electrode, and "A" is a sulfur atom or moiety,
such as generally depicted
below in Structure 27, multiple sulfur atoms may be used to attach the
conductive ofigomer to the
electrode) such as is generally depicted below in Structures 24, 25 and 26. As
will be appreciated by
those in the art, other such structures can be made. In Structures 24, 25 and
26, the A moiety is just a
sulfur atom, but substituted sulfur moieties may also be used.
Structure 24
5 ~ ~ X-nud.ro.W
I
Structure 25
& R
S"'X- nuGlCltaCld
Structure 26
S~ R
S~%-nud.k.dd
It should also be noted that similar to Structure 26, it may be possible to
have a a conductive oiigomer
terminating in a single carbon atom with three sulfur moities attached to the
electrode.
In a preferred embodiment, the electrode is a gold electrode, and attachment
is via a sulfur linkage as
is well known in the art, i.e. the A moiety is a sulfur atom or moiety.
Although the exact characteristics
of the gold-sulfur attachment are not known, this linkage is considered
covalent for the purposes of
this invention. A representative structure is depicted in Structure 27.
Structure 27 depicts the "A"
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linker as comprising just a sulfur atom, although additional atoms may be
present (i.e. linkers from the
sulfur to the conductive oligomer or substitution groups).
Structure 27
s ~v -e --o j--f--v ~t -~,a.W as
In a preferred embodiment, the electrode is a carbon electrode, i.e. a glassy
carbon electrode, and
attachment is via a nitrogen of an amine group. A representative structure is
depicted in Structure 28.
Again, additional atoms may be present, i.e. Z type linkers.
Structure 28
1 5 N~ Y-B--O~ Y~Z~ nuGeicacid
Structure 29
-SI~Y-B-D~Y~2~nuGeicacid
~ ~ ~t
In Structure 29, the oxygen atom is from the oxide of the metal oxide
electrode. The Si atom may also
contain other atoms, i.e. be a silicon moiety containing substitution groups.
Thus, in a preferred embodiment, electrodes are made that comprise conductive
oligomers attached to
nucleic acids for the purposes of hybridization assays, as is more fully
described herein. As will be
appreciated by those in the art, electrodes can be made that have a single
species of nucleic acid, i.e.
a single nucleic acid sequence, or multiple nucleic acid species.
In addition, as outlined herein, the use of a solid support such as an
electrode enables the use of
these gene probes in an array form. The use of oligonucleotide arrays are well
known in the art. In
addition, techniques are known for "addressing" locations within an electrode
and for the surface
modification of electrodes. Thus, in a preferred embodiment, arrays of
different nucleic acids are laid
down on the electrode, each of which are covalently attached to the electrode
via a conductive linker.
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In this embodiment) the number of different probe species of oligonucleotides
may vary widely, from
one to thousands, with from about 4 to about 100,000 being preferred, and from
about 10 to about
10,000 being particularly preferred.
In a preferred embodiment, the electrode further comprises a passivation
agent, preferably in the form
of a monolayer on the electrode surface. As outlined above, the efficiency of
oligonucieotide
hybridization may increase when the oligonucleotide is at a distance from the
electrode. A
passivation agent layer facilitates the maintenance of the nucleic acid away
from the electrode
surface. In addition, a passivation agent serves to keep charge carriers away
from the surface of the
electrode. Thus, this layer helps to prevent electrical contact between the
electrodes and the electron
transfer moieties, or between the electrode and charged species within the
solvent. Such contact can
result in a direct "short circuit" or an indirect short circuit via charged
species which may be present in
the sample. Accordingly, the monolayer of passivation agents is preferably
tightly packed in a uniform
layer on the electrode surface, such that a minimum of "holes" exist.
Alternatively, the passivation
agent may not be in the form of a monolayer, but may be present to help the
packing of the conductive
oligomers or other characteristics.
The passivation agents thus serve as a physical barrier to block solvent
accesibility to the electrode.
As such) the passivation agents themselves may in fact be either (1)
conducting or (2) nonconducting,
i.e. insulating, molecules. Thus, in one embodiment, the passivation agents
are conductive oligomers,
as described herein, with or without a terminal group to block or decrease the
transfer of charge to the
electrode. Other passivation agents which may be conductive include oligomers
of -(CFZ)", -(CHF)~-
and -(CFR)~ . In a preferred embodiment, the passivation agents are insulator
moieties.
An "insulator" is a substantially nonconducting oligomer, preferably linear.
By "substantially
nonconducting" herein is meant that the rate of electron transfer through the
insulator is slower than
the rate of electron transfer through the stacked n-orbitals of double
stranded nucleic acid. Stated
differently, the electrical resistance of the insulator is higher than the
electrical resistance of the nucleic
acid. fn a preferred embodiment, the rate of electron transfer through the
insulator is slower than or
comparable to the rate through single stranded nucleic acid. Similarly, the
rate of electron transfer
through the insulator is preferrably slower than the rate through the
conductive oligomers described
herein. It should be noted however, as outlined in the Examples, that even
oligomers generally
considered to be insulators, such as -(CHZ),s molecules, still may transfer
electrons, albeit at a slow
rate.
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In a preferred embodiment, the insulators have a conductivity, S, of about 10-
' f~-'cm-' or lower, with
less than about 10-8 f2-'cm-' being preferred. See generally Gardner et al.,
supra.
Generally, insulators are alkyl or heteroalkyl oligomers or moieties with
sigma bonds, although any
particular insulator molecule may contain aromatic groups or one or more
conjugated bonds. By
"heteroalkyl" herein is meant an alkyl group that has at least one heteroatom,
i.e. nitrogen, oxygen,
sulfur, phosphorus, silicon or boron included in the chain. Alternatively, the
insulator may be quite
similar to a conductive oligomer with the addition of one or more heteroatoms
or bonds that serve to
inhibit or slow, preferably substantially, electron transfer.
The passivation agents, including insulators, may be substituted with R groups
as defined herein to
alter the packing of the moieties or conductive oligomers on an electrode, the
hydrophilicity or
hydrophobicity of the insulator, and the flexibility, i.e. the rotational)
torsional or longitudinal flexibility of
the insulator. For example, branched alkyl groups may be used. In addition,
the terminus of the
passivation agent, including insulators, may contain an additional group to
influence the exposed
surface of the monolayer. For example, there may be negatively charged groups
on the terminus to
form a negatively charged surface such that when the nucleic acid is DNA or
RNA the nucleic acid is
repelled or prevented from lying down on the surtace, to facilitate
hybridization. Preferred passivation
agent terminal groups include -NH2) -OH, -COOH, -CH3, and (poly)alkyloxides
such as (poly)ethylene
glycol, with -OCHzCHzOH, -(OCH2CH20)ZH and -(OCHZCH20)3H being preferred and
the latter being
particularly preferred.
The length of the passivation agent will vary as needed. As outlined above, it
appears that
hybridization is more efficient at a distance from the surface. Thus, the
length of the passivation
agents is similar to the length of the conductive oligomers, as outlined
above. In addition) the
conductive oligomers may be basically the same length as the passivation
agents or longer than them,
resulting in the nucleic acids being more accessible to the solvent for
hybridization.
The monolayer may comprise a single type of passivation agent, including
insulators) or different
types.
Suitable insulators are known in the art, and include, but are not limited to,
-(CHZ)~ , -(CRH)~ , and
-(CRZ)~ , ethylene glycol or derivatives using other heteroatoms in place of
oxygen, i.e. nitrogen or
sulfur (sulfur derivatives are not preferred when the electrode is gold).
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The passivation agents are generally attached to the electrode in the same
manner as the conductive
oligomer, and may use the same "A" linker as defined above.
It has been found that the present compositions result in excellent
hybridization kinetics of target
sequence hybridizing to probes attached to a surface. Thus, the compositions
and methods of the
present invention may also be used in nucleic acid detection systems that do
not rely on electron
transfer for detection.
Accordingly, in a preferred embodiment, the compositions of the present
invention find use in standard
nucleic acid assays, such as general array-type technologies, i.e. the
electrode may serve just as a
solid support, with detection proceeding using techniques well known in the
art, such as fluoroscence
or radioisotope labelling. in this embodiment, the compositions may comprise a
conductive oligomer
covalently attached to a nucleoside or nucleic acid. It will be recognized by
those in the art that the
conductive oligomers in this embodiment may not be functioning as conductive
oligomers but rather as
linkers that can be used to keep the nucleic acids off the surface. The
conductive oligomer, or linker,
in this case may have the structure depicted in Structures 2, 3, 4, 9 or 10.
However, when the linker
has the structure depicted in Structure 9, preferably at least one of the G
bonds is not alkane,
particularly when m=0.
In a preferred embodiment, the composition comprises (a) a solid support
comprising a monoiayer of
passivation agent; (b) a nucleic acid comprising at least one nucleoside,
wherein the nucleic acid is
covalently attached to the solid support with a linker. The solid support is
the electrode, which is not
necessarily functioning as an electron transfer moiety in this embodiment. The
monolayer of
passivation agent is shown herein to result in excellent hybridization
kinetics and can therefore be
quite useful in both electron-transfer based and traditional nucleic acid
detection schemes. The linkers
are preferably the conductive oligomers of the invention, although as outlined
above, they may not be
functioning as conductive moieties. In this embodiment) the conductive
oligomer, or linker, in this
case, may have the structure depicted in Structures 2, 3, 4, 9 or 10. However,
when the linker has the
structure depicted in Structure 9, preferably at least one of the G bonds is
not alkane, particularly
when m=0.
In this embodiment, it is possible to have each nucleic acid be the same, as
an "anchor sequence",
such that a second sequence can be added which contains the probe sequence and
a sequence
complementary to the anchor sequence. In this way, standard arrays of using
either the same or
different anchor sequences can be made, which then can be used to generate
custom arrays using
novel probe sequences linked to complementary anchor regions.
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Thus, in this embodiment, compositions are provided comprising a conductive
oligomer covalently
attached to an electrode and to a first single stranded anchor sequence. A
second single stranded
nucleic acid is provided, which contains a probe region and a region
substantially complementary to
the anchor sequence, such that a first hybridization complex is formed between
the two
complementary anchor regions, leaving the probe region as a single stranded
region. A target
sequence which is substantially complementary to the probe region is then
added to form a second
hybridization complex. The second hybridization complex is then detected, for
example by labelling
the target nucleic acid as is well known in the art.
As outlined herein, it is also possible to have compositions comprising
electrodes with conductive
oligomers attached to probe nucleic acids, without second electron transfer
moieties, and soluble
second probe sequences with second electron transfer moieties. Upon binding of
the target
sequence, which contains a first target domain for the first probe sequence
and a second target
domain for the second probe sequence) which preferably are adjacent, electron
transfer may occur.
Alternatively, it may be the target sequence which contains the second
electron transfer moiety.
Similar to methods which rely on amplification and labelling of target
sequences, the target nucleic
acid may be labelled with a second electron transfer moiety which then can be
used to effect electron
transfer upon formation of the hybridization complex.
In an alternate embodiment, a hybridization indicator may serve as either the
sole second electron
transfer moiety or as an additional second electron transfer moiety, as is
generally described below.
In a preferred embodiment) the compositions of the present invention comprise
a conductive oligomer,
covalently attached to both an electrode, which serves as a first electron
transfer moiety, and a nucleic
acid, which has at least a second covalently attached electron transfer
moiety. As noted herein, the
conductive oligomer and the second electron transfer moiety may be attached at
any position of the
nucleic acid.
In one embodiment, a nucleic acid is modified with more than two electron
transfer moieties. For
example, to increase the signal obtained from the probe, or alter the required
detector sensitivity, a
plurality of electron transfer moieties may be used. See PCT publication WO
95I15971. For example,
the conductive oligomer may be attached to an internal nucleoside, with second
electron transfer
moieties (ETM) attached both 5' and 3' to the nucleoside containing the
conductive oligomer, as is
generally depicted in Structure 29A. In one embodiment, the two additional
electron transfer moieties
are the same, and are placed the same distance away from the conductive
oligomer, to result in a
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uniform signal. Alternatively, the additional electron transfer moieties may
be different andlor placed at
different distances from the conductive oligomer.
Structure 29A
ITM
( ~ uGeoside)n
F~-X-FZ-nucleoside
(nuGeoside)n
ETM
The terms "electron donor moiety", "electron acceptor moiety", and "electron
transfer moieties" or
grammatical equivalents herein refers to molecules capable of electron
transfer under certain
conditions. It is to be understood that electron donor and acceptor
capabilities are relative; that is, a
molecule which can lose an electron under certain experimental conditions will
be able to accept an
electron under different experimental conditions. It is to be understood that
the number of possible
electron donor moieties and electron acceptor moieties is very large, and that
one skilled in the art of
electron transfer compounds will be able to utilize a number of compounds in
the present invention.
Preferred electron transfer moieties include, but are not limited to,
transition metal complexes, organic
electron transfer moieties, and electrodes.
In a preferred embodiment, the electron transfer moieties are transition metal
complexes. Transition
metals are those whose atoms have a partial or complete d shell of electrons.
Suitable transition
metals for use in the invention are listed above.
The transition metals are complexed with a variety of ligands, L, defined
above, to form suitable
transition metal complexes, as is well known in the art.
In addition to transition metal complexes, other organic electron donors and
acceptors may be
covalently attached to the nucleic acid for use in the invention. These
organic molecules include, but
are not limited to, riboflavin, xanthene dyes, azine dyes, acridine orange,
N,N'-dimethyl-2,7-
diazapyrenium dichloride (DAPZ+), methylviologen, ethidium bromide, quinones
such as N,N'-
dimethylanthra(2,1,9-det..6,5,10-d'e'f')diisoquinoline dichloride (ADIQ2');
porphyrins ([meso-tetrakis(N-
methyl-x-pyridinium)porphyrin tetrachloride], varlamine blue B hydrochloride,
Bindschedler's green;
2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crest blue (3-
amino-9-dimethyl-amino-
10-methylphenoxyazine chloride), methylene blue; Nile blue A
(aminoaphthodiethylaminophenoxazine
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sulfate), indigo-5,5',7,7'-tetrasulfonic acid, indigo-5,5',7-trisulfonic acid;
phenosafranine, indigo-5-
monosulfonic acid; safranine T; bis(dimethyiglyoximato)-iron(II) chloride;
induline scarlet, neutral red,
anthracene, coronene, pyrene, 9-phenylanthracene, rubrene, binaphthyl, DPA,
phenothiazene,
fluoranthene, phenanthrene, chrysene, 1,8-Biphenyl-1,3,5,7-octatetracene,
naphthalene,
acenaphthalene, perylene, TMPD and analogs and subsitituted derivatives of
these compounds.
In one embodiment, the electron donors and acceptors are redox proteins as are
known in the art.
However) redox proteins in many embodiments are not preferred.
The choice of the specific electron transfer moieties will be influenced by
the type of electron transfer
detection used, as is generally outlined below.
In a preferred embodiment, these electron transfer moieties are covalently
attached to the nucleic acid
in a variety of positions. In a preferred embodiment, the attachment is via
attachment to the base of
the nucleoside, or via attachment to the backbone of the nucleic acid,
including either to a ribose of the
ribose-phosphate backbone or to a phosphate moiety. In the preferred
embodiments, the
compositions of the invention are designed such that the electron transfer
moieties are as close to the
"rr-way" as possible without significantly disturbing the secondary and
tertiary structure of the double
helical nucleic acid, particularly the Watson-Crick basepairing.
Alternatively, the attachment can be
via a conductive oligomer, which is used as outlined above with a nucleoside
and an electrode; that is)
an electron transfer moiety may be covalentfy attached to a conductive
oligomer at one end and to a
nucleoside at the other, thus forming a general structure depicted in
Structure 30:
Structure 30
A -X - IuGaoSlde
(nudeoslCe)
/ hucleoslde
/x
ETM
In Structure 30) ETM is an electron transfer moiety) X is a conductive
oligomer, and q is an integer
from zero to about 25, with preferred q being from about 2 to about 10.
Additionally, linker moieties,
for example as are generally described herein as "Z", may also be present
between the nucleoside
and the conductive oligomer, andlor between the conductive oligomer and the
electron transfer
moiety. The depicted nucleosides may be either terminal or internal
nucleosides, and are usually
separated by a number of nucleosides.
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In a preferred embodiment, the second electron transfer moiety is attached to
the base of a
nucleoside, as is generally outlined above for attachment of the conductive
oligomer. This is
preferably done to the base of an internal nucleoside. Surprisingly and
unexpectedly, this attachment
does not perturb the Watson-Crick basepairing of the base to Which the
electron transfer moiety is
attached, as long as the moiety is not too large. In fact, it appears that
attachment at this site actually
results in less perturbation than attachment at the ribose of the ribose-
phosphate backbone) as
measured by nucleic acid melting curves.
Thus, when attachment to an internal base is done, the size of the second
electron transfer moiety
should be such that the structure of double stranded nucleic acid containing
the base-attached
electron transfer moiety is not significantly disrupted, and will not disrupt
the annealing of single
stranded nucleic acids. Preferrably, then, ligands and full second electron
transfer moieties are
generally smaller than the size of the major groove of double stranded nucleic
acid.
Alternatively, the second electron transfer moiety can be attached to the base
of a terminal nucleoside.
Thus, when the target sequence to be detected is n nucleosides long, a probe
can be made which has
the second electron transfer moiety attached at the n base. Alternatively, the
probe may contain an
extra terminal nucleoside at an end of the nucleic acid (n + 1 or n + 2),
which are used to covalently
attach the electron transfer moieties but which do not participate in basepair
hybridization.
Additionally, it is preferred that upon probe hybridization, the terminal
nucleoside containing the
electron transfer moiety covalently attached at the base be directly adjacent
to Watson-Crick
basepaired nucleosides; that is, the electron transfer moiety should be as
close as possible to the
stacked n-orbitals of the bases such that an electron travels through a
minimum of a bonds to reach
the "n-way", or alternatively can otherwise electronically contact the rr-way.
The covalent attachment to the base will depend in part on the second electron
transfer moiety
chosen, but in general is similar to the attachment of conductive oligomers to
bases, as outlined
above. In a preferred embodiment, the second electron transfer moiety is a
transition metal complex,
and thus attachment of a suitable metal ligand to the base leads to the
covalent attachment of the
electron transfer moiety. Alternatively, similar types of linkages may be used
for the attachment of
organic electron transfer moieties, as will be appreciated by those in the
art.
fn one embodiment, the C4 attached amino group of cytosine, the C6 attached
amino group of
adenine, or the C2 attached amino group of guanine may be used as a transition
metal ligand,
although in this embodiment attachment at a terminal base is preferred since
attachment at these
positions will perturb Watson-Crick basepairing.
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Ligands containing aromatic groups can be attached via acetylene linkages as
is known in the art (see
Comprehensive Organic Synthesis, Trost et al., Ed., Pergamon Press, Chapter
2.4: Coupling
Reactions Between sp2 and sp Carbon Centers, Sonogashira, pp521-549, and pp950-
953, hereby
incorporated by reference). Structure 31 depicts a representative structure in
the presence of the
metal ion and any other necessary ligands; Structure 31 depicts uridine,
although as for all the
structures herein, any other base may also be used.
Structure 31
0
15 La is a ligand, which may include nitrogen, oxygen, sulfur or phosphorus
donating ligands or
organometallic ligands such as metallocene ligands. Suitable Le ligands
include, but not limited to,
phenanthrofine, imidazole, bpy and terpy. L, and M are as defined above.
Again, it will be appreciated
by those in the art, a conductive oligomer may be included between the
nucleoside and the electron
transfer moiety.
Similarly, as for the conductive oligomers, the linkage may be done using a
linker, which may utilize an
amide linkage (see generally Telser et al., J. Am. Chem. Soc. 111:7221-7226
(1989); Telser et al., J.
Am. Chem. Soc. 111:7226-7232 ( 1989), both of which are expressly incorporated
by reference).
These structures are generally depicted below in Structure 32, which again
uses uridine as the base,
although as above, the other bases may also be used:
Structure 32
L
M
In this embodiment, L is a ligand as defined above, with L, and M as defined
above as well.
Preferably, L is amino, phen, byp and terpy.
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In a preferred embodiment, the second electron transfer moiety attached to a
nucleoside is a
metallocene; i.e. the L and L~ of Structure 32 are both metallocene ligands,
Lm, as described above.
Structure 33 depicts a preferred embodiment wherein the metallocene is
ferrocene, and the base is
uridine, although other bases may be used:
Structure 33
,o
Preliminary data suggest that Structure 33 may cyclize, with the second
acetylene carbon atom
attacking the carbonyl oxygen, forming a furan-like structure.
Preferred metallocenes include ferrocene, cobaltocene and osmiumocene.
Thus, in a preferred embodiment, the invention provides metallocenes
covalently attached to
nucleosides. In a preferred embodiment, the metallocene is attached to the
base of a nucleoside. In
alternate preferred embodiment, the metallocene is attached to the ribose of a
nucleoside.
Alternatively, the metallocene may be attached to the phosphate of the
backbone, although this is
generally not preferred. If attachment is to the phosphate, generally there
will be no more than about
2-4 atoms between the phosphate atom and a carbon of a ring of the
metallocene. In a preferred
embodiment, the metallocene is ferrocene or substituted ferrocene.
In a preferred embodiment, the second electron transfer moiety is attached to
a ribose at any position
of the ribose-phosphate backbone of the nucleic acid, i.e. either the 5' or 3'
terminus or any internal
nucleoside. As is known in the art, nucleosides that are modified at either
the 2' or 3' position of the
ribose can be made, with nitrogen, oxygen, sulfur and phosphorus-containing
modifications possible.
Amino-modified ribose is preferred. See generally PCT publication WO 95I15971,
incorporated herein
by reference. These modification groups may be used as a transition metal
ligand, or as a chemically
functional moiety for attachment of other transition metal ligands and
organometallic ligands, or
organic electron donor moieties as will be appreciated by those in the art. In
this embodiment, a linker
such as depicted herein for "Z" may be used as well, or a conductive oligomer
between the ribose and
the electron transfer moiety. Preferred embodiments utilize attachment at the
2' or 3' position of the
ribose, with the 2' position being preferred. Thus for example, the conductive
oligomers depicted in
-42-
Structure 13,14 and 15 may be replaced by electron transfer moieties;
alternatively, as is depicted in
Structure 30, the electron transfer moieties may be added to the free terminus
of the conductive
oligomer.
In a preferred embodiment, a metallocene serves as the second electron
transfer moiety, and is
attached via an amide bond as depicted below in Structure 34. The example
outline the synthesis of
a preferred compound when the metallocene is ferrocene.
Image
Amine linkages, or linkages via other heteroatoms, are also possible.
In a preferred embodiment, the second electron transfer moiety is attached to
a phosphate at any
position of the ribose-phosphate backbone of the nucleic acid. This may be
done in a variety of ways.
In one embodiment, phosphodiester bond analogs such as phosphoramide or
phosphoramidite
linkage may be incorporated into a nucleic acid as a transition metal ligand
(see PCT publication WO
95/15971, incorporated by reference). Alternatively, the conductive oligomers
depicted in Structures
16 and 17 may be replaced by electron transfer moieties; alternatively, the
electron transfer moieties
may be added to the free terminus of the conductive oligomer.
Preferred electron transfer moieties for covalent attachment to a single
stranded nucleic acid include,
but are not limited to, transition metal complexes, including metallocenes and
substituted metallocenes
such as metalloceneophanes, and complexes of Ru, Os, Re and Pt. Particularly
preferred are
ferrocene and its derivatives (particularly pentamethylferrocene and
ferroceneophane) and complexes
of transition metals including Ru, Os, Re and Pt containing one or more amine
or polyamine,
imidazole, phenathroline, pyridine, bipyridine and or terpyridine and their
derivatives. For Pt, additional
preferred ligands include the dlimine dithiolate complexes such as quinoxaline-
2,3-dithiolate
complexes.
As described herein, the invention provides compositions containing electrodes
as a first electron
transfer moiety linked via a conductive oligomer to a nucleic acid which has
at least second electron.
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transfer moiety covalently attached. Any combination of positions of electron
transfer moiety
attachment can be made; i.e. an electrode at the 5' terminus, a second
electron transfer moiety at an
internal position; electrode at the 5' terminus, second moiety at the 3' end;
second moiety at the 5'
terminus, electrode at an internal position; both electrode and second moiety
at internal positions;
electrode at an internal position, second moiety at the 3' terminus, etc. A
preferred embodiment
utilizes both the electrode and the second electron transfer moiety attached
to internal nucleosides.
The compositions of the invention may additionally contain one or more labels
at any position. By
"label" herein is meant an element (e.g. an isotope) or chemical compound that
is attached to enable
the detection of the compound. Preferred labels are radioactive isotopic
labels, and colored or
fluorescent dyes. The labels may be incorporated into the compound at any
position. In addition, the
compositions of the invention may also contain other moieties such as cross-
linking agents to facilitate
cross-linking of the target-probe complex. See for example, Lukhtanov et al.,
Nucl. Acids. Res.
24(4):683 (1996) and Tabone et al., Biochem. 33:375 (1994), both of which are
expressly incorporated
by reference.
The compositions of the invention are generally synthesized as outlined below,
generally utilizing
techniques well known in the art. As will be appreciated by those in the art,
many of the techniques
outlined below are directed to nucleic acids containing a ribose-phosphate
backbone. However, as
outlined above, many alternate nucleic acid analogs may be utilized, some of
which may not contain
either ribose or phosphate in the backbone. In these embodiments, for
attachment at positions other
than the base, attachment is done as will be appreciated by those in the art,
depending on the
backbone. Thus, for example, attachment can be made at the carbon atoms of the
PNA backbone, as
is described below, or at either terminus of the PNA.
The compositions may be made in several ways. A preferred method first
synthesizes a conductive
oligomer attached to the nucleoside, with addition of additional nucleosides
followed by attachment to
the electrode. A second electron transfer moiety, if present, may be added
prior to attachment to the
electrode or after. Alternatively, the whole nucleic acid may be made and then
the completed
conductive oligomer added, followed by attachment to the electrode.
Alternatively, the conductive
oligomer and monolayer (if present) are attached to the electrode first,
followed by attachment of the
nucleic acid. The latter two methods may be preferred when conductive
oligomers are used which are
not stable in the solvents and under the conditions used in traditional
nucleic acid synthesis.
In a preferred embodiment, the compositions of the invention are made by first
forming the conductive
oligomer covalently attached to the nucleoside, followed by the addition of
additional nucleosides to
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form a nucleic acid, including, if present, a nucleoside containing a second
electron transfer moiety,
with the last step comprising the addition of the conductive oligomer to the
electrode.
The attachment of the conductive oligomer to the nucleoside may be done in
several ways. In a
preferred embodiment, all or part of the conductive oligomer is synthesized
first (generally with a
functional group on the end for attachment to the electrode), which is then
attached to the nucleoside.
Additional nucleosides are then added as required, with the last step
generally being attachment to the
electrode. Alternatively, oligomer units are added one at a time to the
nucleoside, with addition of
additional nucleosides and attachment to the electrode.
A general outline of a preferred embodiment is depicted in Figure 1, using a
phenyl-acetylene oligomer
as generally depicted in Structure 5. Other conductive oligomers will be made
using similar
techniques, such as heterooligomers, or as known in the art. Thus, for
example, conductive oligomers
using alkene or acetylene bonds are made as is known in the art.
The conductive oligomer is then attached to a nucleoside that may contain one
(or more) of the
oligomer units, attached as depicted herein.
In a preferred embodiment, attachment is to a ribose of the ribose-phosphate
backbone. Thus, Figure
1 depicts attachment via an amide linkage, and Figures 2 and 16 depict the
synthesis of compounds
with amine linkages. In a preferred embodiment, there is at least a methylene
group or other short
aliphatic alkyl groups (as a Z group) between the nitrogen attached to the
ribose and the aromatic ring
of the conductive oiigomer. A representative synthesis is shown in Figure 16.
Alternatively, attachment is via a phosphate of the ribose-phosphate backbone.
Examples of two
synthetic schemes are shown in Figure 4 (synthesis of Structure 16 type
compounds) and Figure 5
(synthesis of Structure 16 type compounds). Although both Figures show
attachment at the 3' position
of the ribose, attachment can also be made via the 2' position. In Figure 5, Z
is an ethylene linker,
although other linkers may be used as well, as will be appreciated by those in
the art.
In a preferred embodiment, attachment is via the base. A general scheme is
depicted in Figure 3)
using uridine as the nucleoside and a phenylene-acetylene conductive oligomer.
As will be
appreciated in the art, amide linkages ace also possible, such as depicted in
Structure 12, using
techniques well known in the art. In a preferred embodiment, protecting groups
may be added to the
base prior to addition of the conductive oligomers, as is generally outlined
in Figures 18 and 19. In
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addition, the palladium cross-coupling reactions may be altered to prevent
dimerization problems; i.e.
two conductive oligomers dimerizing, rather than coupling to the base.
Alternatively, attachment to the base may be done by making the nucleoside
with one unit of the
oligomer, followed by the addition of others.
Once the modified nucleosides are prepared, protected and activated, prior to
attachment to the
electrode, they may be incorporated into a growing oligonucleotide by standard
synthetic techniques
(Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, UK
1984; Eckstein) in
several ways. In one embodiment, one or more modified nucleosides are
converted to the
triphosphate form and incorporated into a growing oligonucleotide chain by
using standard molecular
biology techniques such as with the use of the enzyme DNA polymerise I, T4 DNA
polymerise, T7
DNA polymerise, Taq DNA polymerise, reverse transcriptase, and RNA
polymerises. For the
incorporation of a 3' modified nucleoside to a nucleic acid, terminal
deoxynucleotidyltransferase may
be used. (Ratliff, Terminal deoxynucleotidyitransferase. In The Enzymes, Vot
14A. P.D. Boyer ed. pp
105-118. Academic Press, San Diego, CA. 1981 ). Alternatively, and preferably,
the amino nucleoside
is converted to the phosphoramidite or H-phosphonate form, which are then used
in solid-phase or
solution syntheses of oligonucleotides. In this way the modified nucleoside,
either for attachment at
the ribose (i.e. amino- or thiol-modified nucleosides) or the base, is
incorporated into the
oligonucleotide at either an internal position or the 5' terminus. This is
generally done in one of two
ways. First, the 5' position of the ribose is protected with 4',4-
dimethoxytrityl (DMT) followed by
reaction with either 2-cyanoethoxy-bis-diisopropylaminophosphine in the
presence of
diisopropylammonium tetrazolide, or by reaction with chlorodiisopropylamino 2'-
cyanoethyoxyphosphine, to give the phosphoramidite as is known in the art;
although other techniques
may be used as will be appreciated by those in the art. See Gait, supra;
Caruthers, Science 230:281
(1985), both of which are expressly incorporated herein by reference.
For attachment of an electron transfer moiety to the 3' terminus, a preferred
method utilizes the
attachment of the modified nucleoside to controlled pore glass (CPG) or other
oligomeric supports. In
this embodiment, the modified nucleoside is protected at the 5' end with DMT,
and then reacted with
succinic anhydride with activation. The resulting succinyl compound is
attached to CPG or other
oligomeric supports as is known in the art. Further phosphoramidite
nucleosides are added, either
modified or not, to the 5' end after deprotection. Thus, the present invention
provides conductive
oligomers covalently attached to nucleosides attached to solid oligomeric
supports such as CPG, and
phosphoramidite derivatives of the nucleosides of the invention.
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The growing nucleic acid chain may also comprise at (east one nucleoside with
covalently attached
second electron transfer moiety. As described herein, modified nucleosides
with covalently attached
second electron transfer moieties may be made, and incorporated into the
nucleic acid as outlined
above for the conductive oligomer-nucleosides. When a transition metal complex
is used as the
second electron transfer moiety, synthesis may occur in several ways. in a
preferred embodiment, the
ligand(s) are added to a nucleoside, followed by the transition metal ion, and
then the nucleoside with
the transition metal complex attached is added to an oligonucleotide, i.e. by
addition to the nucleic acid
synthesizer. Alternatively, the ligand(s) may be attached, followed by
incorportation into a growing
oligonucleotide chain, followed by the addition of the metal ion.
In a preferred embodiment, electron transfer moieties are attached to a ribose
of the ribose-phosphate
backbone. This is generally done as is outlined in PCT publication SO
95I15971, using amino-
modified nucleosides, at either the 2' or 3' position of the ribose. The amino
group may then be used
either as a ligand, for example as a transition metal ligand for attachment of
the metal ion, or as a
chemically functional group that can be used for attachment of other ligands
or organic electron
transfer moieties, for example via amide linkages, as will be appreciated by
those in the art. For
example, the examples describe the synthesis of a nucleoside with a
metallocene linked via an amide
bond to the ribose.
In a preferred embodiment, electron transfer moieties are attached to a
phosphate of the ribose-
phosphate backbone. As outlined herein, this may be done using phosphodiester
analogs such as
phosphoramidite bonds, see generally PCT publication WO 95/15971, or can be
done in a similar
manner to that depicted in Figures 4 and 5, where the conductive oligomer is
replaced by a transition
metal iigand or complex or an organic electron transfer moiety.
Attachment to alternate backbones, for example peptide nucleic acids or
alternate phosphate linkages
will be done as will be appreciated by those in the art.
in a preferred embodiment, electron transfer moieties are attached to a base
of the nucleoside. This
may be done in a variety of ways. In one embodiment, amino groups of the base,
either naturally
occurring or added as is described herein (see the fiigures, for example), are
used either as ligands for
transition metal complexes or as a chemically functional group that can be
used to add other ligands,
for example via an amide linkage, or organic electron transfer moieties. This
is done as will be
appreciated by those in the art. Alternatively, nucleosides containing halogen
atoms attached to the
heterocyclic ring are commercially available. Acetylene linked ligands may be
added using the
halogenated bases, as is generally known; see for example, Tzalis et al.,
Tetrahedron Lett.
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36(34):6017-6020 (1995); Tzalis et al., Tetrahedron Lett. 36(2):3489-3490
(1995); and Tzalis et al.,
Chem. Communications (in press) 1996, all of which are hereby expressly
incorporated by reference.
See also the examples, which describes the synthesis of a metallocene attached
via an acetylene
linkage to the base.
In one embodiment, the nucleosides are made with transition metal ligands,
incorporated into a nucleic
acid, and then the transition metal ion and any remaining necessary ligands
are added as is known in
the art. In an alternative embodiment, the transition metal ion and additional
ligands are added prior to
incorporation into the nucleic acid.
In some embodiments, as outlined herein) conductive oligomers are used between
the second
electron transfer moieties and the nucleosides. These are made using the
techniques described
herein, with the addition of the terminal second electron transfer moiety.
Once the nucleic acids of the invention are made, with a covalently attached
conductive oligomer and
optionally a second electron transfer moiety, the conductive oligomer is
attached to the electrode. The
method will vary depending on the type of electrode used. As is described
herein, the conductive
oligomers are generally made with a terminal "A" linker to facilitate
attachment to the electrode. For the
purposes of this application, a sulfur-gold attachment is considered a
covalent attachment.
In a preferred embodiment, conductive oligomers are covalently attached via
sulfur linkages to the
electrode. However, surprisingly, traditional protecting groups for use of
attaching molecules to gold
electrodes are generally ideal for use in bath synthesis of the compositions
described herein and
inclusion in oligonucleotide synthetic reactions. Accordingly, the present
invention provides novel
methods for the attachment of conductive oligomers to gold electrodes,
utilizing unusual protecting
groups, including ethylpyridine, and trimethylsilylethyl as is depicted in the
Figures.
This may be done in several ways. In a preferred embodiment, the subunit of
the conductive oligomer
which contains the sulfur atom for attachment to the electrode is protected
with an ethyl-pyridine or
trimethylsilylethyl group. For the former, this is generally done by
contacting the subunit containing the
sulfur atom (preferably in the form of a sulfhydryl) with a vinyl pyridine
group or vinyl trimethylsilylethyl
group under conditions whereby an ethylpyridine group or trimethylsilylethyl
group is added to the
sulfur atom.
This subunit also generally contains a functional moiety for attachment of
additional subunits, and thus
additional subunits are attached to form the conductive oligomer. The
conductive oligomer is then
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attached to a nucleoside, and additional nucleosides attached. The protecting
group is then removed
and the sulfur-gold covalent attachment is made. Alternatively, all or part of
the conductive oligomer is
made, and then either a subunit containing a protected sulfur atom is added,
or a sulfur atom is added
and then protected. The conductive oligomer is then attached to a nucleoside,
and additional
nucleosides attached. Alternatively, the conductive oligomer attached to a
nucleic acid is made, and
then either a subunit containing a protected sulfur atom is added, or a sulfur
atom is added and then
protected. Alternatively, the ethyl pyridine protecting group may be used as
above, but removed after
one or more steps and replaced with a standard protecting group like a
disulfide. Thus, the ethyl
pyridine or trimethylsilylethyl group may serve as the protecting group for
some of the synthetic
reactions, and then removed and replaced with a traditional protecting group.
By "subunit" of a conductive polymer herein is meant at least the moiety of
the conductive oligomer to
which the sulfur atom is attached, although additional atoms may be present,
including either
functional groups which allow the addition of additional components of the
conductive oligomer, or
additional components of the conductive oligomer. Thus, for example, when
Structure 2 oligomers are
used, a subunit comprises at least the first Y group.
A preferred method comprises 1 ) adding an ethyl pyridine or
trimethylsilylethyl protecting group to a
sulfur atom attached to a first subunit of a conductive oligomer, generally
done by adding a vinyl
pyridine or trimethylsilylethyl group to a sulfhydryl; 2) adding additional
subunits to form the conductive
oligomer; 3) adding at least a first nucleoside to the conductive oligomer; 4)
adding additional
nucleosides to the first nucleoside to form a nucleic acid; 5) attaching the
conductive oligomer to the
gold electrode. This may also be done in the absence of nucleosides, as is
described in the
Examples.
The above method may also be used to attach passivation molecules to a gold
electrode.
In a preferred embodiment, a monolayer of passivation agents is added to the
electrode. Generally,
the chemistry of addition is similar to or the same as the addition of
conductive oligomers to the
electrode, i.e. using a sulfur atom for attachment to a gold electrode, etc.
Compositions comprising
monolayers in addition to the conductive oligomers covalently attached to
nucleic acids (with or
without second electron transfer moieties) may be made in at least one of five
ways: ( 1 ) addition of the
monolayer, followed by subsequent addition of the conductive oligomer-nucleic
acid complex; (2)
addition of the conductive oligomer-nucleic acid complex followed by addition
of the monoiayer; (3)
simultaneous addition of the monolayer and conductive oligomer-nucleic acid
complex; (4) formation of
a monolayer (using any of 1, 2 or 3) which includes conductive oligomers which
terminate in a
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functional moiety suitable for attachment of a completed nucleic acid; or (5)
formation of a monolayer
which includes conductive oiigomers which terminate in a functional moiety
suitable for nucleic acid
synthesis, i.e. the nucleic acid is synthesized on the surface of the
monolayer as is known in the art.
Such suitable functional moieties include, but are not limited to,
nucleosides, amino groups) carboxyl
groups, protected sulfur moieties, or hydroxyl groups for phosphoramidite
additions. The examples
describe the formation of a monolayer on a gold electrode using the preferred
method ( 1 ).
In a preferred embodiment, the nucleic acid is a peptide nucleic acid or
analog. In this embodiment,
the invention provides peptide nucleic acids with at least one covalently
attached chemical substituent.
By "chemical substituent" herein is meant any chemical or biological moiety.
Preferred chemical
substituents include, but are not limited to, chemical functional moieties
such as amino groups, thiol
groups, carbon atoms, etc., which can be used to attach other moieties;
labels; signaling moieties
which can be used for detection; etc. Accordingly, chemical substitutents
include, but are not limited
to, electron transfer moieties, including electrodes, transition metal
complexes, and organic electron
transfer moieties; other transition metal complexes; other labels including
fluoroscent labels,
radioisotope labels and chemiluminescent labels; haptens such as biotin,
avidin, and digoxigenin;
antigens; proteins such as antibodies, ligands, receptors, and enzymes;
conductive oligomers and
other polymers; and other components of binding pairs such as nucleic acids.
In a preferred embodiment, the chemical substituents are covalently attached
to an monomeric subunit
of the PNA. By "monomeric subunit of PNA" herein is meant the -NH-CHZCHZ-
N(COCHZ-Base)-CHZ-
CO- monomer, or derivatives (herein included within the definition of
"nucleoside") of PNA. For
example, the number of carbon atoms in the PNA backbone may be altered; see
generally Nielsen et
al., Chem. Soc. Rev. 1997 page 73, which discloses a number of PNA
derivatives, herein expressly
incorporated by reference. Similarly, the amide bond linking the base to the
backbone may be altered;
phosphoramide and sulfuramide bonds may be used.
In a preferred embodiment, the chemical substituents are attached to an
internal monomeric subunit.
By "internal" herein is meant that the monomeric subunit is not either the N-
terminal monomeric
subunit or the C-terminal monomeric subunit.
In this embodiment, the chemical substituents can be attached either to a base
or to the backbone of
the monomeric subunit. In a preferred embodiment, at least one chemical
substituent is attached to an
internal base. Attachment to the base is done as outlined herein or known in
the literature. In general,
the chemical substituents are added to a base which is then incorporated into
a PNA as outlined
herein. The base may be either protected, as required for incorporation into
the PNA synthetic
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reaction, or derivatized, to allow incorporation, either prior to the addition
of the chemical substituent or
afterwards. Protection and derivatization of the bases is shown in Figures 24-
27. The bases can then
be incorporated into monomeric subunits as shown in Figure 28. Figures 29 and
30 depict two
different chemical substituents, an electron transfer moiety and a conductive
oligomer, attached at a
base. Figure 29 depicts a representative synthesis of a PNA monomeric subunit
with a ferrocene
attached to a uracil base. Figure 30 depicts the synthesis of a three unit
conductive oligomer attached
to a uracil base.
In a preferred embodiment, the chemical substituents are covalently attached
to the backbone of the
PNA monomer. The attachment is generally to one of the unsubstituted carbon
atoms of the
monomeric subunit, preferably the a-carbon of the backbone, as is depicted in
Figures 31 and 32,
although attachment at either of the carbon 1 or 2 positions, or the a-carbon
of the amide bond finking
the base to the backbone may be done. In the case of PNA analogs, other
carbons or atoms may be
substituted as well. In a preferred embodiment, chemical substituents are
added at the a-carbon
atoms, either to a terminal monomeric subunit or an internal one.
In this embodiment, a modified monomeric subunit is synthesized with a
chemical substituent, or a
functional group for its attachment, and then the base is added and the
modified monomer can be
incorporated into a growing PNA chain. Figure 31 depicts the synthesis of a
conductive oligomer
covalently attached to the backbone of a PNA monomeric subunit, and Figure 32
depicts the synthesis
of a ferrocene attached to the backbone of a monomeric subunit.
Once generated, the monomeric subunits with covalently attached chemical
substituents are
incorporated into a PNA using the techniques outlined in Will et al.,
Tetrahedron 51(44):12069-12082
(1995), and Vanderlaan et al., Tett. Let. 38:2249-2252 (1997), both of which
are hereby expressly
incorporated in their entirety. These procedures allow the addition of
chemical substituents to peptide
nucleic acids without destroying the chemical substituents.
In a preferred embodiment, chemical substituents other than electron transfer
moieties and transition
metal complexes are attached to either or both of the bases of the terminal
monomeric subunits. In
this embodiment, preferred chemical substituents include fluoroscent,
radioisotope and
chemiluminescent labels.
As will be appreciated by those in the art, electrodes may be made that have
any combination of
nucleic acids, conductive oligomers and passivation agents. Thus, a variety of
different conductive
oligomers or passivation agents may be used on a single electrode.
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Once made, the compositions find use in a number of applications, as described
herein.
In a preferred embodiment, the compositions of the invention are used as
probes in hybridization
assays to detect target sequences in a sample. The term "target sequence" or
grammatical
equivalents herein means a nucleic acid sequence on a single strand of nucleic
acid. The target
sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA,
RNA including
mRNA and rRNA, or others. It may be any length, with the understanding that
longer sequences are
more specific. As will be appreciated by those in the art, the complementary
target sequence may
take many forms. For example, it may be contained within a larger nucleic acid
sequence, i.e. all or
part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA,
among others. As is
outlined more fully below, probes are made to hybridize to target sequences to
determine the
presence or absence of the target sequence in a sample. Generally speaking,
this term will be
understood by those skilled in the art.
If required, the target sequence is prepared using known techniques. For
example, the sample may
be treated to lyse the cells, using known lysis buffers, electroporation,
etc., with purification andlor
amplification occuring as needed, as will be appreciated by those in the art.
The probes of the present invention are designed to be complementary to the
target sequence, such
that hybridization of the target sequence and the probes of the present
invention occurs. As outlined
below, this complementarity need not be perfect; there may be any number of
base pair mismatches
which will interfere with hybridization between the target sequence and the
single stranded nucleic
acids of the present invention. However, if the number of mutations is so
great that no hybridization
can occur under even the least stringent of hybridization conditions, the
sequence is not a
complementary target sequence.
A variety of hybridization conditions may be used in the present invention,
including high, moderate
and low stringency conditions; see for example Maniatis et al., Molecular
Cloning: A Laboratory
Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed.
Ausubel, et al, hereby
incorporated by referenece. The hybridization conditions may also vary when a
non-ionic backbone,
i.e. PNA is used, as is known in the art. In addition, cross-linking agents
may be added after target
binding to cross-link, i.e. covalently attach, the two strands of the
hybridization complex.
In a preferred embodiment, single stranded nucleic acids are made which
contain a first electron
transfer moiety, an electrode, and at least a second electron tranfer moiety.
Hybridization to a target
sequence forms a double stranded hybridization complex. In a hybridization
complex, at least the
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sequence between the nucleosides containing the electron transfer moieties is
double stranded, i.e.
contains stacked rr-orbitals, such that upon initiation, the complex is
capable of transferring at least
one electron from one of the electron transfer moieties to the other. As will
be appreciated by those in
the art, an electrode may serve as either an electron donor or acceptor, and
the choice of the second
electron transfer species is made accordingly.
In an alternative embodiment, compositions comprising a) a first single
stranded nucleic acid
covalently attached to an electrode via a conductive oligomer and b) a second
single stranded nucleic
acid containing a second electron transfer moiety, are made. In this
embodiment, the first single
stranded nucleic acid is capable of hybridizing to a first target domain, and
the second single stranded
nucleic acid is capable of hybridizing to a second target domain. The terms
"first target domain" and
"second target domain" or grammatical equivalents herein means two portions of
a target sequence
within a nucleic acid which is under examination. The first target domain may
be directly adjacent to
the second target domain, or the first and second target domains may be
separated by an intervening
target domain. Preferably) there are no gaps between the domains; i.e. they
are contiguous. The
terms "first" and "second" are not meant to confer an orientation of the
sequences with respect to the
5'-3' orientation of the target sequence. For example, assuming a 5'-3'
orientation of the
complementary target sequence, the first target domain may be located either
5' to the second
domain) or 3' to the second domain.
In this embodiment, the first single stranded nucleic acid is hybridized to
the first target domain, and
the second single stranded nucleic acid is hybridized to the second target
domain to form a
hybridization complex. As outlined above, the hybridization complex is then
capable of transferring at
least one electron between the electron transfer moieties upon initiation.
As will be appreciated by those in the art, the hybridization complex may
comprise two nucleic acids,
i.e. the probe, attached to the electrode, and the target, with the second
electron transfer moiety being
attached to either. Alternatively, the hybridization complex may comprise
three nucleic acids, i.e. the
first probe, attached to the electrode, a second probe) and the target nucleic
acid, with a second
electron transfer moiety attached to any of them. Similarly, hybridization
complexes can be made with
four or more nucleic acids, etc. What is important is that stacked n-orbitals
exist between the second
electron transfer moiety and the nucleoside to which the electrode is
attached.
In one embodiment, compositions comprising a) a single stranded nucleic acid
covalently attached to
an electrode via a conductive oligomer) and b) a target nucleic acid are made.
In this embodiment,
once hybridization of the target and the probe occurs, a hybridization
indicator is added. Hybridization
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indicators serve as an electron transfer moiety that will preferentially
associate with double stranded
nucleic acid is added, usually reversibly, similar to the method of Millan et
al., Anal. Chem. 65:2317-
2323 (1993); Millan et al., Anal. Chem. 662943-2948 (1994), both of which are
hereby expressly
incorporated by reference. Hybridization indicators include intercalators and
minor and/or major
groove binding moieties. In a preferred embodiment, intercalators may be used;
since intercalation
generally only occurs in the presence of double stranded nucleic acid, only in
the presence of target
hybridization will electron transfer occur. Intercalating transition metal
complex electron transfer
moieties are known in the art. Similarly, major or minor groove binding
moieties, such as methylene
blue, may also be used in this embodiment.
in addition, hybridization indicators may be used in any or all of the other
systems of the invention; for
example, they may be added to facilitate, quench or amplify the signal
generated by the system, in
addition to the covalently attached electron transfer moieties. For example)
it has been shown by
Millan, above, that some hybridization indicators may preferentially bind to
perfectly complementary
double stranded nucleic acids over nucleic acids containing mismatches. This
could serve to
contribute additional information about the system. Similarly, electronic
coupling could be increased
due to hybridization indicator binding. Alternatively, quenching of the
electron transfer signal could be
acheived using hybridization indicators, whereby the electrons would flow
between the second
electron tranfer moiety and the hybridization indicator) rather than the
electrode.
A further embodiment utilizes compositions comprising a) a first single
stranded nucleic acid covalently
attached to an electrode via a conductive oligomer; b) a second single
stranded nucleic acid
containing a second electron transfer moiety; and c) an intervening single
stranded nucleic acid, which
may or may not be labelled or contain an electron transfer moiety. As
generally outlined in PCT WO
95I15971, the first single stranded nucleic acid hybridizes to the first
target domain, the second single
stranded nucleic acid hybridizes to the second target domain, and the
intervening nucleic acid
hybridizes to the intervening target domain, with electron transfer upon
initiation. The intervening
nucleic acid may be any length, taking into consideration the parameters for
the distance between the
electron transfer moieties, although it may be a single nucleoside.
In addition, the first and second, or first, intervening and second, nucleic
acids may be ligated together
prior to the electron transfer reaction) using standard molecular biology
techniques such as the use of
a ligase.
In one embodiment, the compositions of the invention are used to detect
mismatches in a
complementary target sequence. A mismatch, whether it be a substitution,
insertion or deletion of a
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nucleoside or nucleosides, results in incorrect base pairing in a hybridized
double helix of nucleic acid.
Accordingly) if the path of an electron from an electron donor moiety to an
electron acceptor moiety
spans the region where the mismatch lies, the electron transfer will be
reduced such that a change in
the relative impedance will be seen. Therefore, in this embodiment) the
electron donor moiety is
attached to the nucleic acid at a 5' position from the mutation, and the
electron acceptor moiety is
attached at a 3' position, or vice versa.
Electron transfer is generally initiated electronically, with voltage being
preferred. A potential is applied
to a sample containing modified nucleic acid probes. Precise control and
variations in the applied
potential can be via a potentiostat and either a three electrode system (one
reference, one sample and
one counter electrode) or a two electrode system (one sample and one counter
electrode). This
allows matching of applied potential to peak electron transfer potential of
the system which depends in
part on the choice of electron acceptors attached to the nucleic acid and in
part on the conductive
oligomer used. As described herein, ferrocene is a preferred electron transfer
moiety.
Preferably, initiation and detection is chosen to maximize the relative
difference between the
impedances of double stranded nucleic acid and single stranded nucleic acid
systems. The efficiency
of electron transfer through nucleic acid is a function of the impedance of
the compound.
In a preferred embodiment, a co-reductant or co-oxidant (collectively, co-
redoxant) is used, as an
additional electron source or sink. See generally Sato et al., Bull. Chem.
Soc. Jpn 66:1032 (1993};
Uosaki et al.) Electrochimica Acta 36:1799 (1991 ); and Alleman et al., J.
Phys. Chem 100:17050
(1996); all of which are incorporated by reference.
In a preferred embodiment, an input electron source in solution is used in the
initiation of electron
transfer, preferably when initiation and detection are being done using DC
current) and when a
passivation agent monolayer is present on the electrode. This may be done in
several general ways.
In a preferred embodiment, an input electron source is used that has a lower
or similar redox potential
than the second electron transfer moiety (ETM) covalently attached to the
probe nucleic acid. Thus, at
voltages above the redox potential of the input electron source, both the
second ETM and the input
electron source are oxidized and can thus donate electrons; the ETM donates
through the
hybridization complex, through the conductive oligomer, to the electrode, and
the input source donates
to the ETM. For example, ferrocene) as a second ETM attached to the
compositions of the invention
as described in the examples, has a redox potential of roughly 200 mV in
aqueous solution (which
changes slightly depending on what the ferrocene is bound to}. Ferrocyanide,
an electron source,
has a redox potential of roughly 200 mV as well (in aqueous solution).
Accordingly, at or above
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voltages of roughly 200 mV, ferrocene is converted to ferricenium, which then
transfers an electron to
the nucleic acid. If this nucleic acid is double stranded, transfer proceeds
rapidly through the double
stranded nucleic acid, through the conductive oligomer, to the electrode. Now
the ferricyanide can be
oxidized to transfer an electron to the ETM. In this way, the electron source
(or co-reductant) serves
to amplify the signal generated in the system, as the electron source
molecules rapidly and repeatedly
donate electrons to the second ETM attached to the nucleic acid. The rate of
electron donation or
acceptance will be limited by the rate of diffusion of the co-reductant, which
in turn is affected by the
concentration and size, etc.
Alternatively, input electron sources that have lower redox potentials than
the second ETM are used.
At voltages less than the redox potential of the ETM) but higher than the
redox potential of the electron
source, the input source such as ferrocyanide is unable to be oxided and thus
is unable to donate an
electron to the ETM; i.e. no electron transfer occurs. The use of electron
source molecules, however,
is only possible when an insulating or passivation layer is present, since
otherwise the source
molecule will transfer electrons directly to the electrode. Accordingly, in a
preferred embodiment, an
electron source is used in solution to amplify the signal generated in the
presence of hybridized target
sequence.
In an alternate preferred embodiment, an input electron source is used that
has a higher redox
potential than the second electron transfer moiety (ETM) covalently attached
to the probe nucleic acid.
For example, luminoi, an electron source, has a redox potential of roughly 720
mV. At voltages higher
than the redox potential of the ETM, but lower than the redox potential of the
electron source, i.e. 200 -
720 mV, the ferrocene is oxided, and transfers a single electron to the
electrode via the conductive
oligomer. However, the ETM is unable to accept any electrons from the luminol
electron source, since
the voltages are less than the redox potential of the luminol. However, at or
above the redox potential
of luminol, the luminol then transfers an electron to the ETM, allowing rapid
and repeated electron
transfer. In this way, the electron source (or co-reductant) serves to amplify
the signal generated in
the system, as the electron source molecules rapidly and repeatedly donate
electrons to the second
ETM attached to the nucleic acid.
Luminoi has the added benefit of becoming a chemiluminiscent species upon
oxidation (see Jirka et
al., Analytica Chimica Acta 284:345 (1993)), thus allowing photo-detection of
electron transfer through
double-stranded nucleic acid. Thus, as long as the luminol is unable to
contact the electrode directly,
i.e. in the presence of a passivation layer, luminol can only be oxidized by
transferring an electron to
the second electron transfer moiety on the nucleic acid (e.g. ferrocene). When
double stranded
nucleic acid is not present, i.e. when the target sequence is not hybridized
to the composition of the
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invention, the system has a high impedance, resulting in a low photon emission
and thus a low (if any)
signal from the fuminol. In the presence of double stranded nucleic acid, i.e,
target sequence
hybridization, the second electron transfer moieties have low impedance, thus
generating a much
larger signal. Thus, the measure of luminol oxidation by photon emission is an
indirect measurement
of the ability of the second electron transfer moiety to donate electrons to
the electrode. Furthermore,
since photon detection is generally more sensitive than electronic detection,
the sensitivity of the
system may be increased. Initial results suggest that luminescence may depend
on hydrogen
peroxide concentration, pH, and luminol concentration, the latter of which
appears to be non-linear.
Suitable electron source molecules are well known in the art, and include, but
are not limited to,
ferricyanide, and luminol.
Alternatively, output electron acceptors or sinks could be used, i.e. the
above reactions could be run in
reverse, with the ETM such as a metallocene receiving an electron from the
electrode, converting it to
the metallicenium, with the output electron acceptor then accepting the
electron rapidly and
repeatedly. In this embodiment, cobalticenium is the preferred ETM.
Electron transfer through nucleic acid can be detected in a variety of ways. A
variety of detection
methods may be used, including, but not limited to, optical detection, which
includes fluorescence,
phosphorescence, luminiscence, chemiluminescence, electrochemiluminescence)
and refractive
index; and electronic detection, including, but not limited to, amperommetry,
voltammetry, capacitance
and impedence. These methods include time or frequency dependent methods based
on AC or DC
currents, pulsed methods, lock-in techniques, filtering (high pass, low pass,
band pass), and time-
resolved techniques including time-resolved fluoroscence. In some embodiments,
all that is required is
electron transfer detection; in others, the rate of electron transfer may be
determined.
In one embodiment, the efficient transfer of electrons from one end of a
nucleic acid double helix to the
other results in stereotyped changes in the redox state of both the electron
donor and acceptor. With
many electron transfer moieties including the complexes of ruthenium
containing bipyridine, pyridine
and imidazole rings, these changes in redox state are associated with changes
in spectral properties.
Significant differences in absorbance are observed between reduced and
oxidized states for these
molecules. See for example Fabbrizzi et al., Chem. Soc. Rev. 1995 pp197-202).
These differences
can be monitored using a spectrophotometer or simple photomultiplier tube
device.
In this embodiment, possible electron donors and acceptors include all the
derivatives listed above for
photoactivation or initiation. Preferred electron donors and acceptors have
characteristically large
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spectral changes upon oxidation and reduction resulting in highly sensitive
monitoring of electron
transfer. Such examples include Ru(NH3)4py and Ru(bpy)Zim as preferred
examples. It should be
understood that only the donor or acceptor that is being monitored by
absorbance need have ideal
spectral characteristics. That is, the electron acceptor can be optically
invisible if only the electron
donor is monitored for absorbance changes.
In a preferred embodiment, the electron transfer is detected fluorometrically.
Numerous transition
metal complexes, including those of ruthenium, have distinct fluorescence
properties. Therefore, the
change in redox state of the electron donors and electron acceptors attached
to the nucleic acid can
be monitored very sensitively using fluorescence) for example with Ru(4,7-
biphenyl2-phenanthroline)3z+
. The production of this compound can be easily measured using standard
fluorescence assay
techniques. For example, laser induced fluorescence can be recorded in a
standard single cell
fluorimeter, a flow through "on-line" fluorimeter (such as those attached to a
chromatography system)
or a multi-sample "plate-reader" similar to those marketed for 96-well immuno
assays.
Alternatively, fluorescence can be measured using fiber optic sensors with
nucleic acid probes in
solution or attached to the fiber optic. Fluorescence is monitored using a
photomuitiplier tube or other
light detection instrument attached to the fiber optic. The advantage of this
system is the extremely
small volumes of sample that can be assayed.
in addition, scanning fluorescence detectors such as the Fluorlmager sold by
Molecular Dynamics are
ideally suited to monitoring the fluorescence of modified nucleic acid
molecules arrayed on solid
surfaces. The advantage of this system is the large number of electron
transfer probes that can be
scanned at once using chips covered with thousands of distinct nucleic acid
probes.
Many transition metal complexes display fluorescence with large Stokes shifts.
Suitable examples
include bis- and trisphenanthroline complexes and bis- and trisbipyridyl
complexes of transition metals
such as ruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V.
84, p. 85-277, 1988).
Preferred examples display efficient fluorescence {reasonably high quantum
yields) as well as low
reorganization energies. These include Ru(4,7-biphenyl2-phenanthroline)32',
Ru(4,4'-Biphenyl-2,2'-
bipyridine)32+ and platinum complexes {see Cummings et al., J. Am. Chem. Soc.
118:1949-1960
(1996), incorporated by reference).
Alternatively, a reduction in fluorescence associated with hybridization can
be measured using these
systems. An electron transfer "donor" molecule that fluoresces readily when on
single stranded
nucleic acid (with an "acceptor" on the other end) will undergo a reduction in
fluorescent intensity when
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complementary nucleic acid binds the probe allowing efficient transfer of the
excited state electron.
This drop in fluorescence can be easily monitored as an indicator of the
presence of a target sequence
using the same methods as those above.
In a further embodiment, electrochemiluminescence is used as the basis of the
electron transfer
detection. With some electron transfer moieties such as Ru2'(bpy)3, direct
luminescence accompanies
excited state decay. Changes in this property are associated with nucleic acid
hybridization and can
be monitored with a simple photomultiplier tube arrangement (see Blackburn, G.
F. CJin. Chem. 37:
1534-1539 (1991); and Juris et al., supra.
In a preferred embodiment, electronic detection is used, including
amperommetry, voltammetry,
capacitance, and impedence. Suitable techniques include, but are not limited
to, electrogravimetry;
coulometry {including controlled potential coulometry and constant current
coulometry); voltametry
(cyclic voltametry, pulse voltametry (normal pulse voltametry, square wave
voltametry, differential
pulse voltametry, Osteryoung square wave voltametry, and coulostatic pulse
techniques); stripping
analysis (aniodic stripping analysis, cathiodic stripping analysis, square
wave stripping voltammetry);
conductance measurements (electrolytic conductance, direct analysis); time-
dependent
electrochemical analyses (chronoamperometry, chronopotentiometry, cyclic
chronopotentiometry and
amperometry, AC polography, chronogalvametry, and chronocoulometry); AC
impedance
measurement; capacitance measurement; AC voltametry; and
photoelectrochemistry.
In a preferred embodiment, monitoring electron transfer through nucleic acid
is via amperometric
detection. This method of detection involves applying a potential (as compared
to a separate
reference electrode) between the nucleic acid-conjugated, electrode and a
reference {counter)
electrode in the sample containing target genes of interest. Electron transfer
of differing efficiencies is
induced in samples in the presence or absence of target nucleic acid; that is,
the presence or absence
of the target nucleic acid alters the impedance of the nucleic acid (i.e.
double stranded versus single
stranded) system which can result in different currents.
The device for measuring electron transfer amperometrically involves sensitive
current detection and
includes a means of controlling the voltage potential, usually a potentiostat.
This voltage is optimized
with reference to the potential of the electron donating complex on the
nucleic acid. Possible electron
donating complexes include those previously mentioned with complexes of iron,
osmium, platinum,
cobalt) rhenium and ruthenium being preferred and complexes of iron being most
preferred.
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In a preferred embodiment, alternative electron detection modes are utilized.
For example,
potentiometric (or voltammetric) measurements involve non-faradaic (no net
current flow) processes
and are utilized traditionally in pH and other ion detectors. Similar sensors
are used to monitor
electron transfer through nucleic acid. In addition, other properties of
insulators (such as resistance)
and of conductors (such as conductivity, impedance and capicitance) could be
used to monitor
electron transfer through nucleic acid. Finally, any system that generates a
current (such as electron
transfer) also generates a small magnetic field, which may be monitored in
some embodiments.
It should be understood that one benefit of the fast rates of electron
transfer observed in the
compositions of the invention is that time resolution can greatly enhance the
signal-to-noise results of
monitors based on absorbance, fluorescence and electronic current. The fast
rates of electron
transfer of the present invention result both in high signals and stereotyped
delays between electron
transfer initiation and completion. By amplifying signals of particular
delays, such as through the use
of pulsed initiation of electron transfer and "lock-iri" amplifiers of
detection, between two and four
orders of magnitude improvements in signal-to-noise may be achieved.
In a preferred embodiment, electron transfer is initiated using alternating
current (AC) methods.
Without being bound by theory, it appears that nucleic acids, bound to an
electrode, generally respond
similarly to an AC voltage across a resistor and capacitor in series.
Basically, any methods which .
enable the determination of the nature of these complexes, which act as a
resistor and capacitor, can
be used as the basis of detection. Surprisingly, traditional electrochemical
theory, such as exemplified
in Laviron et al., J. Electroanal. Chem.~J7:135 (1979) and Laviron et al., J.
Electroanal. Chem. 105:35
(1979), both of which are incorporated by reference, do not accurately model
the systems described
herein, except for very small EAR (less than 10 mV) and relatively large
numbers of molecules. That is,
the AC current (I) is not accurately described by Laviron's equation. This may
be due in part to the
fact that this theory assumes an unlimited source and sink of electrons, which
is not true in the present
systems.
Accordingly, alternate equations were developed, using the Nernst equation and
first principles to
develop a model which more closely simulates the results. This was derived as
follows. The Nernst
equation, Equation 1 below, describes the ratio of oxidized (O) to reduced (R)
molecules (number of
molecules = n) at any given voltage and temperature, since not every molecule
gets oxidized at the
same oxidation potential.
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Equation 1
EDC-Ea+ nF pn ~R~ C 1 )
Ep~ is the electrode potential, Efl is the formal potential of the metal
complex, R is the gas constant, T
is the temperature in degrees Kelvin, n is the number of electrons
transferred, F is faraday's constant,
[O] is the concentration of oxidized molecules and [R] is the concentration of
reduced molecules.
The Nernst equation can be rearranged as shown in Equations 2 and 3:
Equation 2
ED~'Eo= RT pn [O] { 2 )
nF [RJ
Ep~ is the DC component of the potential.
Equation 3
exp R (Eon - Eo) - [O] { 3 )
[R]
Equation 3 can be rearranged as follows, using normalization of the
concentration to equal 1 for
simplicity, as shown in Equations 4, 5 and 6. This requires the subsequent
multiplication by the total
number of molecules.
Equation 4 [O] + [R] = 1
Equation 5 [O] = 1 - [R)
Equation 6 (R] = 1 - [O]
Plugging Equation 5 and 6 into Equation 3, and the fact that nF/RT equals 38.9
V'', for n=1, gives
Equations 7 and 8, which define [O] and [R], respectively:
Equation 7
38.9(E-Eo)
O exp 4
[ ] - 1 38.9(E-Eo)
+ exp
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Equation 8
R _ 1
[ ~ 1 + eXp38.9 (E - Eo)
Taking into consideration the generation of an AC faradaic current, the ratio
of [O]/[R] at any given
potential must be evaluated. At a particular Eoc with an applied EAC, as is
generally described herein,
at the apex of the EAC more molecules will be in the oxidized state, since the
voltage on the surface is
now (Epc + EAC); at the bottom, more will be reduced since the voltage is
lower. Therefore, the AC
current at a given Eoc will be dictated by both the AC and DC voltages, as
well as the shape of the
Nernstian curve. Specifically, if the number of oxidized molecules at the
bottom of the AC cycle is
subtracted from the amount at the top of the AC cycle, the total change in a
given AC cycle is
obtained) as is generally described by Equation 9. Dividing by 2 then gives
the AC amplitude.
Equation 9
iAC ~ .electrons at fE~c ~ ~"c 1L(electrons at [Epc-E~c~
2
Equation 10 thus describes the AC current which should result:
Equation 10
1AC = C~ FGJ ~z U~~Euc ' Eec ~~]EnC - E~c~ { 6 )
As depicted in Equation 11, the total AC current will be the number of redox
molecules C), times
faraday's constant (F), times the AC frequency {w), times 0.5 (to take into
account the AC amplitude),
times the ratios derived above in Equation 7. The AC voltage is approximated
by the average, EAC2/n.
Equation 11
38.9 [Epc + 2E~c - Eo] 38.9 [EDC _ 2E~c - Epl
~c = C~ Fc.~ ~ exp " ) - exp "
2E 2E
38.9 [Epc + ec _ Eo) 38.9 [Eoc - ~C _ E
1 + exp " 1 + exp "
Using Equation 11, simulations were generated using increasing overpotential
(AC voltage). Figure
22A shows one of these simulations, while Figure 228 depicts a simulation
based on traditional theory.
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Figures 23A and 23B depicts actual experimental data using the Fc-wire of
Example 7 plotted with the
simulation, and shows that the model fits the experimental data very well. In
some cases the current
is smaller than predicted, however this has been shown to be caused by
ferrocene degradation which
may be remedied in a number of ways. However, Equation 11 does not incorporate
the effect of
electron transfer rate nor of instrument factors. Electron transfer rate is
important when the rate is
close to or lower than the applied frequency. Thus, the true iA~ should be a
function of all three, as
depicted in Equation 12.
Equation 12
iA~ = f(Nernst factors)f(kET)f{instrument factors)
These equations can be used to model and predict the expected AC currents in
systems which use
input signals comprising both AC and DC components. As outlined above,
traditional theory
surprisingly does not model these systems at all, except for very low
voltages.
In general) a single stranded probe nucleic acid system has a high impedance,
and a double stranded
nucleic acid system (i.e. probe hybridized to target to form a hybridization
complex) has a lower
impedance. This difference in impedance serves as the basis of a number of
useful AC detection
techniques, as outlined below, but as will be appreciated by those in the art,
a wide number of
techniques may be used. In addition, the use of AC input and output signals
enables the identification
of different species based on phase shifting between the AC voltage applied
and the voltage or current
response. Thus, AC detection gives several advantages as is generally
discussed below, including an
increase in sensitivity, the ability to monitor changes using phase shifting,
and the ability to "filter out"
background noise.
Accordingly, when using AC initiation and detection methods, the frequency
response of the system
changes as a result of hybridization to form a double-stranded nucleic acid.
By "frequency response"
herein is meant a modification of signals as a result of electron transfer
between the electrode and the
second electron transfer moiety. This modification is different depending on
signal frequency. A
frequency response includes AC currents at one or more frequencies, phase
shifts, DC offset
voltages, faradaic impedance, etc.
In a preferred embodiment, a target sequence is added to a probe single
stranded nucleic acid.
Preferably, the probe single stranded nucleic acid comprises a covalently
attached first electron
transfer moiety comprising an electrode, and a covalently attached second
electron transfer moiety as
described above. However, as outlined herein, it is also possible to use a
variety of other
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configurations in the system) including a second electron transfer moiety
attached to the target nucleic
acid, a second probe nucleic acid containing a second electron transfer
moiety, intervening nucleic
acids, etc.
In a preferred embodiment, the single stranded nucleic acid is covaiently
attached to the electrode via
a spacer. By "spacer" herein is meant a moiety which holds the nucleic acid
off the surface of the
electrode. In a preferred embodiment, the spacer is a conductive oligomer as
outlined herein,
although suitable spacer moieties include passivation agents and insulators as
outlined above. The
spacer moieties may be substantially non-conductive, although preferably (but
not required) is that the
rate of electron transfer through the spacer is faster than the rate through
single stranded nucleic acid,
although substantially conductive spacers are generally preferred. In general,
the length of the spacer
is as outlined for conductive polymers and passivation agents. Similarly)
spacer moieties are attached
as is outlined above for conductive oligomers, passivation agents and
insulators, for example using
the same "A" tinker defined herein.
The target sequence is added to the composition under conditions whereby the
target sequence, if
present, will bind to the probe single stranded nucleic acid to form a
hybridization complex, as outlined
above.
A first input electrical signal is then applied to the system, preferably via
at least the sample electrode
(containing the complexes of the invention) and the counter electrode, to
initiate electron transfer
between the electrode and the second electron transfer moiety. Three electrode
systems may also be
used, with the voltage applied to the reference and working electrodes. The
first input signal
comprises at least an AC component. The AC component may be of variable
amplitude and
frequency. Generally, for use in the present methods, the AC amplitude ranges
from about 1 mV to
about 1.1 V, with from about 10 mV to about 800 mV being preferred, and from
about 10 mV to about
500 mV being especially preferred. The AC frequency ranges from about 0.01 Hz
to about 10 MHz,
with from about 1 Hz to about 1 MHz being preferred, and from about 1 Hz to
about 100 kHz being
especially preferred
Surprisingly, the use of combinations of AC and DC signals allows the
differentiation between single-
stranded nucleic acid and double stranded nucleic acid, as is outlined herein.
In addition, signals
comprised of AC and DC components also allow surprising sensitivity and signal
maximization.
in a preferred embodiment, the first input signal comprises a DC component and
an AC component.
That is) a DC offset voltage between the sample and counter electrodes is
swept through the
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electrochemical potential of the second electron transfer moiety (for example,
when ferrocene is used,
the sweep is generally from 0 to 500 mV). The sweep is used to identify the DC
voltage at which the
maximum response of the system is seen. This is generally a# or about the
electrochemical potential
of the second electron transfer moiety. Once this voltage is determined,
either a sweep or one or
more uniform DC offset voltages may be used. DC offset voltages of from about -
1 V to about +1.1 V
are preferred, with from about -500 mV to about +800 mV being especially
preferred, and from about
300 mV to about 500 mV being particularly preferred. In a preferred
embodiment, the DC offset
voltage is not zero. On top of the DC offset voltage, an AC signal component
of variable amplitude
and frequency is applied. If the nucleic acid has a low enough impedance to
respond to the AC
perturbation, an AC current will be produced due to electron transfer between
the electrode and the
second electron transfer moiety.
For defined systems, it may be sufficient to apply a single input signal to
differentiate between single
stranded and double stranded (i.e. the presence of the target sequence)
nucleic acid. Alternatively, a
plurality of input signals are applied. As outlined herein, this may take a
variety of forms, including
using multiple frequencies, multiple DC offset voltages, or multiple AC
amplitudes, or combinations of
any or all of these.
Thus) in a preferred embodiment, multiple DC offset voltages are used,
although as outlined above,
DC voltage sweeps are preferred. This may be done at a single frequency, or at
two or more
frequencies .
In a preferred embodiment, the AC amplitude is varied. Without being bound by
theory, it appears that
increasing the amplitude increases the driving force. Thus, higher amplitudes,
which result in higher
overpotentials give faster rates of electron transfer. Thus, generally, the
same system gives an
improved response (i.e. higher output signals) at any single frequency through
the use of higher
overpotentials at that frequency. Thus, the amplitude may be increased at high
frequencies to
increase the rate of electron transfer through the system, resulting in
greater sensitivity. In addition,
this may be used, for example, to induce responses in slower systems such as
single stranded nucleic
acids for identification, calibration and/or quantification. Thus, the amount
of unhybridized single
stranded nucleic acid on an electrode may be compared to the amount of
hybridized double stranded
nucleic acid to quantify the amount of target sequence in a sample. This is
quite significant to serve
as an internal control of the sensor or system. This allows a measurement
either prior to the addition
of target or after, on the same molecules that will be used for detection,
rather than rely on a similar
but different control system. Thus, the actual molecules that will be used for
the detection can be
quantified prior to any experiment. For example, a preliminary run at 1 Hz or
less, for example, will
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quantify the actual number of molecules that are on the surface of the
electrode. The sample can then
be added, an output signal determined, and the ratio of bound/unbound
molecules determined. This is
a significant advantage over prior methods.
In a preferred embodiment) measurements of the system are taken at at least
two separate amplitudes
or overpotentials, with measurements at a plurality of amplitudes being
preferred. As noted above,
changes in response as a result of changes in amplitude may form the basis of
identification,
calibration and quantification of the system. In addition) one or more AC
frequencies can be used as
well.
In a preferred embodiment, the AC frequency is varied. At different
frequencies, different molecules
respond in different ways. As wil! be appreciated by those in the art,
increasing the frequency
generally increases the output current. However, when the frequency is greater
than the rate at which
electrons may travel between the electrode and the second electron transfer
moiety, higher
frequencies result in a loss or decrease of output signal. For example, as
depicted in Figure 11, a
response may be detected at 1 Hz for both single stranded nucleic acid and
double stranded nucleic
acid. However, at the higher frequencies, such as 200 Hz and above, the
response of the single
stranded nucleic acid is absent, while the response of the double stranded
nucleic acid continues to
increase. At some point, the frequency will be greater than the rate of
electron transfer through even
double-stranded nucleic acid, and then the output signal will also drop. Thus)
the different frequency
responses of single stranded and double stranded nucleic acids, based on the
rate at which electrons
may travel through the nucleic acid (i.e. the impedance of the nucleic acid),
forms the basis of
selective detection of double stranded nucleic acids versus single stranded
nucleic acids.
In one embodiment, detection utilizes a single measurement of output signal at
a single frequency.
That is, the frequency response of a single stranded nucleic acid can be
previously determined to be
very low at a particular high frequency. Using this information, any response
at a high frequency, for
example such as 10 to 100 kHz, where the frequency response of the single
stranded nucleic acid is
very low or absent, will show the presence of the double stranded
hybridization complex. That is, any
response at a high frequency is characteristic of the hybridization complex.
Thus, it may only be
necessary to use a single input high frequency, and any frequency response is
an indication that the
hybridization complex is present, and thus that the target sequence is
present.
In addition, the use of AC techniques allows the significant reduction of
background signals at any
single frequency due to entities other than the covalently attached nucleic
acids, i.e. "locking out" or
"filtering" unwanted signals. That is, the frequency response of a charge
carrier or redox active
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molecule in solution will be limited by its diffusion coefficient and charge
transfer coefficient.
Accordingly, at high frequencies, a charge carrier may not diffuse rapidly
enough to transfer its charge
to the electrode, andlor the charge transfer kinetics may not be fast enough.
This is particularly
significant in embodiments that do not utilize a passivation layer monolayer
or have partial or
insufficient monoiayers, i.e. where the solvent is accessible to the
electrode. As outlined above, in DC
techniques, the presence of "holes" where the electrode is accessible to the
solvent can result in
solvent charge carriers "short circuiting" the system. However, using the
present AC techniques, one
or more frequencies can be chosen that prevent a frequency response of one or
more charge carriers
in solution, whether or not a monoiayer is present. This is particularly
significant since many biological
fluids such as blood contain significant amounts of redox active molecules
which can interfere with
amperometric detection methods.
In a preferred embodiment, measurements of the system are taken at at least
two separate
frequencies, with measurements at a plurality of frequencies being preferred.
A plurality of
frequencies includes a scan. For example, measuring the output signal, e.g.,
the AC current, at a low
input frequency such as 1 - 20 Hz) and comparing the response to the output
signal at high frequency
such as 10 -100 kHz will show a frequency response difference between double
stranded nucleic
acids with fast electron transfer rates and single stranded nucleic acids with
slow electron transfer
rates. In a preferred embodiment, the frequency response is determined at at
least iwo, preferably at
least about five, and more preferably at least about ten frequencies.
After transmitting the input signal to initiate electron transfer, an output
signal is received or detected.
The presence and magnitude of the output signal will depend on the
overpotential/amplitude of the
input signal; the frequency of the input AC signal; the composition of the
intervening medium, i.e. the
impedance) between the electron transfer moieties (i.e. single stranded versus
double stranded, etc.);
the DC offset; the environment of the system; the nature of the second
electron transfer moiety; and
the solvent. At a given input signal) the presence and magnitude of the output
signal will depend in
general on the impedance of the medium between the two electron transfer
moieties and the character
of the input signal. Double stranded nucleic acids, i.e. hybridization
complexes, have relatively low
impedance as compared to single stranded nucleic acids, and thus result in
greater output signals.
However) as noted herein, single stranded nucleic acids, in the absence of the
complementary target,
can result in electron transfer between the electron transfer moieties. Thus,
upon transmitting the
input signal, comprising an AC component and a DC offset, electrons are
transferred betvueen the first
electron moiety, i.e. the electrode) and the second electron moiety covalently
attached to the nucleic
acid, when the impedance is low enough, the frequency is in range, and the
amplitude is sufFcient,
resulting in an output signal.
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In a preferred embodiment, the output signal comprises an AC current. As
outlined above, the
magnitude of the output current will depend on a number of parameters. By
varying these parameters,
the system may be optimized in a number of ways.
In general, AC currents generated in the present invention range from about 1
femptoamp to about 1
milliamp, with currents from about 50 femptoamps to about 100 microamps being
preferred, and from
about 1 picoamp to about 1 microamp being especially preferred.
In a preferred embodiment, the output signal is phase shifted in the AC
component relative to the input
signal. Without being bound by theory, it appears that surprisingly, the
systems of the present
invention are sufficiently uniform to allow phase-shifting based detection.
That is, the complex
biomolecules of the invention through which electron transfer occurs react to
the AC input in a
homogeneous manner, similar to standard electronic components, such that a
phase shift can be
determined. This may serve as the basis of detection between single-stranded
and double stranded
nucleic acids, but more importantly, may allow the detection of mismatches,
since small changes in
impedance, such as would be assumed from a mismatch present in the
hybridization complex, may
effect the output AC phase in a greater manner than the frequency response.
The output signal is characteristic of electron transfer through the
hybridization complex; that is, the
output signal is characteristic of the presence of double stranded nucleic
acid. In a preferred
embodiment, the basis of the detection is a difference in the faradaic
impedance of the system as a
result of the formation of the hybridization complex. Faradaic impedance is
the impedance of the
system between the two electron transfer moieties, i.e. between the electrode
and the second electron
transfer moiety. Faradaic impedance is quite different from the bulk or
dielectric impedance, which is
the impedance of the bulk solution between the electrodes. Many factors may
change the faradaic
impedance which may not effect the bulk impedance, and vice versa. Thus,
nucleic acids in this
system have a certain faradaic impedance, that will depend on the distance
between the electron
transfer moieties, their electronic properties, and the composition of the
intervening medium, among
other things. Of importance in the methods of the invention is that the
faradaic impedance between
the electron transfer moieties is signficantly different depending on whether
the intervening nucleic
acid is single stranded or double stranded. Thus, the faradaic impedance of
the system changes upon
the formation of a hybridization complex, and it is this change which is
characteristic of the
hybridization complex.
Accordingly, the present invention further provides apparatus for the
detection of nucleic acids using
AC detection methods. The apparatus includes a test chamber which has at least
a first measuring or
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sample electrode, and a second measuring or counter electrode. Three electrode
systems are also
useful. The first and second measuring electrodes are in contact with a test
sample receiving region,
such that in the presence of a liquid test sample, the two electrodes may be
in electrical contact.
In a preferred embodiment, the first measuring electrode comprises a single
stranded nucleic acid
covalently attached via a spacer, and preferably via a conductive oligomer,
such as are described
herein. In one embodiment, the second electron transfer moiety may be attached
to the probe single
stranded nucleic acid, or it may be attached to a second probe nucleic acid,
the target nucleic acid, or
may be added separately, for example as an intercalator. In a preferred
embodiment, the second
electron transfer moiety is covalently attached to the probe single stranded
nucleic acid.
The apparatus further comprises an AC voltage source electrically connected to
the test chamber; that
is, to the measuring electrodes. Preferably, the AC voltage source is capable
of delivering DC offset
voltage as well.
In a preferred embodiment, the apparatus further comprises a processor capable
of comparing the
inpu# signal and the output signal. The processor is coupled to the electrodes
and configured to
receive an output signal, and thus detect the presence of the target nucleic
acid.
Thus, the compositions of the present invention may be used in a variety of
research, clinical, quality
control, or field testing settings.
In a preferred embodiment, the probes are used in genetic diagnosis. For
example, probes can be
made using the techniques disclosed herein to detect target sequences such as
the gene for
nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene
associated with a
variety of cancers, the Apo E4 gene that indicates a greater risk of
Alzheimer's disease, allowing for
easy presymptomatic screening of patients, mutations in the cystic fibrosis
gene, or any of the others
well known in the art.
In an additional embodiment, viral and bacterial detection is done using the
complexes of the
invention. In this embodiment, probes are designed to detect target sequences
from a variety of
bacteria and viruses. For example, current blood-screening techniques rely on
the detection of anti-
HIV antibodies. The methods disclosed herein allow for direct screening of
clinical samples to detect
HIV nucleic acid sequences, particularly highly conserved HIV sequences. In
addition, this allows
direct monitoring of circulating virus within a patient as an improved method
of assessing the efficacy
of anti-viral therapies. Similarly, viruses associated with leukemia, HTLV-l
and HTLV-II, may be
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detected in this way. Bacterial infections such as tuberculosis, clymidia and
other sexually transmitted
diseases, may also be detected.
In a preferred embodiment, the nucleic acids of the invention find use as
probes for toxic bacteria in
the screening of water and food samples. For example, samples may be treated
to lyse the bacteria
to release its nucleic acid, and then probes designed to recognize bacterial
strains, including, but not
limited to, such pathogenic strains as, Salmonella, Campylobacter, Vibrio
cholerae, Leishmania,
enterotoxic strains of E. coli, and Legionnaire's disease bacteria. Similarly,
bioremediation strategies
may be evaluated using the compositions of the invention.
In a further embodiment, the probes are used for forensic "DNA fingerprinting"
to match crime-scene
DNA against samples taken from victims and suspects.
In an additional embodiment, the probes in an array are used for sequencing by
hybridization.
The present invention also finds use as a unique methodology for the detection
of mutations or
mismatches in target nucleic acid sequences. As a result, if a single stranded
nucleic acid containing
electron transfer moieties is hybridized to a target sequence with a mutation,
the resulting perturbation
of the base pairing of the nucleosides will measurably affect the electron
transfer rate. This is the case
if the mutation is a substitution, insertion or deletion. Alternatively, two
single stranded nucleic acids
each with a covalently attached electron transfer species that hybridize
adjacently to a target
sequence may be used. Accordingly, the present invention provides for the
detection of mutations in
target sequences.
Thus, the present invention provides for extremely specific and sensitive
probes, which may, in some
embodiments, detect target sequences without removal of unhybridized probe.
This will be useful in
the generation of automated gene probe assays.
In an alternate embodiment the electron transfer moieties are on separate
strands. In this
embodiment, one single stranded nucleic acid has an electrode covalently
attached via a conductive
oligomer. The putative target sequences are labelled with a second electron
transfer moiety as is
.. generally described herein, i.e. by incorporating an electron transfer
moiety to individual nucleosides of
a PCR reaction pool. Upon hybridization of the two single-stranded nucleic
acids, electron transfer is
detected.
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Alternatively, the compositions of the invention are useful to detect
successful gene amplification in
PCR, thus allowing successful PCR reactions to be an indication of the
presence or absence of a
target sequence. PCR may be used in this manner in several ways. For example,
in one
embodiment, the PCR reaction is done as is known in the art, and then added to
a composition of the
invention comprising the target nucleic acid with a second ETM, covalently
attached to an electrode
via a conductive oligomer with subsequent detection of the target sequence.
Alternatively, PCR is
done using nucleotides labelled with a second ETM, either in the presence of,
or with subsequent
addition to, an electrode with a conductive oligomer and a target nucleic
acid. Binding of the PCR
product containing second ETMs to the electrode composition will allow
detection via electron transfer.
Finally, the nucleic acid attached to the electrode via a conductive polymer
may be one PCR primer,
with addition of a second primer labelled with an ETM. Elongation results in
double stranded nucleic
acid with a second ETM and electrode covalently attached. In this way, the
present invention is used
for PCR detection of target sequences.
The present invention provides methods which can result in sensitive detection
of nucleic acids. In a
preferred embodiment, less than about 10 X 106 molecules are detected, with
less than about 10 X 105
being preferred, less than 10 X 104 being particularly preferred, less than
about 10 X 103 being
especially preferred, and less than about 10 X 102 being most preferred. As
will be appreciated by
those in the art, this assumes a 1:1 correlation between target sequences and
reporter molecules; if
more than one reporter molecule (i.e. second electron transfer moeity) is used
for each target
sequence, the sensitivity will go up.
While the limits of detection are currently being evaluated, based on the
published electron transfer
rate through DNA, which is roughly 1 X 10s electrons/seclduplex for an 8 base
pair separation (see
Meade et al., Angw. Chem. Eng. Ed., 34:352 (1995)) and high driving forces, AC
frequencies of about
100 kHz should be possible. As the preliminary results show, electron transfer
through these systems
is quite efficient, resulting in nearly 100 X 103 electrons/sec) resulting in
potential femptoamp sensitivity
for very few molecules.
In an additional embodiment, the present invention provides novel compositions
comprising
metallocenes covalently attached via conductive oligomers to an electrode,
such as are generally
depicted in Structure 35:
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Structure 35
~Y-B-D j-j-Y-f--Z-Lm
_ 5 ,'.~,
Structure 35 utilizes a Structure 4 conductive oligomer, although as will be
appreciated by those in the
art, other conductive oligomers such as Structures 2, 3, 9 or 10 types may be
used. Preferred
embodiments of Structure 35 are depicted below.
Structure 36
4"
n ~%r
Structure 37
R R R, R
I1 "~r
, ~M
\\ .''4.
\R - R~m
Preferred R groups of Structure 37 are hydrogen.
Structure 38
Fe
3
These compositions are synthesized as follows. The conductive oligomer finked
to the metallocene is
made as described herein; see also, Hsung et al., Organometallics 14:4808-4815
(1995); and Bumm
et al., Science 271:1705 (1996), both of which are expressly incorporated
herein by reference. The
conductive oligomer is then attached to the electrode using the novel
ethylpyridine protecting group,
as outlined herein.
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Once made, these compositions have unique utility in a number of applications,
including
photovoltaics, and infrared detection. A preferred embodiment utilizes these
compounds in calibrating
a potentiostat, serving as an internal electrochemistry reference in an array
of the invention.
The following examples serve to more fully describe the manner of using the
above-described
invention, as well as to set forth the best modes contemplated for carrying
out various aspects of the
invention. It is understood that these examples in no way serve to limit the
true scope of this invention,
but rather are presented for illustrative purposes. All references cited
herein are incorporated by
reference.
EXAMPLES
Example 1
Synthesis of Conductive Oligomer linked via an amide to a nucleoside
This synthesis is depicted in Figure 1, using uridine as the nucleoside and a
Structure 4 phenyl-
acetylene conductive oligomer.
Compound # 1: To a solution of 10.0 gm (40 mmol) of 4-iodothioanisole in 350
mL of dichforomethane
cooled in an ice-water bath was added 10.1 gm of mCPBA. The reaction mixture
was stirred for half
hour and the suspension was formed. To the suspension was added 4.0 gm of
powered Ca(OH)2, the
mixture was stirred at room temperature for 15 min and filtered off and the
solid was washed once with
mL of dichloromethane. To the combined filtrate was added 12 mL of
trifluoroacetic anhydride and
the reaction mixture was refluxed for 1.5 h under Argon. After removing the
solvents, the residue was
dissolved in 200 mL of a mixture of TEA and methanol (ratio = 50 : 50) and
concentrated to dryness.
25 The residue was dissolved in 100 mL of dichloromethane and the solution was
washed once with 60
mL of the saturated ammonim chloride solution. The aqueous layer was extracted
twice with
dichloromethane (2 x 70 mL). The organic extracts were combined and dried over
anhydrous sodium
sulfate and immediately concentrated to dryness as quickly as possible. The
residue was dissolved in
120 mL of benzene, followed by adding 5.3 mL of 4-vinylpyridine. The reaction
mixture was refluxed
30 under Argon overnight. The solvent was removed and the residue was
dissolved in dichloromethane
for column chromatography. Silica gel (150 gm) was packed with 20 % ethyl
acetate / hexane
mixture.The crude product solution was loaded and the column was eluted with
20 to 60 % ethyl
acetate / hexane mixture. The fractions was identified by TLC (EtOAc : Hexane
= 50 : 50, Rf = 0.24) .
and pooled and concentrated to dryness to afford 7.4 gm (54.2%) of the solid
title compound.
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Compound # 2: To a solution of 3.4 gm (9.97 mmol) of Compound # 1 in 70 mL of
diethylamine was
added 200 mg of bis(triphenylphosphine)palladium (II) chloride, 100 mg of
cuprous iodide and 1.9 mL
of trimethylsilylacetyiene under Argon. The reaction mixture was stirred for 2
h. After removing the
diethylamine, the residue was dissolved in dichloromethane for column
chromatography. Silica gel
(120 gm) was packed with a cosolvent of 50 % ethyl acetate / 50 % hexane. The
crude sample
solution was loaded and the column was eluted with the same cosolvent. After
removing the solvents,
the liquid title compound (2.6 gm, 83.7 %) was obtained.
Compound # 3: To a solution of 2.6 gm of Compound # 2 in 150 mL of
dichloromethane coiled in an
ice-water bath was added 9.0 mL of 1 N tetrabutylammonium fluoride THF
solution. The reaction
mixture was stirred for 1 h. and washed once with water and dried over
anhydrous Na2S04. After
removing the solvent, the residue was used for column separation. Silica gel
(50 gm) was packed with
a coslovent of 50 % ethyl acetate / 50 % hexane. The crude product solution
was loaded and the
column was eluted with the same solvents. The removal of the solvents gave the
solid title compound
(1.87 gm, 94.1 %).
Compound # 4: To a glass bottle were added 1.80 gm (7.52 mmol) of Compound #
3, 160 mg of
bis(triphenylphosphine)palladium (II) chloride, 80 mg of cuprous iodide and
2.70 gm (9.0 mmol) of 1-
trimethylsilyl-2-(4-iodophenyl)acetylene. The bottle was sealed and bubbled
with Argon. Diethyiamine
was introduced by a syringe. The reaction mixture was heated at 50 ~C under
Argon for 1 h. The
amine was removed and the residue was dissolved in dichloromethane for the
separation. Silica gel
(100 gm ) was packed with 60 % ethyl acetate / hexane. The crude mixture was
loaded and the
column was eluted with the same solvents. The fractions were identified by TLC
(EtOAc : Hexane =
50 : 50, the product emitted blue light) and pooled. The removal of the
solvents gave the solid title
product (2.47 gm, 79.8 %).
Compound # 5: To a solution of 2.47 gm of Compound # 4 in 130 mL of
dichloromethane cooled in
an ice-water bath was added 8.0 mL of 1 N tetrabutylammonium fluoride THF
solution. The reaction
mixture was stirred for 1 h. and washed once with water and dried over
anhydrous NazS04. After
removing the solvent, the residue was used for column separation. Silica gel
(60 gm) was packed with
a coslovent of 50 % ethyl acetate / 50 % CHZCIz. The crude solution was loaded
and the column was
eluted with the same solvents. The removal of solvents gave the solid title
product (1.95 gm, 95.7 %).
Compound # 6: To a glass bottle were added 0.23 gm (0.68 mmol) of Compound #
5, 0.5 gm (0.64
mmol) of 2'-deoxy-2'-(4-iodophenylcarbonyl) amino-5'-O-DMT uridine, 60 mg of
bis(triphenylphosphine)palladium (II) chloride, 30 mg of cuprous iodide. The
bottle was sealed and
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bubbled with Argon. Pyrrodine(15 mL) and DMF(15 mL) were introduced by a
syringe. The reaction
mixture was heated at 85 ~C overnight. The solvents were removed in vacuo and
the residue was
dissolved in 300 mL of dichloromethane. The solution was washed three times
with water and dried
over sodium sulfate. After removing the solvent, the residue was subjected to
column purification.
Silica gel (30 gm) was packed with 1 % TEA/1 % methanoll CH2CI2 and the sample
solution was
loaded. The column was eluted with 1 % TEAI1 % methanoI/CH2Cl2 and 1 % TFr4l2
methanoI/CH2Clz. The fractions were identified and concentrated to dryness.
The separated product
was subjected to another reverse-phase column purification. Reverse-phase
silica gel(C-18, 120 gm)
was packed with 60 % CH3CNI40 % H20 and the sample was dissolved in very small
amount of THF
and loaded. The column was eluted with 100 mL of 60 % CH3CN/40 % H20, 100 mL
of 70
CH3CNI30 % HzO, 100 mL of 60 % CH3CN/10 % THF/30 % HzO, 200 mL of 50 %
CH3CN/20
THFI30 % H20 and 500 mL of 35 % CH3CN/35 % THF I30 % H20. The fractions were
identified by
HPLC (0.1 mM TEAA : CH3CN = 20 : 80, flow rate = 1.0 mL/min). and concentrated
to dryness to
afford a pure title compound.
Compound # 7: To a solution of 100 mg(0.1 mmol) of pure compound # 6 in 40 mL
of pyridine were
added 50 mgm of DMAP and 1.0 gm {10 mmol) of succinic anhydride. The reaction
mixture was
stirred under Argon for 40 h. After removing pyridine, the residue was
dissolved in 300 mL of
dichloromethane, followed by adding 150 mL of 5 % aqueous NaHC03 solution. The
mixture was
vigorously stirred for 3 h and separated. The organic layer was washed once
with 1 % citric acid
solution and dried over anhydrous sodium sulfate and concentrated to dryness
to give 110 mgm of
Compound # 7. Without further purification, the Compound # 7 was used for the
preparation of the
corresponding CPG.
Conductive oligomer-Uridine-CPG: To 1.4 gm of LCAA-CPG(500 _) in 100 mL round
bottom flask
were added 110 mgm(101 Nmol) of the Compound # 7, 100 mgm (230 Nmol) of BOP
reagent, 30
mgm (220 Nmol) of HBT, 70 mL of dichloromethane and 2 mL of TEA. The mixture
was shaken for
three days. The CPG was filtered off and washed twice with dichloromethane and
transferred into
another 100 mL flask. Into CPG were added 50 mL of pyridine, 10 mL of acetic
anhydride and 2mL of
N-methylimidizole. The CPG was filtered off, washed twice with pyridine,
methanol, dichloromethane
and ether, and dried over a vacuum. The loading of the nucleoside was measured
according to the
standard procedure to be 7.1 Nmol/gm.
2'-Deoxy-2'-(4-iodophenylcarbonyl)amino-5'-O-DMT uridine: To a solution of 5.1
gm(9.35 mmol)
of 2'-deoxy-2'-amino-5'-O-DMT uridine in 250 mL of pyridine cooled in an ice-
water bath was added 3
mL of chlorotrimethylsilane. The reaction mixture was warmed up to room
temperature and stirred for
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1 h. To the prepared solution were added 0.1 gm of DMAP and 3.0 gm (10.9 mmol)
4-iodobezoyl
chloride and the reaction mixture was stirred overnight. To this solution was
added 30 mL of
concentrated ammonium hydroxide solution and the mixure was stirred for exact
15 min. The solvents
were removed in vacuo. The residue was dissolved in 15 mL of dichloromethane
for column
separation. Silica gel (125 gm) was packed with 1 % TEAI2 % CH30H/CH2C12.
After loading the
sample, the column was eluted with 300 mL of 1 % TEA/2 % CH30H/CHZCI2, and 500
mL of 1
TEA/4 % CH30H/CHZCIz. The fractions were identified by TLC (CH30H : CH2CIz =
10 : 90) and pooled
and concentrated to dryness to give 6.2 gm (85.5 %) of the pure title
compound.
Synthesis of the Phosphormidite (Compound # 8).
To a solution of 0.2 gm of Compound # 6 and 30 mg of diisopropylammonium
tetrazolide in 10 mL of
dry dichloromethane is added 0.12 gm of 2-cyanoethyl N, N, N', N'-
tetraisopropyiphosphane under
Argon. The solution was stirred for 5 h and diluted by adding 60 mL of
dichloromethane. The solution
was washed twice with 2.5 % w/v sodium bicarbonate solution, once with the
brine and dried over
sodium sulfate. After removing the solvent, residue was dissolved in 5 mL of
dichloromethane,
followed by adding slowly 100 mL of hexane. The suspension was stored at - 20
~C for 1 h. The
supernatant was decanted and the residue was dried over a high vacuum
overnight to afford 0.19 gm
79.0 %) of the title product, which will be used for DNA synthesis.
In addition, this procedure was done to make a four unit wire.
Example 2
Synthesis of conductive oligomers linked to the ribose
of a nucleoside via an amine linkage
Example 2A:
Synthesis of 2'-(4-iodophenyl)amino-2'-deoxy-5'-O-DMT-uridine (Product 4):
This synthesis is
depicted in Figure 2, and reference is made to the labelling of the products
on the figure. To a solution
of 5.0 gm of 5'-O-DMT-uridine (Product 1 ) and 2.7 gm of dimethylaminopyridine
in 200 mL of
acetonitrile was added 3.3 gm of p-iodophenyl isocyalide dichloride portion by
portion under Argon.
The reaction mixture was stirred overnight. The mixture was diluted by adding
550 mL of
dichloromethane and washed twice with 5 % sodium bicarbonate aqueous solution
and once with the
brine solution, and then dried over sodium sulfate. The removal of the solvent
in vacuo gave the crude
Product 2. Without further purification, Product 2 was dissolved in 50 mL of
dry DMF and the
solution was heated at 150 ~C foe 2 h. After distillation of DMF, the residue
was dissolved in 300 mL
of dichloromethane, washed once with 5 % sodium bicarbonate solution, once
with the brine solution
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and dried over sodium sulfate. The removal of the solvent gave the crude
Product 3. Without
purification, the Product 3 was dissolved 100 mL of a mixture of 50 %Dioxane
and 50 % Methanol.
To this solution was added 43 mL of 1 N NaOH solution. The reaction mixture
was stirred overnight.
The mixture was diluted by adding 800 mL of dichloromethane and washed twice
water and dried over
Na2S04. After removing the solvent, the residue was dissolved in 15 mL of
dichloromethane for the
column separation. Silica gel (100 gm) of packed with 1 % TEA I 2 % Ethanol /
CHZCI2, after loading
the sample solution, the column was eluted with 1 % TEA / 2 - 3 % Ethanol /
CHZCIz. The fractions
were identified by TLC (CH30H : CH2CI2 = 1 : 9) and pooled and concentrated to
give 2.0 gm (29.2
%) of the Product 4.
Additional conductive oligomer units can then be added to product 4 as
outlined herein, with additional
nucleotides added and attachment to an electrode surtace as described herein.
Example 2B:
Benzylamino-uridine was synthesized as shown in Figure 16.
Synthesis of Compound C2: To a solution of 8.3 gm (15.7 mmol) of
cyclonucleoside C1 in 200 mL
of dichloromethane was added 2.80 gm of carbonyldiimidazole under Argon. After
the solution was
stirred for 7 h, into this solution were added 4.3 gm of 4-iodobenzylamine and
10 mL of
diisopropylethylamine. The mixture was stirred overnight under Argon
atmosphere. The solution was
washed twice with 5 % Citric acid solution and dried over sodium sulfate.
After concentration, the
residue was dissolved in a small amount of dichgloromethane for the column
separation. Silica gel
(150 gm) was packed with 1 % TEA I 2 % CH30H I CHZCI2, upon loading the sample
solution, the
column was eluted with 1 % TEA / 2-10 % CH30H / CHZCI2. The fractions were
identified by TLC
(CH30H : CHZCIz = 7 : 93) and pooled and concentrated to afford 9.75 gm (78.8
%) of the product C2.
Synthesis of Compound C3:A mixture of 9.75 gm (12.4 mmol) of the compound C2
and 1.0 mL of
DBU in 250 mL of dry THF was stirred at 50 ~C under Argon for two days. THF
was removed by a
rotavapor and the residue was dissolved 20 mL of dichloromethane for the
purification. Silica gel (130
gm) was packed with 1 % TEA I 25 % EtOAc I CH2CIz, after loading the sample
solution, the column
was eluted with same solvent mixture. The fractions containing the desired
product was pooled and
concentrated to give 6.46 gm (66.3 %) of the product C3.
Synthesis of the Final Compound C4: The compound C3 (6.46 gm) was dissolved in
a mixture of
150 mL of 1,4-dioxane and 100 mL of methanol, followed by adding 100 mL of 4.0
M aqueous sodium
hydroxide. The mixture was stirred at room temperature overnight. The solution
was diluted by
adding 500 mL of dichloromethane and 500 mL of the brine solution. The mixture
was shaken well
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and the organic layer was separated and washed once with the 500 mL of the
brine solution and dried
over sodium sulfate. The dichloromethane was removed by a rotavapor and the
dixoxane was
removed by a high vacuum. The residue was dissolved in 20 mL of
dichloromethane for the
separation. Silica gel (80 gm) was packed with 1 % TEA I 25 % EtOAc I CHzCl2
and the sample
solution was loaded. The column was eluted with 1 % TEA I 25-50 % EtOAc I
CH2CI2. The right
fractions were combined and concentrated to give 4.1 gm (65.7 %) of the final
product C4.
Example 3
Synthesis of a conductive oiigomer with an R group attached to
the Y aromatic group
This synthesis is depicted in Figure 6.
Synthesis of 2-Acetyl-5-iodotoluene (P 1 ). To a suspension of 20 gm of
aluminum trichloride in 500
mL of dichloromethane was added 10.2 mL of acetyl chloride under Argon. After
the reaction mixture
was stirred for 15 min, 3-iodotoluene (20 gm) was added through a syringe. The
mixture was stirred
overnight under Argon and poured into 500 gm of ice-water. Organic layer was
separated and
washed once with the saturated ammonium chloride solution, and washed once
with 10 % sodium
thiosulfate solution and dried over sodium sulfate. After removing the
solvent, the residue was
dissolved in hexane for the column purification. Silica gel (260 gm) was
packed with hexane, after
loading the sample solution, the column was eluted with 750 mL of hexane, 750
mL of 1 % v/v ether /
hexane, 750 mL of 2 % v/v ether I hexane and 1500 mL of 3 % v/v ether I
hexane. The fractions
containing the right isomer were identified by GC-MS and'H NMR and pooled and
concentrated to
dryness to afford 12.2 gm (51.2 %) of the title product (P 1).
lodo-3-methyl-4-(ehynyl trimethylsilyl) benzene (P2). Under inert atmosphere
500 ml bound bottom
flask was charged with 25 ml of dry THF, cooled to -78~C and 14 ml of 2.0 M
LDA solution
(heptanelethylbenzenel THF solution) was added by syringe. To this solution
6.34 gr (24.38 mmole) of
iodo-3-methyl-4-acetyl benzene in 25 ml of THF was added dropwise and the
reaction mixture was
stirred for 1 hr at -78 ~ C, then 4.0 ml ( 19.42 mmole) of
diethylchlorophosphate were added by syringe.
After 15 min cooling bath was removed and the reaction mixture was allowed to
heat up to RT and
stirred for 3 hrs. The resulted mixture was cooled again to -78~C and 29 ml of
2.0 M LDA solution
_ were added dropwise. At the end of the addition the reaction mixture was
allowed to warm up to RT
and stirred for additional 3 hrs. After that period of time it was cooled
again to -20~C, 9.0 ml (70.91
mmole) of trimethylsilyl chloride were injected and the stirring was continued
for 2 hrs at RT. The
reaction mixture was poured into 200 ml of icelsodium bicarbonate saturated
aqueous solution and
300 ml of ether were added to extract organic compounds. The aqueous phase was
separated and
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extracted again with 2x100 ml of ether. The ether fractions were combined,
dried over sodium sulfate
and evaporated. The resulted liquid residue was purified by silica gel
chromatography (100% n-
hexane as eluent). 4.1 gr (54% yield) were obtained.
Synthesis of Product (P 3). To a solution of 1.14 gm of Compound # 3 (as
described above) and
1.60 gm of P 2 in 100 mL of diethylamine were added 0.23 gm of [1,1'-
bis(diphenylphosphino)ferrocene]palladium (II) chloride and 0.1 gm of copper
(I) iodide under Argon.
The reaction mixture was stirred at 55 ~C for 1 h and stirred at room
temperature overnight. After
removing the solvent, the residue was dissolved in dichloromethane for column
separation. Silica gel
(120 gm) was packed with 20 % ethyl acetate I CHzCl2. The sample solution was
loaded and the
column was eluted with 20 - 50 % ethyl acetate / CHZCI2. The fractions were
identified by TLC (EtOAC
CH2C12 = 50 : 50) and pooled and concentrated to give 1.70 gm (84.0 %) of TMS-
derivative of P 3.
To a solution of 0.74 gm of TMS-derivative of P 3 in 70 mL of dichloromethane
at 0 ~C was added 2.2
mL of 1.0 M (nBu)4NF THF solution. After stirring for 30 min, the solution was
washed once with
water and dried over sodium sulfate. The solvent was removed, the residue was
used for column
separation. Silica gel (20 gm) was packed with 20 % ethyl acetate / CH2CI2,
the column was eluted
with 20 - 40 % ethyl acetate I CHZCIz. The fractions containing the
fluorescent compound were
combined and concentrated to dryness to afford 0.5 gm (81.3 %) of the pure P
3.
Synthesis of P 4: To a solution of 0.5 gm of P 3 and 0.63 gm of P 2 in 50 mL
of dry DMF and 10 mL
of TEA were added 100 mgm of [1,1'-bis(diphenylphosphino)ferrocene]palladium
(II) chloride and 50
mgm of copper (I) iodide under Argon. The reaction mixture was stirred at 55
~C for 1 h and stirred at
35 ~C overnight. The solvents were removed in vacuo and the residue was
dissolved in 10 mL of
CHZCIZ for column separation. Silica gel (100 gm) was packed with 20 % ethyl
acetate / CHZCIz, after
loading the sample, the column was eluted with 20 - 40 % ethyl acetate /
CHZCI2. The fractions were
identified by TLC (EtOAC : CHZCIZ = 50 : 50) and pooled and concentrated to
give 0.47 gm (61.3 %) of
TMS-derivative of P 4.
To a solution of 0.47 gm of TMS-derivative of P 4 in 70 mL of dichloromethane
at 0 ~C was added 1.0
mL of 1.0 M (nBu)4NF THF solution. After stirring for 30 min, the solution was
washed once with
water and dried over sodium sulfate. The solvent was removed, the residue was
used for column
separation. Silica gel (20 gm) was packed with 20 % ethyl acetate / CHZCIz,
the column was eluted
with 20 - 40 % ethyl acetate / CH2Clz. The fractions containing the
fluorescent compound were
combined and concentrated to dryness to afford 0.32 gm (78.7 %) of the pure P
4.
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Other conductive oligomers with R groups are depicted in Figure 17, which were
made using the
techniques outlined herein.
Example 4
Synthesis of a nucleoside with a metallocene second electron transfer
moiety attached via a ribose
Synthesis of 5'-O-DMT-2'-deoxy-2'-(ferrocenecarbonyl)amino Uridine (UAF): To a
solution of 2.5
gm(10.9 mmol) of ferrocene monocarboxylic acid in 350 mL of dichloromethane
were added 2.25 gm
(10.9 mmol) of DCC and 1.27 gm (10.9 mmol) of N-hydroxysuccinimide. The
reaction mixture was
stirred for 3 h and the precipitate was formed. The precipitate was filtered
off and washed once with
dichloromethane. The combined filtrate was added into 4.5 gm (8.25 mmol) of 2'-
deoxy-2'-amino-5'-O-
DMT uridine, followed by adding 2 mL of triethylamine. The reaction mixture
was stirred at room
temperature for 8 days. After removing the solvent, the residue was dissolved
in dichloromethane for
separation. Silica gel (120 gm) was packed with 1 % TEA I 2 % CH30H I CHZCI2.
After loading the
sample solution, the column was eluted with 2-7 % CH30HI1 % TEA / CH2CI2. The
fraction was
identified by TLC(CH30H : CH2C12 = 1 : 9) and pooled and concentrated to
dryness to afford 1.3
gm(22.0 %) of the title compound.
Synthesis of UAF Phosphoramidite:
Preparation of Diisopropylaminochloro(p-cyano)ethoxyphosphine: To a solution
of 0.54 mL(4.0
mmol) of dichloro(~i-cyano)ethoxyphosphine in 40 mL of dichloromethane cooled
in an ice-water bath
was added 10 mL of diisopropylethylamine, followed by adding 0.64 mL (4.0
mmol) of
diisopropylamine under Argon. The reaction mixture was warmed up to room
temperature and stirred
for 2 h. After adding 0.1 gm of DMAP into the solution, the reaction mixture
is ready for the next step
reaction.
Preparation of UAF phosphoramidite: To a solution of 1.30 gm (1.72 mmol) of 5'-
O-DMT-5-
ferrocenylacetylenyl-2'-deoxy uridine in 40 mL of dichloromethane cooled in an
ice-water bath was
added 10 mL of diisopropylethylamine. The prepared phosphine solution was
transferred into the
nucleoside solution through a syringe. The reaction mixture was warmed up to
room temperature and
stirred overnight. The solution was diluted by adding 100 mL of
dichloromethane and washed once
with 200 mL od 5 % aqueous NaHC03 solution, and once with the brine (200 mL)
and dried over
NaZS04 and concentrated to dryness. Silica gel(47 gm) was packed with 2 %
TEA/1
CH30H/CHZCI2. The residue was dissolved in 10 mL of dichloromethane and
loaded. The column
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was eluted with 150 mL of 1 % TEA I 1 % CH30H I CHZCIZ and 250 mL of 1 % TEA
/2 % CH30H I
CHZCI2. The fractions were pooled and concentrated to give 0.5 gm (30.3 %) of
the title compound.
Nucleotides containing conductive oligomers and second electron transfer
moieties were incorporated
into nucleic acids using standard nucleic acid synthesis techniques; see
"Oligonucleotides and
Analogs, A Practical Approach", Ed. By F. Eckstein, Oxford University Press,
1991, hereby
incorporated by reference.
Example 5
Synthesis of a nucleoside with a metallocene second electron transfer
moiety attached via the base
Synthesis of 5'-O-DMT-5-ferrocenylacetylenyl-2'-deoxy uridine (UBF): In a
flask were added 4.8
gm(13.6 mmol) of 5-iodo-2'-deoxy uridine, 400 mg of
bis(triphenylphosphine)palladium (li) chloride,
100 mg of cuprous iodide, 95 mL of DMF and 10 mL of TEA. The solution was
degassed by Argon
and the flask was sealed. The reaction mixture was stirred at 50 ~C overnight.
After removing
solvents in vacuo, the residue was dissolved in 140 mL of dry pyridine,
followed by adding 0.2 gm of
DMAP and 5.0 gm (14.8 mmol) of DMT-CI. The reaction mixture was stirred at RT
overnight. After
removing the solvent, the residue was dissolved in 300 mL of dichloromethane
and washed finrice with
5 % aqueous NaHC03 (2 x 200 mL), twice with the brine (2 x 200 mL) and dried
over sodium sulfate.
The solvent was removed and the residue was coevaporated twice with toluene
and dissolved in 15
mL of dichloromethane for column separation. Silica gel (264 gm) was packed
0.5 % TEA/CH2C12.
After loading the crude product solution, the column was eluted with 300 mL of
1 % TEA/2
CH30H/CHzCl2, 400 mL of 1 ~!~ TEAl5 % CH30H/CH2CIz, and 1.2 L of 1 % TEAl7 %
CH30H/CH2Ct2.
The fractions were identified by TLC(CH30H : CHZCIZ = 10 : 90) and pooled and
concentrated to
dryness to give 7.16 gm (71.3 %) of the title compound.
Synthesis of UBF Phosphoramidite:
Preparation of Diisopropylaminochloro((3-cyano)ethoxyphosphine: To a solution
of 1.9 mL(13.8
mmol) of dichloro(p-cyano)ethoxyphosphine in 40 mL of dichloromethane cooled
in an ice-water bath
was added 10 mL of diisopropylethylamine, followed by adding 2.3 mL (13.8
mmol) of
diisopropylamine under Argon. The reaction mixture was warmed up to room
temperature and stirred
for 2 h. After adding 0.1 gm of DMAP into the solution, the reaction mixture
is ready for next step
reaction.
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Preparation of UBF phosphoramidite: To a solution of 3.42 gm (4.63 mmol) of 5'-
O-DMT-5-
ferrocenylacetylenyl-2'-deoxy uridine in 40 mL of dichloromethane cooled in an
ice-water bath was
added 10 mL of diisopropylethylamine. The prepared phosphine solution was
transferred into the
nucleoside solution through a syringe. The reaction mixture was warmed up to
room temperature and
stirred overnight. The solution was diluted by adding 150 mL of
dichloromethane and washed once
with 200 mL of 5 % aqueous NaHC03 solution, and once with the brine (200 mL)
and dried over
NazS04 and concentrated to dryness. Silica gel(92 gm) was packed with 2 %
TEA/1
CH30H/CHzCIz. The residue was dissolved in 10 mL of dichloromethane and
loaded. The column
was eluted with 500 mL of 1 % TEA/2 % CH30H/CHZCI2. The fractions were pooled
and concentrated
to give 3.0 gm (69.0 %) of the title compound.
Nucleotides containing conductive oligomers and second electron transfer
moieties were incorporated
into nucleic acids using standard nucleic acid synthesis techniques; see
"Oligonucleotides and
Analogs, A Practical Approach", Ed. By F. Eckstein, Oxford University Press,
1991, hereby
incorporated by reference.
Example 6
Synthesis of an electrode containing nucleic acids containing
conductive oligomers with a monolayer of (CHZ),s
Using the above techniques, and standard nucleic acid synthesis, the uridine
with the phenyl-
acetylene conductive polymer of Example 1 was incorporated at the 3' position
to form the following
nucleic acid: ACCATGGACTCAGCU-conductive polymer of Example 1 (hereinafter
"wire-1").
HS-(CH2)16-OH (herein "insulator-2") was made as follows.
16-Bromohexadecanoic acid. 16-Bromohexadecanoic acid was prepared by refluxing
for 48 hrs 5.0
gr (18.35 mmole) of 16-hydroxyhexadecanoic acid in 24 ml of 1:1 v/v mixture of
HBr (48% aqueous
solution) and glacial acetic acid. Upon cooling, crude product was solidified
inside the reaction vessel.
It was filtered out and washed with 3x100 ml of cold water. Material was
purified by recrystalization
from n-hexane, filtered out and dried on high vacuum. 6.1 gr {99% yield) of
the desired product were
obtained.
16-Mercaptohexadecanoic acid. Under inert atmosphere 2.0 gr of sodium metal
suspension (40% in
mineral oil) were slowly added to 100 ml of dry methanol at 0~C. At the end of
the addition reaction
mixture was stirred for 10 min at RT and 1.75 ml (21.58 mmole) of thioacetic
acid were added. After
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additional 10 min of stirring, 30 ml degassed methanolic solution of 6.1 gr
(18.19 mmole) of 16-
bromohexadecanoic acid were added. The resulted mixture was refluxed for 15
hrs, after which,
allowed to cool to RT and 50 ml of degassed 1.0 M NaOH aqueous solution were
injected. Additional
refluxing for 3 hrs required for reaction completion. Resulted reaction
mixture was cooled with ice bath
and poured, with stirring, into a vessel containing 200 ml of ice water. This
mixture was titrated to
pH=7 by 1.0 M HCI and extracted with 300 ml of ether. The organic layer was
separated, washed with
3x150 ml of water, 150 ml of saturated NaCI aqueous solution and dried over
sodium sulfate. After
removal of ether material was purified by recrystalization from n-hexane,
filtering out and drying over
high vacuum. 5.1 gr (97% yield) of the desired product was obtained.
16-Bromohexadecan-1-ol. Under inert atmosphere 10 ml of BH3~THF complex (1.0 M
THF solution)
were added to 30 ml THF solution of 2.15 gr (6.41 mmole) of 16-
bromohexadecanoic acid at -20~C.
Reaction mixture was stirred at this temperature for 2 hrs and then additional
1 hr at RT. After that
time the resulted mixture was poured, with stirring, into a vessel containing
200 ml of ice/saturated
sodium bicarbonate aqueous solution. Organic compounds were extracted with
3x200 ml of ether. The
ether fractions were combined and dried over sodium sulfate. After removal of
ether material was
dissolved in minimum amount of dicloromethane and purified by silica gel
chromatography (100%
dicloromethane as eluent). 1.92 gr (93% yield) of the desired product were
obtained.
16-Mercaptohexadecan-1-ol. Under inert atmosphere 365 mg of sodium metal
suspension (40% in
mineral oil) were added dropwise to 20 ml of dry methanol at 0~C. After
completion of addition the
reaction mixture was stirred for 10 min at RT followed by addition of 0.45 ml
(6.30 mmole) of
thioacetic acid. After additional 10 min of stirring 3 ml degassed methanolic
solution of 1.0 gr (3.11
mmole) of 16-bromohexadecan-1-of were added. The resulted mixture was refluxed
for 15 hrs,
allowed to cool to RT and 20 ml of degassed 1.0 M NaOH aqueous solution were
injected. The
reaction completion required additional 3 hr of reflux. Resulted reaction
mixture was cooled with ice
bath and poured, with stirring, into a vessel containing 200 ml of ice water.
This mixture was titrated to
pH=7 by 1.0 M HCI and extracted with 300 ml of ether. The organic layer was
separated, washed with
3x150 ml of water, 150 ml of saturated NaCI aqueous solution and dried over
sodium sulfate. After
ether removal material was dissolved in minimum amount of dicloromethane and
purified by silica gel
chromatography (100% dicloromethane as eluent). 600 mg (70% yield) of the
desired product were
obtained.
A clean gold covered microscope slide was incubated in a solution containing
100 micromolar HS-
(CH2),6-COON in ethanol at room temperature for 4 hours. The electrode was
then rinsed throughly
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with ethanol and dried. 20-30 microliters of wire-1 solution (1 micromolar in
1XSSC buffer at pH 7.5)
was applied to the electrode in a round droplet. The electrode was incubated
at room temperature for
4 hours in a moist chamber to minimize evaporation. The wire-1 solution was
then removed from the
electrode and the electrode was immersed in 1 XSSC buffer followed by 4 rinses
with 1 XSSC. The
electrode was then stored at room temperature for up to 2 days in 1XSSC.
Alternatively, and preferably, either a "two-step" or "three-step" process is
used. The "two-step"
procedure is as follows. The wire-1 compound, in water at ~ 5-10 micromolar
concentration, was
exposed to a clean gold surface and incubated for - 24 hrs. It was rinsed well
with water and then
ethanol. The gold was then exposed to a solution of - 100 micromolar insulator
thiol in ethanol for --
12 hrs, and rinsed well. Hybridization was done with complement for over 3
hrs. Generally, the
hybridization solution was warmed to 50~C, then cooled in order to enhance
hybridization.
The "three-step" procedure uses the same concentrations and solvents as above.
The clean gold
electrode was incubated in insulator solution for ~ 1 hr and rinsed. This
procedure presumably results
in an incomplete monolayer, which has areas of unreacted gold. The slide was
then incubated with
wire-1 solution for over 24 hrs (generally, the longer the better). This wire-
1 still had the ethyl-pyridine
protecting group on it. The wire-1 solution was 5% NH40H, 15% ethanol in
water. This removed the
protecting group from the wire and allowed it to bind to the gold (an in situ
deprotection). The slide
was then incubated in insulator again for ~ 12 hrs, and hybridized as above.
In general, a variety of solvent can be used including water, ethanol,
acetonitrile, buffer, mixtures etc.
Also, the input of energy such as heat or sonication appears to speed up all
of the deposition
processes, although it may not be necessary. Also, it seems that longer
incubation periods for both
steps, for example as long as a week, the better the results.
Hybridization efficiency was determined using 32P complementary and
noncomplementary 15 mers
corresponding to the wire-1 sequence. The electrodes were incubated with 50
microliters of each of
the labelled non-complementary (herein "A5") or complementary (herein "S5")
target sequences
applied over the entire electrode in 1XSSC as depicted in Table 1. The
electrodes were then
incubated for 1-2 hours at room temperature in a moist chamber, and rinsed as
described above. The
amount of radiolabelled ANA was measured for each electrode in a scintllation
counter, and the
electrodes were dried and exposed to X-ray film for 4 hours.
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Table 1
hybridized with: total 32P counts3zP counts
added hybridized
to
surface
A5, 20% specific activity,46,446 152
DNA
concentration 1 nM, 1 hour
incubation
S5, 30% specific activity,39,166 10,484
DNA
concentration 1 nM, 1 hour (27% hybridized)
incubation
A5, 14% specific activity,182,020 172
DNA
concentration 5 nM, 2 hour
incubation
S5, 20% specific activity,96,284 60,908
DNA
concentration 5 nM, 2 hour
incubation (63% hybridized)
Example 7
Synthesis of compositions containing ferrocene linked to an electrode
It has been shown in the literature that cyclic voltametry, and other DC
techniques, can be used to
determine the electron transfer rate of surface bound molecules. Surface bound
molecules should
show perfectly symetric oxidation and reduction peaks if the scan speed of the
voltammagram is
sufficiently slow. As the scan rate is increased, these peaks are split apart
due to the kinetics of
electron transfer through the molecules. At a given scan speed, a poorly
conducting molecule should
exhibit greater splitting than a good conductor. As the speed is increased,
the poor conductor will be
split even more.
Accordingly, to test the conductivity of the conductive polymer as compared to
a traditional insulator)
two molecules were tested. The synthesis of ferrocene attached via a
conductive oligomer to an
electrode (herein "wire-2") was made as follows) as depicted in Figure 7.
Synthesis of compound #11 was as follows. 2.33 gr (5.68 nmole) of compound #10
(made as
described in Hsung et al., Organometallics 14:4808-4815 (1995), incorporated
by reference), 90 mg
(0.47 mmole) of Cul and 80 mg (0.11 mmole) of PdClz(PPh3)2 were dissolved in
100 ml of pyrrolidine
under inert atmosphere and heated for 20 hrs at 50~C. All volatile components
were removed on high
vacuum and resulted crude residue was dissolved in minimum amount of
dichloromethane. The
desired compound was purified by silica gel chromatography (50% ethyl acetate
+ 50%
dichloromethane as eluent). 3.2 gr (90% yield) of the pure product were
obtained.
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Compound #12. To 200 mg (0.32 mmole) of suspension of MG#1 in 200 ml of
acetone (sonication was
applied in order to get better results) 3 ml of Mel were added and the
reaction mixture was stirred for
20 hrs at RT. After that time volume of the resulted solution was reduced by
rotovap evaporation to 50
ml and then 400 ml of n-hexane were added. Formed precipitate was filtered
out, washed with 3x200
ml of n-hexane and dried on high vacuum. Quantitative yield of the desired
compound was obtained.
Compound #13. To 100 mg (0.13 mmole) of suspension of MG#2 in 200 ml of
acetone (sonication was
applied in order to get better results) 10 ml of triethyl amine were added and
the reaction mixture was
stirred for 20 hrs at RT. After that time volume of the resulted solution was
reduced by rotovap
evaporation to 50 ml and then 400 ml of n-hexane were added. Formed
precipitate was filtered out,
washed with 3x200 ml of n-hexane and dried on high vacuum. The desired
compound was extracted
from this precipitate with 3x50 ml of THF. Evaporation of the THF fractions
gave 35 mg (52%) of the
compound #13. This was then added to a gold electrode as known in the art.
HS-(CH2)15NHC0-Fc (herein "insulator-1") was made as described in Ward et al.,
Anal. Chem.
66:3164-3172 (1994), hereby incorporated by reference (note: the Figure 1 data
has been shown to be
incorrect, although the synthesis of the molecule is correct).
Monolayers of each were made as follows. Insulator: Gold covered microscope
slides were immersed
in a mixture of insulator-1 and HS-(CH2)15-OH (insulator-2) in neat ethanol.
Insulator-2 molecule is
added to the mixture to prevent the local concentration of ferrocene at any
position from being too
high, resulting in interactions between the ferrocene molecules. The final
solution was 0.1 mM
insulator-1 and 0.9 mM insulator-2. The mixture was sonicated and heated (60-
80~C) for 1-10 hours.
The electrodes were rinsed thoroughly with ethanol, water and ethanol. The
electrodes were
immersed in a 1 mM thiol solution in neat ethanol and let stand at room
temperature for 2-60 hours.
The electrodes were then rinsed again. This procedure resulted in 1-10%
coverage of insulator-1 as
compared to calculated values of close packed ferrocene molecules on a
surface. More or less
coverage could easily be obtained by altering the mixture concentration and/or
incubation times.
Wires: The same procedure was followed as above, except that the second step
coating required
between 10 and 60 hours, with approximately 24 hours being preferable. This
resulted in lower
coverages, with between 0.1 and 3% occurring.
Cyclic voltametry was run at 3 scan speeds for each compound: 1 Vlsec, 10
V/ec, and 50 V/sec. Even
at 1 V/sec, significant splitting occurs with insulator-1, with roughly 50 mV
splitting occuring. At higher
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speeds, the splitting increases. With wire-2, however, perfectly symmetrical
peaks are observed at
the lower speeds, with only slight splitting occurring at 50 Vlsec.
It should be noted that despite a significant difference in electron transfer
rate, electron transfer does
still occur even in poorly conducting oligomers such as (CHZ),5, traditionally
called "insulators". Thus
the terms "conductive oligomer" and "insulator" are somewhat relative.
Example 8
Synthesis and analysis of nucleic acid with both a conductive
oligomer and a second electron transfer moiety
The following nucleic acid composition was made using the techniques above: 5'-
ACCATGGAC[UBF]CAGCU-conductive polymer (Structure 5 type, as outlined above)
herein "wire-3",
with UBF made as described above. Thus, the second electron transfer moiety,
ferrocene, is on the
sixth base from the conductive oligomer.
Mixed monolayers of wire-3 and insulator-2 were constructed using the
techniques outlined above.
The compositions were analyzed in 0.2 M NaCl04 in water using cyclic
voltametry (CV) and square
wave voltametry (SW), in the absence (i.e. single stranded) and presence (i.e,
double stranded) of
complementary target sequence.
The results of SW show the absence of a peak prior to hybridization, i.e. in
the absence of double
stranded nucleic acid. In the presence of the complementary target sequence, a
peak at 240 mV,
corresponding to ferrocene, was seen.
A mediator as described herein was also used. 6 mM ferricyanide (Fe(CN)6) was
added to the
solution. Ferricyanide should produce a peak at 170 mV in a SW experiment.
However, no peak at
170 mV was observed, but the peak at 240 mV was greatly enhanced as compared
to the absence of
ferricyanide.
Alternatively, CV was done. No peaks were observed in the absence of target
sequence. Once
again, the chip was incubated with pertectly complimentary nucleic acid in
order to hybridize the
surface nucleic acid. Again, the chip was scanned under the same conditions.
An increased signal
was observed. Finally) the chip was soaked in buffer at 70~C in order to melt
the compliment off the
surtace. Previous experiments with radioactive probes have shown that 15-mers
hybridized on a very
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similar surface melted at approximately 45~C. Repeating the scan after the
heat treatment shows a
reduced signal) as in the first scan prior to hybridization.
Example 9
AC detection methods
Electrodes containing four different compositions of the invention were made
and used in AC detection
methods. In general, all the electrodes were made by mixing a ratio of
insulator-2 with the sample as
is generally outlined above.
Sample 1, labeled herein as "Fc-alkane", contained a mixed monolayer of
insulator-2 and insulator-1.
Sample 2, labeled herein as "Fc-amido-alkane", contained a mixed monolayer of
insulator-2 and a
derivative of insulator-1 which has an amido attachment of the ferrocene to
the alkane. Sample 3,
labeled herein as "Fc-wire", contained a mixed monolayer of insulator-2 and
wire-2. Sample 4 was the
same as Sample 3, with the exception that a new in situ deprotection step was
used, described below.
Sample 5, labeled herein as "ssDNA" (AGCTGAGTCCA{UBF)GGU-conductive oligomer),
contained a
mixed monolayer of insulator-2 and wire-3. Sample 6, labeled herein as
"dsDNA", contained a mixed
monolayer of insulator-2 and wire-3, wherein the complement of wire-3 was
hybridized to form a
double stranded wire-3. Sample 7 was a solution of ferrocene in solution. As
is shown herein, the rate
of electron transfer, from fast to slower, is as follows: Sample 3 > Sample 8
> Sample 1 > Sample 2 >
Sample 5. Generally, Sample 1 models ssDNA, and Sample 3 models dsDNA.
The experiments were run as follows. A DC offset voltage between the working
(sample) electrode
and the reference electrode was swept through the electrochemical potential of
the ferrocene, typically
from 0 to 500 mV. On top of the DC offset, an AC signal of variable amplitude
and frequency was
applied. The AC current at the excitation frequency was plotted versus the DC
offset.
Figure 8 depicts an experiment with Sample 1, at 200 mV AC amplitude and
frequencies of 1, 5 and
100 Hz. Sample 1 responds at all three frequencies, and higher currents result
from higher
frequencies, which is simply a result of more electrons per second being
donated by the ferrocene at
higher frequencies. The faster the rate, the higher the frequency response,
and the better the
detection limit. Figure 9 shows overlaid AC voltammograms of an electrode
coated with Sample 3.
Four excitation frequencies were applied: 10 Hz, 100 Hz, 1 kHz, 10 kHz, all at
25 mV overpotential.
Figure 10, shows the frequency response of samples 1, 2 and 3 by measuring the
peak currents vs.
frequency. Sample 3 response to increasing frequencies through 10 kHz (the
detector system limit),
while Sample 1 lose its responses at between 20 and 200 Hz. Thus, to
discriminate between Sample
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1 and Sample 3, one could simplify the methods by analyzing it at 1 Hz and
1000 Hz and compare the
responses, although as will be appreciated, this is only one method of a
variety of possible methods.
This should be similar to the dsDNA and ssDNA system. Figure 11 shows Sample 5
and Sample 6)
plotted as a function of normalized current (with the highest current being 1
for both cases; the actual
current of dsDNA is much higher than that of ssDNA, so the graph was
normalized to show both). The
lines are modeled RC circuits, as described above, and not a fit to the data.
At 1 Hz, both ssDNA and
dsDNA respond; at 200 Hz, the ssDNA signal is gone. Figure 12 shows that
increasing the
overpotential will increase the output signal for slow systems like samples 1
and 2. Figures 13A and
13B show that the overpotential and frequency can be tuned to increase the
selectivity and sensitivity.
For example, a low overpotential and high frequence can be used to minimize
the slower species
(Sample 1 or Sample 5). Then the overpotential can be increased to induce a
response in the slower
species for calibration and quantification.
Figure 14 shows that the ferrocene added to the solution (Sample 7) has a
frequency response related
to diffusion that is easily distinguishable from the frequency response of
attached ferrocene. This
indicates that by varying frequency, signals from bound molecules, particular
fast bound molecules
such as dsDNA) can be easily distinguished from any signal generated by
contaminating redox
molecules in the sample.
Figures 15A and 15B shows the phase shift that results with different samples.
Figure 15A shows the
model compounds, and 15B shows data with dsDNA and ssDNA. While at this
frequency, the phase
shift is not large) a frequency can always be found that results in a 90~
shift in the phase.
Example 10
Synthesis of conductive oligomers attached via a base
Representative syntheses are depicted in Figures 18 and 19. When using
palladium coupling
chemistry, it appears that protecting groups are required on the base, in
order to prevent significant
dimerization of conductive oligomers instead of coupling to the iodinated
base. In addition, changing
the components of the palladium reaction may be desirable also. Also, for
longer conductive
oligomers, R groups are preferred to increase solubility.
Example 11
The use of trimethylsilylethyl protecting groups
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The use of an alternate protecting group for protection of the sulfur atom
prior to attachment to the
gold surtace was explored.
oompowa a i
- s -
ou~~.~m¢
1
SN
compound a:
To 0.5 gm of molecular sieve (3 A) was added 3 ml of dry THF and 2.5 ml of 1.0
tetrabutyiammonium
fluoride. After stirring for 20 minutes, 100 mg of compound #1 was added under
Argon. The reaction
mixture was stirred for 1 hour and poured into 100 ml of 5% citric acid
solution and the aqueous
solution was shaken well and extracted twice with either (2 X 100 ml). The
combined ether solution
was dried over Na2S04 and concentrated. The residue was purified by column
chromatography using
10% CHZCIZ/Hexane as eluent. The purified product was analyzed by'HNMR which
should 50% of
compound #2 and 50% of the corresponding disulfide.
The use of this protecting group in synthesizing base-attached conductive
oligomers is depicted in
Figures 20 and 21.
Example 12
Preparation of Peptide Nucleic Acids with Electron Transfer Moieties
The synthesis of a peptide nucleic acid monomeric subunit with a conductive
oligomer covalently
attached to the a-carbon is depicted in Figure 31.
4-lodophenylalanine: Into a solution of 40.15 gm (0.243 mol) of phenylalanine
in a mixture of 220 mL
of acetic acid and 29 mL of concentrated HZSO4 was added 24.65 gm (0.097 mol)
of powered iodine
and 10. 18 gm(0.051 mol) of powered Na103 while stirring. The reaction mixture
was stirred at 70 ~C
for 21 h) during this time, two portions of 1 gm of Na103 were added. The
mixture was cooled and the
acetic acid was removed by using rotavapor while temperature was maintained at
35~C and the
residue oil was diluted by adding 400 mL of water. The aqueous solution was
extracted once with 100
mL ether and once with 100 of dichloromethane. After decolorization with 5 gm
of Norit, the aqueous
solution was neutralized by adding solid NaOH to precipitate the crude
product, which, after chilling,
was filtered and rinsed with 800 mL of water and 300 mL of ethanol. The wet
product was
recrystallized from 200 mL of acetic acid to produce 37.5 gm of 4-iodo-L-
phenylalanine.
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Methyl 4-lodophenyl Alaninate Hydrochloride: To 10 mL of methanol cooled in an
ice-water bath was
added dropwise 10.2 gm of thionyl chloride. Into the cold solution was added
5.0 gm of 4-
iodophenylalanine and the yellow solution was formed and refluxed for 2 h.
After removing the
solvent, the white solid was obtained and recrystallized from 10 mL of
methanol by addition of 50 mL
of ether. The title product(5.4 gm) was prepared.
Methyl N-Amidocarboxylethyl-4-lodophenyl Alaninate: To a solution of 5.0 gm
(14.6 mmol) of methyl 4-
iodophenylalaninate hydrochloride in 100 mL of acetonitrile was added 6 mL of
triethylamine and 1.1
gm (15.4 mmol) of acrylamide. The solution was stirred overnight. After
removing the solvent, the
residue was dissolved in 200 mL of dichloromethane and the solution was washed
once with 5
NaHC03 solution and dried over sodium sulfate. The product was purified by
column separation.
Methyl N-Aminoethyl-4-lodophenyl Alaninate: To a solution of 3.46 gm (8 mmol)
of [I,I-
bis(trifloroacetoxy)iodo]benzene in 24 mL of acetonitrile was added 12 mL of
the glass-distilled water,
followed adding 2.98 gm(8 mmol) of methyl N-amidocarboxylethyl alninate. The
mixture was stirred
for 6 h at room temperature and diluted by 150 mL of water and 16 mL of
concentrated HCI solution.
The aqueous solution was extracted once with 150 mL of ether and concentrated
to about one third of
the original volume. The concentrated NaOH solution was used to adjust pH of
the aqueous solution
to greater than 12 and the basic water solution was extracted 6 times with
CHZCIz (6 x 200 mL). The
combined extracts were dried over anhydrous sodium sulfate and concentrated to
dryness and further
dried over a high vacuum line and the product was used for next step without
further purification.
Methyl N-(2-Nitrobenzenesulfonyl)-4-lodophenyl Alaninate: To a two-necked
flash was added 19.0 g
(55.8 mmol) of methyl N-aminoethyl-4-iodophenyl alaninate and 600 mL of dry
DMF. The resulting
solution was cooled in an ice-water bath. Into the cold solution was added 20
mL of TEA, followed by
adding 13.5 gm (60.9 mmol) of 2-nitrobenzenesulfonyl chloride portion by
portion. The mixture was
stirred at low temperature for 30 min. and warmed up to room temperature and
stirred for another 4 h.
The precipitate was formed and filtered off and washed once with DMF. After
removing DMF on the
high vacuum, the residue was dissolved in the 500 mL of dichloromethane. The
organic solution was
washed twice with the brine and dried ver Na2S04 and then concentrated. The
residue was dissolved
in a small amount of dichloromethane for the column purification. Silica gel
(250 gm) was packed with
CH2CL2, the sample solution was loaded and the column was eluted with CHZCL2.
The fractions were
identified by TLC (CHzCl2 as developing solvent} and pooled and concentrated
to afford 24.1 gm
(88.1 %) of the title compound.
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Methyl N-(2-MMT-aminoethyl)-N-(2-Nitrobenzenesulfonyl)-4-lodophenyl Alaninate:
To a solution of
16.5 gm (49.5 mmol) of 2-MMT-amino ethanol, 20.0 gm (40.8 mmol) of methyl N-(2-
nitrobenzensulfonyl)-4-iodophenyl alaninate and 13 gm (49.5 mmol) of
triphenylphosphine in 250 mL
of dry THF cooled in an ice-water bath was added 7.8 mL (49.5 mmol) of diethyl
azodicarboxylate
under Argon. The solution was warmed up to room temperature and stirred
overnight. After removing
THF, the residue dissolved in the small amount of the CHzCl2 for column
separation. TLC (CH2C12:
Hexane = 9:1 ) of the sample solution indicated two products, i.e., the early
spot is the desired product,
the later spot is triphenylphosphine oxide. Silica gel (300 gm) was packed
with 1 % TEA/hexane. The
sample solution was loaded and the column was eluted with 500 mL of 1 %
TEA/hexane, 100 mL of
1 % TEA/25% CHzCIZ/hexane and 1000 mL of 1 % TEA/50% CHZCIZ/hexane. The
fractions were
identified by TLC (CHZCI2:Hexane = 9:1 ). The fractions containing the pure
early spot were pooled
and concentrated to give 17 gm of the title compound. The overlapping
fractions were pooled,
concentrated and repurified to give another 3.0 gm of the title compound. The
total yield is 62.0%.
Methyl N-(2-MMT-aminoethyl)-4-iodophenyl Alaninate: To a suspension of 17.0 gm
(21 mmol) of
methyl N-(2-MMT-aminoethyl)-N-(2nitrobenzenesulfonyl)-4-iodophenyl alaninate,
11.6 gm (84 mmol)
of Potassium Carbonate in 150 mL of DMF was added 2.6 mL {25.8 mmol) of
thiophenol under Argon.
The reaction mixture was stirred at room temperature for 1.3 h. and diluted by
adding 1.2 L of the
brine. The aqueous solution was extracted three times by ether (2x 500 mL) and
the combined
extracts was washed once with the diluted NaOH solution and dried over sodium
sulfate. After the
removal of the solvent, the residue was used for column separation. Silica Gel
(220 gm) was packed
with 1 % TEAJhexane, upon loading the sample solution, the column was eluted
with 500 mL of 1
TEAlhexane, 1000 mL of 1 % TEA/25% ether/hexane, and 1000 mL of 1 % TEA/50%
ether/hexane.
The fractions were identified by TLC (Ether:Hexane) and pooled and
concentrated to afford 5.6 gm
(43.1%) of the title product.
Methyl N-(2-MMT-aminoethyl)-N-[(Thymin-1-yl)acetyl]-4-lodophenyl Alaninate: To
a solution of 3.37 g
(5.43 mmol) of methyl N-{2-MMT-aminoethyl)-4-iodophenyl alaninate in DMF (10
mL) was added 3,4-
Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (.884 g, 5.43 mmol) and 4-
ethylmorpholine (1.38 mL,
10.86 mmol). A solution of thymine acetic acid (1.00 g, 5.43 mmol) in DMF (10
mL) was then added,
followed by N,N'-diisopropylcarbodiimide (1 mL, 6.5 mmol). The reaction
mixture was left stirring
overnight at room temperature for 20.5 h. The solvent was removed in vacuo.
The residue was
dissolved in 600 mL of CHzCl2 and the solution was washed with twice with 500
mL of water and once
with 500 mL of brine and dried in NaZS04. After the removal of the solvent,
the crude residue was
dissolved in ~10mL of CHZCI2 for column separation. Silica gel (135 gm) was
packed with 1%
TEA/CHZCI2, upon loading the sample solution, and the column was eluted with 1
% TEA/CHzCIz. The
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fractions were identified by TLC (CH2CIz:CH30H=95:5). The fractions containing
the desired product
were pooled and concentrated to dryness. The solid product was dissolved in
minimum amount of
EtOAc and left in the freezer to precipitate out n,n'diisopropylurea. The
precipitate was filtered and the
filtrate was concentrated to afford 3.56 g (83.3%) of the title compound.
N-(2-MMT-aminoethyl)N-[(Thymin-1-yl)acetyl]-4-lodophenyl Alaninate: 3.5 g
(4.45 mmol) of methyl N-
(2-MMT-aminoethyl)-N[(thymin-1-yl)acetyl]-4-iodophhenyl alaninate was
dissolved in dioxane (20 mL)
and water (4 mL). The solution was cooled to 0~C and 1 M NaOH was added
dropwise until the
pH=12. After 1 h., the reaction mixture was warmed to room temperature and
more 1 M NaOH was
added and the pH remained at 12. The reaction was monitored by TLC
(CHzCI2:CH30H=95:5). After
the hydrolysis was complete, the pH of the reaction mixture was adjusted to 5
with 2 M KHS04. Then
it was diluted by adding 300 mL of CHZCI2. The organic layer was separated and
the aqueous layer
was extracted twice with 250 mL of CHZCIz. The combined organic extracts were
dried over Na2S04
and concentrated. The residue was dissolved in minimum amount of CHZCIz for
column purification.
Silica gel (52 gm) was packed with 1 % TEA/2% CH30H/CHzCl2, after loading the
sample, the column
was elute with 700 mL of 1 % TEA/2%CH30H/CHZCIZ and 1 L of 1 % TEA/5%
C~I30H/CHZC12. The
fractions were identified by TLC (CHzCI2:CH30H=95:5). The removal of the
fractions containing the
desired product gave 2.9 g(84.6%) of the title compound.
PNA-Backbone-Wire: A mixture of 1g (1.29 mmol) of N-(2-MMT-aminoethyl)-N-
[(thymin-1-yl}acetyl]-4-
iodophenyl alaninate, 0.5 g (1.29 mmol) of trimethyl silyl ethyl protected 3-
unit wire, 44.6 mg (0.077
mmol) of Pd(dba)2, 91.6 mg (0.349 mmol) of triphenylphosphine, and 44.6 mg
(0.17 mmol) of copper
(I) iodide 120 mL of DMF and 62 mL of pyrrolidine was degassed well and
stirred at 60~C for 5h. The
solvent was removed and the residue was dissolved in 250 mL of CHzCl2 and 200
mL of saturated
EDTA solution. This mixture was stirred for 30 min. The organic layer was
separated" dried over
sodium sulfate and concentrated. The crude product was dissolved in minimum
CH2CI2 for column
separation. Silica gel (22 gm) was packed with 1 % TEA/CH2CIz, upon loading
the sample solution, the
column was eluted with 1 L of 1 % TEA/2%CH30H/CHzCIz and 1 % TEA/5%
CH30H/CH2CI2 until
finishing the separation. The fractions were identified by TLC
(CHZCIz:CH30H=95:5). The right
fractions were combined and concentrated to afford 0.55 g of yellow-orange
solid, which was
dissolved in 150 mL of CHZCI2 and diluted by adding 50 mL of water and 50 mL
of 10%
tetrabutylamine hydroxide. The mixture was placed in a separatory funnel and
shaken for 5 min. The
organic layer was separated and the aqueous layer was extracted once more with
50 mL of CHzCl2
and the combined organic layer was dried in Na2S04. The solvent was removed to
afford 0.8 g (46.5%
of the title product.
CA 02270633 1999-OS-04
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Example 13
Preparation of Peptide Nucleic Acids with Electron Transfer Moieties
The synthesis of a peptide nucleic acid monomeric subunit with a ferrocene
electron transfer moiety
covaiently attached to the base is depicted in Figure 32.
Synthesis of Y1: 5-lodo uracil (100.0 gm) was suspended in 250 ml of dry DMF.
1.68 gms of sodium
hydride was added in portions. The reaction mixture was then stirred at room
temperature for 40
minutes. Then 6.16 ml of t-butyl bromoacetate ws added and the reaction
mixture was stirred for an
additional two hours at room temperature. The reaciton mixture was quenched
with 5 ml of methanol
containing C02. The solvent was then removed and the residue was dissolved in
dichloromethane
and washed with water. The precipitate was formed during the wash and then
filtered and dried. The
reaction yielded 9.33 g of product Y1.
Synthesis of Y2: To a solution of 6.33 g of Y1 in 140 ml of dichloromethane
was added 35 ml of
triethylamine, 0.55 g of 4-dimethylaminopyridine, and 5.89 g of 2-
mesitylenesulfonyl chloride. The
reaction mixture was stirred for 40 minutes and then 0.40 g of 1.4-
diazobicyclo[2,2,2] octane and 4.34
ml of 2,4-dimethylphenol were added and stirred for 2 hours. The reaction
mixture was then diluted by
adding 200 ml of dichloromethane and the solution was washed with a 5% sodium
bicarbonate
solution, dried over sodium sulfate and concentrated. The residue was
dissolved in 5 ml of
dichloromethane and loaded onto a 200 g silica gel column packed with
dichloromethane. The column
was eluted with 1-5% methanol/dichloromethane. The fractions containing the
diesired product was
pooled and concentrated to give 2.5 g of Y2.
Synthesis of Y3: A mixture of 2.5 g of Y2, 1.38 g of ferrocene acetylene, 200
mg of Pd(pph3)CIz and
208 mg of copper iodide in 100 ml of dimehtylformamide (DMF) and 100 ml of
triethylamine was
degassed well and stirred at 55~C for 2 hours. Upon removing solvent the
residue was dissolved in
dichloromethane and the solution was washed with a 5% sodium bicarbonate
solution, dried over
sodium sulfate and concentrated. The crude residue was dissolved in 5 ml of
dichloromethane and
loaded onto a 200 g silica gel column packed with dichloromethane. The column
was eluted with 2
5% methanol/CHzCl2. The right fractions were pooled and evaporated to yield
2.98 g of Y3.
Synthesis of Y4: To a solution of 2.50 g of Y3 in 40 ml of dichloromethane
cooled in an ice bath was
added 7.1 ml of trimethylsilane, followed by adding 17.5 ml of trifluoroacetic
acid. The resulting
reaction mixture was warmed to room temperature after 5 min of stirring at the
same temperature The
reaction mixture was stirred at room temp for 7.5 hours. The solvent was
removed. The residue was
CA 02270633 1999-OS-04
WO 98I20162 PCT/US97/20014 ~ -
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dissolved in 5 ml of dichloromethane and loaded onto a column containing 25 g
of silica gel packed
with dichloromethane. The column was eluted with 0-2.5% methanoI/CH2C12. The
fractions were
pooled and evaporated to yield 2.18 g of Y4.
Synthesis of Y5: 0.98 g of methyl N-(2-MMT-aminoethyl) glycinate was dissolved
in 7 ml of
dimethylformaide (DMF). To this solution was added 0.329 g of 3,4-dihydro-3-
hydorxy-4-oxo-1,2,3-
benzotriazine and 0.51 ml of 4-ethylmorpholine. A solution of 1.0 g of Y4 in 7
ml of DMF was added to
the reaction mixture, followed by adding 0.38 ml of N,N'-
diisopropyfcarbodiimide. The reaction was
stirred at room temperature for 20 hours. The solvent was then removed and the
residue was
dissolved in dichloromethane. The solution was washed with a saturated sodium
chloride solution,
and dried over sodium sulfate. The solvent was evaporated to about 5 ml for
column chromatography.
The crude mixture was loaded onto a 20 g silica gel column packed with 1 %
TEA/CH2CIz. The column
was eluted with 0-2% methanol/1 %TEA/CHzCIz. Evaporation of the solvent
afforded 0.97 g of Y5.
Synthesis of Y6: 0.97 g of Y5 was dissolved in 10m1 of dioxane and 2ml water.
The pH of the
mixture was adjusted to 11 with 1 M NaOH. The reaction was stirred for two
hours at 0~C. The
hydrolysis reaction was monitored by TLC (CH30H:CHZCIz). Upon the completeness
of the hydrolysis,
the pH of the mixture was adjusted to 5 with 2M potassium hydrogen sulfate.
The mixture was
extracted three times with CHzCl2 (3 X 200 ml) and the combined extracts dried
over sodium sulfate.
The solution was evaporated to about 5 ml for column chromatography. Silica
gel (20 gm) was
packed with 1 % triehtylamine in dichloromethane. The sample solution was
loaded and the column
was eluted with a 5-10% methanol./1 % TEA/dichloromethane. The fractions
containing the right
product was pooled, evaporated and co-evaporated with pyridine and toluene in
order to remove the
triehthylamine to give 0.8 g of Y6.
30
Synthesis of Y7: To a solution of 0.8 g of Yfi in 80 ml of acetonitrile was
added 0.61 g of 2-
mitrobenzaldoxime and 0.37 g of 1,1,3,3-tetramethylguandine. The resulting
solution was stirred at
room temperature for 6 hours. The solvent was removed. The residue was
dissolved in
dichloromethane and washed with a saturated NaCI solution. Silica gel (20 g)
was packed with 1
triehtylamine in dichloromethane. The crude residue was dissolved in 5 ml of
dichloromethane and
loaded onto the column. The column was eluted with 0-5% methanoll1
%TEA/CH2C12. The fractions
containing the product were pooled and concentrated to give 150 mg of product.
The product was
then dissolved in 100 ml of dichloromethane. The solution was washed with 10
ml of water and 10 ml
of 10% tetrabutylammonium hydroxide. The organic layer was separated and dried
over sodium
sulfate and evaporated to give 200 mg of Y7.
