Note: Descriptions are shown in the official language in which they were submitted.
,` .. ` 7282Z !. '
JEY-R
L~BE~ING OF NUCLEIC ACIDS WIT~I FLUORESCENT M~RKERS ..
FIELD OF Tl-l~ INVENTION
The present invention relates to DN~ markers and,
particularly, nucleic acid labeling techniques. More
specifically, this invention contemplates a protocol which
permits the covalent introduction of single or multiple
fluorescent markers or other probes such as spin labels and
drug analogues into DNA fragments and oligodeoxynucleotides.
The instant technique, particularly employing multiple '~
fluorescent markers, allows high sensitivity detection of
nucleic acids (without the use of sophisticated detection
devices) in the low femtomolar (10 15 moles) range and
additionally permits the placement ~f markers and probes at
specific locations within the macromolecule. The present
invention can be used with high detection sensitivity for DNA
sequencing and hybridization procedures including a host of
diagnostic and therapeutic procedures. The present technique
can also be employed as a tool for the study of nucleic acid
dynamics through recognition and evaluation of fluorescence
energy transfer and electron spin resonance, and the study of
structure, conformation and dynamics of biopoIymers.
Specific labeling procedures allow the introduction of a ;~
probe or other entlty for the location of desired sequences `
or the delivery of the probe to a specific sequence. This
process is fundamental to the emerging fields of DNA
diagnostics and therapeutics.
BACKGROUND OF THE INVENTION
The determination of the presence of nucleic acid
fragments has typically relied on the use of radioisotopic
3 labeling techniques. The enormous utility of these
techniques has largely been a function of the high
sensitivity associat~ed with their detection. Such
sensitivity has allowed the location of quantities of
1 material in amounts in the low femtomolar range (10 lS
moles). However, the use of radioisotopes is rendered less
than ideal by the associated problems of safety and disposal.
Fluorescent rather than radioisotopic labeling
procedures are an attractive option which avoids these
liabilities, but fluorescent labeling procedures have
previously been compromised by their greatly reduced
sensitivity. Fluorescent dyes as well as spin labels are
also useful in many aspects of biophysics since the
properties of a given marker can vary substantially with
changes in the immediate microenvironment. Such probes can
be useful for the study of structure, conformation and
dynamics in biopolymers providing that they can easily be
placed at specific locations within the desired
macromolecule.
In order for fluorescent labeling procedures to
compete effectively with and replace radioisotopic labeling
techniques for the detection of macromolecules during various
biochemical assays, the fluorescent labeling must result in
high detection sensitivity, rapid and simple procedures for
the introduction of the fluorescent marker to the
macromolecule of interest must be available, and the results
must be reproducible. By meeting these criteria and with the
additional advantage of reduced health hazards, fluorescent
labeling techniques could then replace the use of
radioisotopes in a number of biochemical assays.
Intercalative dyes such as ethidium bromide
generally meet these criteria and in many cases have
completely replaced radioisotopic labeling procedures for the
3 detection of double stranded DNA. However, a number of
assays, including DNA sequencing and hybridization
_~ 3
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techniques, cannot benefit from intercalative fluorescent
1 labeling. These procedures require that the fluorescent
marker be covalently bound to the nucleic acid, and the
intercalative dye is unable to meet this requirement.
All prior studies for the covalent attachment of
fluorescent markers to nucleic acids, until the present
invention, suffered from at least one of two disadvantagesO
First, attachment of only a single label to the nucleic acid
(usually at one o~ the termini) severely compromised its
detection. Secondly, although multiple labeling techniques
10 can enhance detection sensitivity, they have gen~rally J '~
requixed the time-consuming synthesis of a modified
nucleoside derivative containing a fluorophore or one which
can be modified with a fluorophore. In addition to
fluorophores, the use of biotin as a non-radioactive labeling
technique has also been considered.
The use of single labels, usually at the terminus
of the nucleic acid fragment, is the conventional state of
the art primarily because it is chemically and enzymatically
easier to exploit modification reactions at a nucleic acid
terminus rather than at a specific point in the internal
regions of the sequence. Additionally, the placement of the
marker at one of these termini also removes the marker from
the "site of action" when monitoring protein binding or any
process where an essentially native DNA sequence is required.
It has commonly been difficult to detect fragments containing
a single fluorescent marker with the high sensitivity
available with a radioisotopic label. Although problematic,
labeling with a single fluorophore has been accomplished
using both chemical and enzymatic techniques. DNA sequencing
3 has been attempted using such labeling techniques but
requires sophisticated electronic detection, and then only
has evidenced limited success.
.. . . .
Several methods have been reported for the
1 incorporation of multiple labels into nucleic acids. Most of
these rely on an enzymatic polymerization reaction in order
to introduce a modified nucleoside carrying the desired label
or one which can be easily modified with the 1uorescent
marker at numerous positions. sase~specific reactions have
also been employed, such as modification of guanine ~esidues
with N~acetoxy-2-acetylaminofluorene followed by detection
with tetramethylrhodamine-labeled antibodies raised against
the modifying reagent. Multiple labeling techniques have
commonly resulted in enhanced detection sensitivity with
respect to single labels and have been reasonably
reproducible. However, these techniques have previously not
been simple or rapid to employ. The modified nucleoside has
previously only been obtained by time-consuming chemical
syntheses.
~ nother prior approach involves the use of biotin
labeling. While biotln itself is not a fluorescent
chromophore, biotin labeling when combined with
immunochemical, histochemical or affinity detection systems
provides another alternative to radioisotopic labeling of
nucleic acids. Biotin-labeled nucleic acids have been used
in hybridization studies, gene mapping studies employing
electron microscopy and gene enrichment in cesium chloride
gradients. Biotin labeling has been typically approached in
conceptually the same manner as fluorescent labeling
techniques in which either a single label at the nucleic acid
terminus or multiple labels requiring the synthesis of a
biotin labeled dNTP derivative are employed. Generally, each
of the existing techniques suffers from the requirements of
3 arduous chemical synthesis and/or limited detectability. ~
:.
Conventional techniques when applied to DNA
1 sequencing procedures add additional complications since the
DNA fragments prepared during sequencing techniques must be ,
resolved by electrophoresis in a polyacrylamide gel matrix.
Since electrophoresis procedures resolve nucleic acid
fragments on the basis of size (or molecular weight), the
addition of one or more fluorescent labels to the fragments
prior to electrophoresis results in anomalous migration of ~;
the DNA within the gel and undue complications in the
analysis of the sequence. The most desirable procedure for
employing fluorescent labeling techniques in DNA sequencing
and hybridization procedures would involve the incorporation
of multiple labels into the nucleic acid or hybridization
probe (to enhance detection sensitivity), before or after
electrophoretic resolution of such fragments or before or
after hybridization of the probe onto a nitrocellulose
membrane ("pre-assay" or "post-assay" labeling). Multiple
covalent labeling of nucleic acids with fluorophores in a
"post-assay" manner has not been previously contemplated or
described.
SUMM~RY OF T~IE INVENTION
Accordingly, one object of the present invention is
to provide an improved method for labeling nucleic acids.
Another obiect of this invention is to provide an
improved method of fluorescently labeling nucleic acids.
A further object of the present invention is to
provide new probes for use in DNA labeling and related
techniques.
A still furth~r object of this invention is to
provide a new detection product which constitutes a
3 phosphorothiolate diester covalently complexed with a
nucleotidic residue, and which is also complexed with a
detectable marker.
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Another object of this invention is to provide
1 multiple sites, i.e., internally within the macromolecule,
for the attachment of fluorophores and other markers and/or
probes to the ~ucleic acid thereby enabling multiple labeling
techniques.
A further object of the present invention is to
selectively introduce fluorescent markers and other markers
and probes at specifically desired sites of the
macromolecule. These markers or reporter groups include
fluorophores, biotin, spin labels, drugs or their analogues,
hydrolytic reagents, chiral metal complexes and the like.
Another ob}ect of this invention is to selectively
introduce fluorescent markers and other probes after the
molecule of interest has been treated with any one of various
desired biochemical assays, i.e., in a "post-assay"
procedure.
Still another object of this invention is to
selectively introduce fluorescent markers and other probes
before the molecule of interest has been treated with any one
of various desired biochemical assays, i.e., in a
"pre-assay" procedure.
Yet another object of the present invention is to
provide an improved process for DNA sequencing, DNA .:: :
hybridization tec~niques and DNA diagnostics and DNA :
therapeutics.
A still further other ob~ect of this invention is
to provide a new detection procedure which eliminates the use
of radioisotopes and the disadvantages associated with such
conventional methods.
These and other objects of the present invention
3 are achieved by providing a protocol which permits the
covalent introduction of single or multiple markers,
particularly fluorescent markers, and other probes into DNA
fragments and oligodeoxynucleotides at selective sites. More
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specifically, according to the present invention, nucleic
1 acids are labeled with markers such that, e.g., the
fluorescent marker or any other type of probe can be placed ;
into a specific location in the nucleic acid. By the
technique of the present invention, various sites for the
attachment of the desired probes or markers are generated by
employing phosphorothioate diesters in place of native
phosphodiesters which are chemically or enzymatically
introduced at the desired site within a nucleic acid and
subsequently marked with the desired reporter group. The
present methodology not only permits multiple labeling and
high sensitivity in a simple technique ln the absence of
sophisticated detection devices, but also permits the'
introduction of a particular probe or marker after
conventional biochemical assays, i.e., I'post-assay.'' The
advantages of the novel detection products of this invention
also allow the labeling of DNA fragments in conventional DNA
sequencing or hybridization assays. Such assays further
permit a host of therapeutic procedures where a DNA
hybridization probe with attached phosphorothioate diester(s)
is employed in vivo or in vitro to locate a sequence within
genomic DNA and which is subsequently reacted with, e.g., a
label for detection or identification, a reactive molecule
for degradation, or other toxic therapeuti~ agents. The
novel product also allows study of the structure and dynamics
of nucleic acids as well as protein-nucleic acid complexes.
The novel product of the present invention includes a
nucleotidic residue covalently complexed with a
phosphorothioate diester and further complexed to a marker
enabling detection of the product.
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BRIEF DESCRIPTION OF TI~E VR~WINGS
1 Fig. 1 sets forth the structure of the
phosphorothioate triester composed of the nucleotidic residue
and phosphorothioate dies-ter complexed with the bimane label
(bimane-Tp(S)T triester).
Eig. 2 is a graphic illustration of the stability
of the bimane-Tp(S)T triester at ambient temperature measured
during a total time period of 20 hours at pH values between
3-11.
Fig. 3 is a graphic depiction of an HPLC analysis
of the reaction mixture containing the octamer d[GC(s)CCGGGC]
(0.3 mM) and monobromobimane (3.0 T~M) after reaction for
5 hours at ambient temperature.
Fig. 4 is a photographic reproduction of a ~ -
polyacrylamide gel (6%) illustrating "post-assay" labeling of
DNA fragments with monobromobimane.
Fig. 4(A) represents an HpaII restriction
endonuclease digest of an M13mpl8 DNA template, which has
been elongated with DNA polymerase I ~E. coli) using dNTPs
and then treated with the endonuclease.
Fig. ~B) represents an AvaI restriction
endonuclease digest of an M13mpl9 DNA template, which was ~
elongated with DNA polymerase I (E. coli) using dNTPs and --
then treated with endonuclease.
Fig. 5 represents phosphorothioate triester
oligodeoxynucleotides carrying (a) a PROXYL spin label. (b)
a derivative of the dihydropyrroloindole subunit of CC-1065,
(c) a sulfonamide-linked dansyl fluorophore, and (d) an
N-linked dansyl fluorophore.
DET~ILED DESCRIPTION OF T~IE INVENTION
3 The present invention contemplates the selective
labeling of nucleic acids with fluorescent molecules and
other probes such as, for example, biotin, which are useful
in DNA sequencing and DNA hybridization assays. The present
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invention also contemplates other probes such as, for
l e~ample, spin labels which are useful in the analysis of
nucleic acid structure and dynamics. The convenient labeling
methodology of this invention further permits a broad range
of DN~ therapeutic and diagnostic procedures and is
particularly characterized by the selective covalent
introduction of single or multiple markers and probes into
DNA fragments and oligodeoxynucleotides. The novel detection
product of this invention is characterized by a nucleotidic
residue covalently complexed with a phosphorothioate diester
which is mutually covalently complexed with a selected
marker. The probe is selectively introduced into a single
site of choice or into multiple sites as desired.
The present invention preferably employs a
phosphorothioate diester [for example, Tp(s)T, -
phosphorothioate diester derivative of TpT (thymidyl~3'-~5')
thymidine)] which is selectively incorporated into a DNA
fragment or oligodeoxynucleotide at any and each nucleotide
residue desired.
Specifically, the probe of the present invention, a
phosphorothioate diester derivative, is prepared by
introducing the phosphorothioate diester into the nucleic
acid fragment either enzymatically, e.g., according to the
method o~ Potter and Eckstein (Potter, B. and Eckstein, F.,
J. Biol. Chem., 259: 14243-14248, 1984), or chemically,
e.g., according to the method of Connolly, et al. (~onnolly,
et al., Biochemistry, 23: 3443-3453, 1982).
The enzymatic technique of Potter and Eckstein
employs the desired dNTP~ S 2'-deoxynucleoside-5'-0-
~l-thiotriphosphate), a suitable enzyme Wit}l polymerizing
3 characteristics such as DNA polymerase~or reverse
transcriptase, a DNA template and a primer. The enzyme
employed, uses dNTP S as a substrate to synthesize nucleic
acids of varying chain length, and upon enzymatic reaction, a
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phosphorothioate diester is incorporated between two
1 nucleoside residues, along with the concurrent liberation of
pyrophosphate.
The phosphorothioate diester may be introduced
chemically into the nucleic acid by the method of Connolly,
et al. (or Stec, et al., J. Am. Chem. Soc., 106: 6077-6079).
-
This is generally a three step procedure. First, a phosphitetriester (nucleoside phosphite triester) is formed by
reacting a nucleoside phosphoramidite in the presence of a
weak acid such as tetrazole. Second, the phosphite triester
is oxidized in the presence of elemental sulfur (S8), CS2 and
lutidine, to form a phosphorothioate triester complex.
Third, in the presence of a base such as ammonial the
phosphorothioate triester is hydrolyzed to the desired
phosphorothioate diester.
The selective introduction of the phosphorothioate
diester derivative into the DNA fragment or
oligodeoxynucleotide, is determined by the choice of
oxidation procedures at any given position. As explained
above, the phosphorothioate diester is obtained by oxidation
in the presence of S8, CS2 and lutidine. The native
phosphate diester is obtained by oxidation of the phosphite `
triester with a mixture of I2, THF (tetrahydrofuran), H2O and
lutidine followed by hydrolysis of the triester to yield a
phosphate diester. The appropriate choice of either set of
conditions allows the placement of the phosphorothioate
diester in the desired position with respect to the native
phosphate diester. This technique allows for selective
reactivity at a specific nucleotidyl site, and avoids
nonspeci~ic reaction with other functional groups available
3 in the nucleic acid.
.; .
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.
The complex formed is described below:
1 3'-Nucleoside
o `:
--S--P=O
0-5'-Nucleoside
~Internucleotidic Phosphorothioate Diester)
The phosphorothioate diester can subsequently be
alkylated with fluorescent molecules or other probes such as,
for example, biotin. In this procedure, the complex which
results i5 referred to as a "phosphorothioate triester"
~which comprises an internucleotidic residue, a
phosphorothioate diester and a detectable marker). The means ;
by which this procedure occurs, e.g., alkylation, refers to
the displacement of the functional group (such as the bromine
in monobromobimane) and the formation of a sulfur-carbon bond
between the fluorescent marker and the phosphorothioate
diester.
For purposes of fluorescent labeling techniques
herein contemplated, various fluorophores can be employed,
for example, monobromobimane (MsB), bromomethylcoumarin, as
well as a variety of chromophores carrying bromoacetamides,
iodoacetamides, aziridinosulfonamides or ~-bromo~ -unsatu~
rated carbonyls; monobromobimane is preferred.
One of the most surprising advantages of this
invention is that the present methodology permits the
introduction of fluorescent dyes or other probes in a
"post-assay" procedure. By "post-assay" procedure is meant,
generally, that the phosphorothioate diester-containing DNA
is used in the assay of choice, for example, in
polyacrylamide gel electrophoresis, and the fluorescent
3 molecule or other marker or probe can bq introduced at a -
later time, for example, while the nucleic acid is embedded -;
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in the polyacrylamide gel matrix. The assay procedures
1 contemplated by the present invention in this context
include, for example, gel electrophoresis, Southern
hybridization, and DNA sequencing techniques such as are
described by Sanger, et al. ~Sanger, et al., Proc. Natl.
~cad. Sci., 74: 5436-5467, 1977). -
Gel electrophoresis as used here is typically
performed by running DNA samples down speci~Eic lanes in a gel
(e.g., a polyacrylamide gel or agarose gel), under controlled
current and temperature conditions for a short period of
time. This procedure leaves the DNA embedded in the gel
matrix.
Southern hybridization involves the use of a
blotting membrane to remove the fractionated nucleic acid
from the gel and allows for hybridization of labeled probes
to the nucleic acid on the surface of the blotting membrane.
Radioisotopic labeling (32p) has been commonly employed for ~`
the detection of nucleic acids resolved by electrophoresis or
after hybridization techniques. ;
San~er DNA sequenciny ~also known as "dideoxy
sequencing") has previously been done using 35S labeling.
This typically involves two steps. The labeling reaction is
initiated after annealing of the primer to t~e template. A
low concentration of dTTP, dGTP, dCTP and ~-[ S]dATP is
employed in order to elongate the primer and incorporate some
radioisotope. The second step involves adding the
termination mixture, which is a higher concentration of all
four dNTP derivatives plus one of the dideoxy derivatives
(ddNTP).
Post-assay fluorescent labeling techniques as
3 described herein permit the introduction of multiple
fluorescent molecules or other appropriate markers into the
;
, ~
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, ,
nucleic acid, e.g., after electrophoresis and "post-assay"
1 labeling of detecting oligodeoxynucleotides and DN~ fragments
can be detected on the basis of, e.g., fluorescence, with
high sensitivity. "
Detection of fluorescent markers can be achieved by
5 use of e.g., a standard long-wavelength ultra~iolet
transilluminator, to view the DNA in the gel, -
The labeling procedure is particularly useful in
conventional enzymatic procedures for the sequencing of DNA.
Instead of radioisotopic labeling as described in the Sanger
10 sequencing technique the four dNTPd S derivatives used in the
sequencing reaction can be substituted such that the DNA
fragments produced will contain phosphorothioate diesters at
ali internucleotidic positions which can allow multiple -
labeling and ultimately allow reading of large and small DNA
15 fragments. The labeling procedure is also applicable to site ,
specific identification of nucleotides by introducing at
least one phosphorothioate diester selectively into an
internucleotidic residue or DN~ fragment or oligodeoxy-
nucleotide, labeling said phosphorothioate diester with a
20 marker and detecting said marker.
The aforesdescribed labeling technique can also be
applicable to hybridization studies using, e.g.,
membrane-bound nucleic acids.
A fiuorescently labeled cloned DNA probe can be
25 used to localize specific nucleic acid sequences in mixtures
of DNA restriction fragments fractionated b~,gel - -
electrophoresis. A replica of the gel is made by
transferring all of the fractionated DNA fragments to a sheet
o~ nitrocellulose paper or similar me~brane (the "blotting
30 membrane") by diffusion or electrophoresis. The
hybridization probe can be labeled before or after the
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14
-.
hybridization assay occurs. The locations of the fragments
1 that hybridize to fluorescently labeled DNA probes are then
identifiea by their fluorescence. Similarly, nitrocellulose
paper replicas can be made of crowded colonies of bacteria
growing on an agar surface so that hybridization of the paper
with a specific labeled probe can be used to identify the few
cells carrying a newly cloned specific DNA fragment.
The labeling and d~tection techniques herein
discussed, can also surprisingly be easily employed in DNA
diagnostics and DNA therapy. The present advantage, relative
to art recognized techniques, is particularly manifest in
that the presence of the phosphorothioate diester does not
effectively alter the biophysical nature of the DNA and yet
selectively introduces a nucleophilic site which is readily `
modified and exploited for diagnostic and therapeutic
purposes. For example, the phosphorothioate diester can be
introduced into the DNA and subsequently hybridized to a gene
of interest ln vitro or ln vlVO, and then followed by
specific introduction of a probe to that gene. The probe to
the particular gene can then be used to discover the location
oE the gene. This leads to detection of the presence or
absence of the gene under diagnostic investigation. The
probe can then be used in DNA therapeutics to inactivate or
destroy that particular gene or i~ necessary, to activate
that gene. For example, diagnosing genetic disorders and
direction of drug delivery ~e.g., anticancer or antiviral
drugs).
Another surprising advantage of the present
invention is that the DNA-containing phosphorothioate diester ;~
is largely resistant to nucleases and therefore is very
3 stable when introduced into complex biological systems found `
in vitro and in vivo.
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The present invention can be used in spectroscopic
1 analysis te.g., Nuclear Magnetic Resonance studies, and in
particular, the Nuclear Overhauser Enhancement [NOE~) to
measure distances within nucleic acids by use of probes which
can label specific phosphorothioate diesters.
The present invention can also be applied to
Electron Spin Resonance studies, which previously relied upon
the use of non-specific labeling. The simple and rapid
procedures described here will allow the preparation and
study of nucleic acid fragments containing spin labels,
attached at well-characterized locations. The proceudre
described herein can also be used for the specific attachment
of hydrolytic reagents (e.g., ferric ion complexes),
intercalators and proteins to nucleic acids.
Additionally, the present invention can also be
used to probe the structure of DNA fragments or
oligodeoxynucleotides by using chiral metal complexes (e.g.,
the ~-isomer or ~ -isomer of tris-~4,7-diphenylphenan-
throline) cobalt tIII)) as the one marker of choice to be
attached to the phosphorothioate diester.
In order to use the phosphorothioate diester
effectively in a procedure for detecting nucleic acids, it is
advantageous to assess the stability, particularly with
respect to p~l, of the labeled phosphorothioate diester-
fluorescent marker product. An HPLC analysis can be used
employing a reversed phase column. This assays the stability
of the labeled phosphorothioate derivative ~triester) over a
broad pH range during an incubation period at ambient
temperature.
In another aspect of the present invention, high
3 detection sensitivity of fluorescent labeled nucleic acids
can be facilitated by the introduction of multiple
~ , ,,. : : , '~ . .
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~::
fluorescent markers to a corresponding multiple number of
1 phosphorothioate diesters earlier introduced at the selected
internucleotidic sltes; the labeling reaction must occur at
adjacent phosphorothioate diesters such that, to achieve
maximum sensitivity, a nucleic acid fragment carries a
fluorophore at each and every internucleotidic phosphorus
residue. Surprisingly, experimentation indicates that there -;
is no steric hindrance or other difficulty in placing
fluorescent labels on adjacent phosphorothioate diesters,
thus permitting maximization of this technique.
As earlier discussed, "post-assay" labeling
procedures are useful for a variety o biochemical assays
one of the most important specific applications involves the
detection of nucleic acids resolved by gel electrophosesis
techniques. One "post-assay" labeling procedure, for
example, can be accomplished using short oligodeoxynucleotide
fragments resolved by a given assay (e.g., gel
electrophoresis) and then soaking the gel containing the
small nucleic acid fragment with a solution which contains
the ~luorescent marker of choice. Small fragments with
several labeled phosphorothioate diesters are quantitatively
compared with the fluorescence exhibited by a nucleic acid
fragment with a single fluorophore. There is a concomitant -
increase in detection sensitivity with an increase in the -
number of labeled phosphorus residues.
Longer DNA fragments containing phosphorothioate
diesters can be prepared by enzymatic synthesis when the
normal dNTP substrates are replaced by ~-thio derivatives
~dNTP ~S). In order to generate fragments of defined length,
a~l oligodeoxynucleotide primer can be extended using a
3 template (e.g., M13mpl~ or M13mpl9 or other single-stranded
DNA) and then the resulting material can be hydrolyzed with
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7-
an appropriate restriction endonuclease. The amount of DNA
1 fragment which can be visuali~ed is approximated based upon
the maximum amount of template present in the reaction
mixture or as the result of internal standardization via
radioisotopic labeling. The variety of bancls produced can be
visualized by "post-assay" fluorescent label:ing procedures.
The results show a further increase in sensitivity relative
to the increased sensitivity in small nucleic acid fragments.
Various fluorophores are available and many can be
employed in the present process. Any fluorophore can be
utilized for the "post-assay" fluorescent labeling procedures
contemplated by the present invention ,which reasonably
possess the following properties: high quantum yield
solubility in aqueous (or largely aqueous) solutions; ~-~
relatively small size to allow diffusion throu~h the gel
matrix; high fluorescence only after reaction with a sulfur
residue; and removal of the excitation maximum from the
absorbance maxi~um of the nucleic acids. One preferred
fluorophore which meets these criteria is monobromobimane.
Other fluorophores of choice can include, for example,
bromomethylcoumarin! or fluorophores carrying bromo- or
iodoacetamides, or aziridinosulfonamides. The fluorophores
of choice have the ability to alkylate the phosphorothioate
diester. The phosphorothioate diester is more nucleophilic
than any other site on the nucleic acid and results in
formation of a stable phosphorothioate triester when labeled
with the fluorophore of choice.
In particular, two widespread assays which can be
employed in conjunction with the "post-assay" fluorescent
labeling of this invention are DNA sequencing using, e.g.,
3 the Sanger dideoxy method and DNA hybridization (using e.g.,
the Southern technique).
3j
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l. DN~ Sequencinq
l Post-assay labeling is most amenable to enzymatic
dideoxy sequenclng procedures. This approach incorporates
phosphorothioate diesters in place of native phosphate
diesters in the DN~ fragments generated. After gel
electrophoresis, multiple fluorophores, such as MBB, can be
attached to the DNA via alkylation of ~he sulfur residue of
the phosphorothioate diesters.
Current technology of Sanger sequencing utilizes
the dNTP derivatives. The Sanger sequencing technique
commonly utili~es a single C~-[535]dNTP derivative to
introduce the readioactive label. However, by using all four -
dNTP ~S derivatives in the present invention, DNA fragments
can be generated by this technique which can contain hundreds
of phosphorothioate diesters. The "post-assay" labeling of
this invention can be directly applied to the detection of
these fragments.
The "post-assay" fluorescent labeling technique
provides the sensitivity necessary to visualize DNA
sequenciny ladders in the absence of radioisotopes. The
technique as described here employs all four dNTP ~S
derivatives plus one of the dideoxy derivatives (ddNTP) in
the elongation and then termination of the DNA primer. ;
Sequencing ladder6 can be generated with dNTP ~S substrates
in the like manner to the methodology with dNTP derivatives.
It is then desirable to vary the elongation and
te~mination conditions such that in the initial fluorescence ;`
labeling the amount of DNA in each band may be varied. Then
the amount of DNA that appears in the bands can be maximized,
e.g., ranging from approximately 300 to 500 base pairs.
3 Fragments of this size can be resolved, and 300 to 500
fluorophores or other types of markers can be incorporated
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~-- --19--
into such fragments. The distribution of the fragments can
1 be altered by changing the relative ratios of the
dideoxynucleotide/deoxynucleotides triphosphates.
~ ddNTP/dNTP~S ratio of about 1:10 may be used to
obtain a distribution of small and large fragments. A
decrease in this ratio is effected to allow for more
efficient polymerization in a stepwise manner to as low as
about 1:500 in order to shift the distribution to longer
fragments.
The use of ~-[35S]dATP as a method for introducing
the radioisotopic label has been reported and is commonly
employed. Dideoxy sequencing using 35S labeling typically
involves two steps. After annealing of the primer to the
template the labeling reaction is initiated. A low
concentration of dTTP, dGTP, dCTP and ~-[35S]dATP is
employed in order to elongate the primer and incorporate some
radioisotope. The second step involves adding the
termination mixture which is a higher concentration of all
four dNTP derivates plus one of the dideoxy derivatives
(ddNTP). It is a simple procedure to then substitute the
four dNTP ~S derivatives in both reactions (actually there is
only one reaction since no radioisotopic labeling is
involved) such that the DNA fragments produced will contain
phosphorothioate diesters at all internucleotidic positions.
For internal standardization, radioisotopic
labeling can be used in combination with fluorescent markers
to monitor the limits of detection sensitivity. To obtain
fragments which have been labeled to a known specific
activity a "minus-dCTP" labeling reaction is employed. This
uses a primer and template of known sequence, for example, of
3 the following sequences:
. ! ' ~ ~ '~ ; ' :
; ' '; ' ~ : ,~ ' ' . ~, ' ., . '`.: .- ' . . :
' ' . "'..... , : ~, :'' , . : ' ' .
~, .: ', ' :..... -
:: ' , ' ': , ;. .: ': , . ':
-20-
Ml3mpl8 3'...C~AAAGGGTC~GTGCTGCAACATTTTGCT...5'
lprimer 5'-GTTTTCCCAGTCACGAC-3'
The labeling reaction can now be performed with low
concentration of the dTTP ~S, dGTP~ S and ~-[35S]dATP. The
elongation of the primer proceeds until the first dG present
in the template and then terminates resulting in the
following sequence containing four S labels:
~113mpl8 3'... CAAAAGGGTCAGTGCTGC~CATTTTGCT... S' ; -
elongated primer 5'-~TTTTCCCAGTCACGACGTTCTAAAA-3'
**** .: . . .
The termination reaction uses all four dNTP~ S
derivatives at concentrations some two orders of magnitude
higher than the labeling reaction such that any remaining
radioactive ~-[35S]dATP is diluted and the quantity
available for incorporation becomes insignificant. The
amount of material present (based upon the known specific
activity of the ~-~35S~dATP) in a given band can now be -
easily determined by excising the band, lyophilizing the gel
and determining the radioactivity present by scintillation ;,
counting. By adjusting the concentrations of the template
and primer as well as the ratio of the ddNTP to dNTP~ S, the
amount of DNA present in a given fragment can be altered. In
addition, distribution of fragments can be shifted to those
of higher or lower molecular weight. Optimization of
detection can allow "reading" of smaller fragments ~smaller ;
25 than 300 nucleotide residuesj. DNA sequencing in the -
abscence of radioisotopes can then be effectuated by
detecting the hundreds of labeled, e.g., bimane-labeled
phosphorothioate triesters by utilization of single or
sophisticated electronic techniques.
. .
~ :
-21-
2. DN~ ~3ybr dlzation
l In another embodiment of the present invention, the
post-assay fluorescent labeling technique can also be applied
to hybridization studies using nucleic acids. The stability
of a native DN~ duplex is first tested against nucleic acid
containing a num~er of phosphorothioate diesters and the
effect of this stability when the phosphorothioate diesters
are alkylated by a fluorophore is determined. For example,
the results for the detection of a 21-mer fragment containing
20 phosphorothioate diesters shows that in the absence of
electronic instrumentation it can readily be detected
visually. Nucleic acids with one label can be detected and
dètection of single nucleotides can be facilitated. Such
visibility is increased proportionatly with the proportionate
number of markers.
~ 21-mer fragment is one example of a small
hybridization probe which can be used to detect nucleic acid
sequences. This is utilized in the following manner: DNA
fragments or oligodeoxynucleotides of reproducible size are
qenerated by selective chemical means, such as by a
restriction endonuclease enæyme. These nuc]eic acids are
resolved by a biochemical assay such as polyacylamide or
aqarose gel electrophoresis. The nucleic acid resolved in
this manner is then transferred to a blotting membrane, e.g.,
nitrocellulose membrane and the DNA probe is hybridized to
the nucleic acid. Although the DNA probe at this point has
the phosphorothioate diester or diesters incorporated into
it, the marker of choice, e.g., a fluorescent marker, may be
introduced before or after the hybridization assay.
Following these steps, the marker can be detected using
3 simple or sophisticated detection techniques.
~~ -22-
One of the primary differences between "post-assay"
1 fluorescent labeling within a gel matrix and labeling on a
blotting membrane is that the latter occurs primarily on the
surface of the membrane and not within a three dimensional
matri~ ith such surface phenomena it is possible to also
5 use biotin labeled hybridization probes and detection with -~
fluorescent protein complexes which could not be used for
labels embedded in a gel matrix (the proteins involved are of
large molecular weight and would not readily diffuse through
the pores of the gel matrix). The phosphorothioate diester
lO can be employed to allow efficient multiple ~and specific) ~ ;
labeling with a biotin derivative. For example, the
bromoacetamido group can be used to modify the
phosphorothioate diester. A biotin derivative containing
this functional group can be prepared quite simply by
techniques available to one of ordinary skill in the art.
Biotin labeling in this manner is considered an effective
method for detecting nucleic acids when combined with
immunochemical, histochemical or affinity detection systems.
Two similar proteins, avidin and streptavidin, bind biotin
very strongly and when coupled to fluorescent markers,
enzymes or electron-dense proteins, can be e~ploited for the
detection of nucleic acids. The use of fluorescent labeled
antibodies raised against biotin can also be employed for
detection. The biotin-labeled hybridization prohe may be `
detected by use of a commercially available kit used for the
detection of fluorescently labeled antibodies or by use of a
transilluminator to detect the fluorescent group or protein.
Hybridization assays require the hybridization
probe ~orm stable Watson-Crick base pairs in order to
3 localize the probe at a given sequence.~ The addition of
biotin derivatives to the internucleotidic phosphorus
-23-
, .
residues can result in some destabilization of the double
1 stranded hybridization product. A series of biotin labeled
probes can be prepared containing from one to approximately
five biotin labels and the stability of the .luplexes formed
can be examined with biotin modified oligodeoxynucleotideS in
comparison with those unmodified. This can be accomplished
by labeling of the oligodeoYynucleotides containing the
correctly positioned (and number of) phosphorothioate
diester~s) and isolation of the product using HPLC
techniquss. Duplex stability can be monitored by thermal
denaturation experiments and circular dichroism spectra.
The ability of the biotin labeled oligodeoxy-
nucleotide to function as a hybridization probe can then be
examined using, for èxample, the 21-mer previously described.
The sensitivity to detection of probes containing a varying
number of biotin labels can be examined using commercially
available fluorescent labeled proteins. "Spacing" the labels
every two, three or more phosphorus residue-; can be the
simplest route to enhance detection sensitivity.
In a second approach involving "post-assay"
labeling, the phosphorothioate-containing probe is hybridized
in one step; this avoids problems with the instability ~if
any) of the biotin labeled hybridization product.
Subsequently, modification with the biotin label occurs, and
after removal of the excess label, the protein solution is
added for detection. This approach is conceptually similar
to the one described for the visualization of DN~ sequencing
ladders and may also be the simplest approach to
hybridization assays. `
Hybridization experiments can also be performed
3 with relatively long DNA fragments obtained from restriction
digests and multiple phosphorothioate diesters can be
:
24-
incorporated into such a fragment using DNA polymerase and
1 nick-translation procedures. Radioisotopic labeling is
accomplished by introducing "nicks" in the DNA with a dilute ,~
solution of DNase I and then elongating the nicked sites
using DNA polymerase and the ~-~32P]dNTP substrates. The
radioisotopic derivatives can then be replacecl with the
dNTP S derivatives and then hundreds of phosphorothioate
diesters can be incorporated into the fra~ment. The simplest
system to test hybridization can be one involving the M13 DNA
being use~ in the sequencing reactions. For example, M13 RF
(replicative form) DNA can be prepared in the conventional
manner and then cleaved out a 444-mer to use as a
hybridization probe. The 444-mer can then undergo
nick-translation to incorporate the phosphorothioate diesters
and then the modified and native sequences resolved by gel
electrophoresis. A second sample of the M13 RF DNA, for
example, can be digested such that the complementary 4~44-mer ~;
restriction fragment (in additon to others) is produced and
trans~erred from an agarose gel to nitrocellulose or similar
blotting membrane. 5'he hybridization can then proceed
20 followed by post-assay fluorescent labeling using, e.g., `
monobromobimane; fluorescent labeling with hundreds of
markers provides the desired detection sensitivity. Since
the monobromobimane is largely non-fluorescent until it
alkylates a sulfur containing functionality, the membrane
background fluorescence is relatively low. The labeled
marker can then be detected with relative ease.
In another embodiment of the present invention, DNA
probes are generated from mRNA. Again, one can simply use
the dNTP S derivatives, which function as substrates for
3~ reverse transcriptase, to form the complementary DNA strand
for use as a hybridization probe. The use of the new
,
-25-
labeling approach provides well-characterized hybridization
1 probes which can be used for the detection of specific DNA
sequences, in the absence of radioisotopes, for example, in
Southern blots, Northern blots, colony screening or plaque
screening.
3. Specific ~odification of Nucleic Acids wlth
Fluorescent Markers or Spin Labels
In a further aspect of this invention, the labeling
of specific phosphorothioate diesters is also valuable for
structural studies involvlng fluorescent energy transfer
techniques and electron spin resonence ~ESR) techniques.
The application of these two spectroscopic
techniques has long suffered from the difficulty in
specifically attaching the desired probe to the nucleic acid
fragment. The present procedure permits simple and rapid
synthesis of a variety of nucleic acid sequences which can be
easily modified with fluorescent markers or spin labels for
spectroscopic studies.
Fluorescent Energy Transfer Techniques allow for a .
simple and rapid means for measurement of longer distances
within the nucleic acid structure, complementing NMR
techniques such as that of the Nuclear Overhauser Enhancement
(NOE) which can only measure small distances in the nucleic
acid.
The.disadvantages of the energy transfer technique
have previously been in the difficulty of easily placing the
donor and acceptor chromophores in specific positions, and
the questionable accuracy of the technique when the
o~ientation of the chromophores is unknown.
These two shortfalls are eliminated by the labeling
3o of specific phosphorothioate diesters pursuant to the
methodology of the present invention. By controlling the
-26-
.
position of the phosphorothioate diester, the placement of a
1 specific label becomes as rapid as it is simple. Since the
label is oriented on the outer surface of the macromolecule a
freely rotating chromophore is likely.
ESR spectra can be valuable for the study of -
biopolymer dynamics providing that the appropriate spin label
can be specifically bound to the macromolecule of interest.
In general, the technique has suffered a similar disadvantage
to energy transfer experiments in the difficulty of ;
specifically placing the label on the macromolecule. The use
of the phosphorothioate diester can again be valuable in this
respect. Nucleic acid fragments can be prepared with spin
labels by eY~actly the same approach as described above for
fluorescent markers. Specifically labeled probes can be
designed and prepared for these ESR studies.
Other procedures which can be used in association
with the instant technique involve optimization of
fluorescerlce detection. These include, Eor example,
1) altering the microenvironment of the labeled nucleic acid
fragments in the gel matrix to increase the quantum yield of
the fluorophore, 2) adjusting the excitation light energy to
optimally fit the excitation spectrum of the dye and using
filters to screen out all light energy (largely excitation
wavelengths) other than the desired emission energy, and
3) examining electronic detection as a means of automating
the reading of the information present. The first two
approaches together can be expected to increase the detection
sensitivity by roughly one order of maqnitude. Electronic
methods can be expected to provide one or more additional
orders of magnitude enhancement.
3 The following examples would assist in further
detailing the SU~D ject invention herein.
.
' . ,
' ', , ', ;' ` . ' . ,, , ; , . . ,,, , . , ~ ' .' , ! ~ : ' . . ~ ' ' ; , ,
EXAMPLES
1) Chemical OligodeoxYnucleotide S~nthesis
Tp(s)T, the phosphorothioate diester derivative of
TpT, is an example of the simplest phosphorothioate diester
amenable to the labeling procedures described.
The (dT)15 with phosphorothioate diesters 3' to
thymidine residues 7, 8, and 9 were synthesized by using the
phosphite triester methodology (Beaucage & Caruthers,
Tetrahedron Lett., 22: 1859-1863, 1981) on a solid-phase CPG
lO support. The synthesis was interrupted prior to the ~.
oxidation step when the incorporation of a phosphorothioate
diester was desired. The normal oxidation step with 0.1 M I2
in tetrahydrofuran/distilled water/lutidine (40:1:10) was
replaced with a solution of 2.5 M sulfur in CS2/lutidine ,
15 (1:1). The sulfur oxidation solution was injected directly ;
onto the column with a syringe. After a reaction time of 1 h
at ambient temperature, the column was washed with a 1:1
solution of CS, and lutidine to remove the residual sulfur.
The column was then replaced on the machine, and the
synthesis cycle was resumed. The 21-mer
d(GCTATCG~AGATCTCATAAG) was synthesized in an analogous
manner. The synthesis was interrupted at every oxidation -~
step to allow oxidation with the sulfur solution.
Both oligodeoxynucleotides were deprotected in -
ammonia at 50C for 18 h. Isolation was done by
reverse-phase HPLC on a 9.4 x 250 mm column of MOS-Hypersil
using a buffer of 50mM triethylammonium acetate, pH 7.0 with
a gradient of 20-653 acetonitrile in 40 min.
,.
i "" ,"
: ~.
;
~",',' ', '' ,:
: ' .'' '",
.' ', ',' '
-28-
2) Solution Fluorescent Labelinq Studles
1 The fluorophore of choice in this example,
monobromobimane (kl~B), was dissolved in acetonitrile, and
stock solution ~lOOmM) was stored in the dark at -20C.
Typically, the oligodeoxynucleotides of interest
were treated with an excess of monobromobimane, and the
reaction was monitored by HPLC. Specifically, a solution of
Tp(s)T 13.6 ~) in water was allowed to react overnight
(18 h~ with a 6-fold excess of monobromobimane (22 mM). The
octamer (0.3 mM) in water was allowed to react with either a
5-fold excess of MBB (1.5 m~l) or a 10-fold excess of MBB
(3.0 mM). The fragment Tp(s)Tp(s)Tp(S)T (0.43 mM, a
phosphorothioate diester concentration of 1.29 mM) was
treated with an 8-fold excess (with respect to the
phosphorothioate diesters) of MBB (10.5 m~l). Covalent
fluorescent labeling of the 15-mer in solution (0.8 mM) with
MBB was achieved at 7.5 mM MBB (3-fold excess for 2.4 mM
phosphorothioate diester).
The bimane-labeled Tp(s)T (see Figure 1) was
isolated by reverse-phase }TPLC on a 4.6 x 250 mm column of
ODS-llypersil with 50 mM triethylammonium acetate, pH 7.0, and
a gradient of 0~70% acetonitrile in 1 h. The other labeling
reactions were monitored by reverse-phase HPLC on a 4.5 x 250
mm column of ODS-Hypersil with either 20 mM KH2PO4j pH 5.5,
and a gradient of 0-70~ methanol in 30 min (the octamer and
tetramer) or 50 mM triethylammonium acetate, pH 7.0, and a
gradient of 0-35~ acetonitrile in 1 h (15-mer).
Thin-layer chromatography studies were performed on
silica gel thin-layer plates with a mobile phase of
dichloromethane/methanol (9:1).
,, : , ' ' ' :
:' ,,. :' , ,,: ': :, ' ''
' ,, . : ' ' . , ~
-29-
.
3) pll Stability Studies
l Duplicate reaction mixtures of 6 nmol of
bimane-labeled Tp(s)T were incubated at ambient temperature
in 50 mM buffer at the appropriate pH values. The following
buffers were used: pH values 3, 4 and 5, acetic
acid/potassium acetate; pH values 6 and 7, K2PO4/K2HPO4; pH
values 8 and 9, Tris-HCl; pH values 10 and 11~ CAPS. At
various reaction times, the samples were analyzed by HPLC on
a 4.6 x 250 mm column of ODS-~ypersil using 0.02 M potassium
phosphate, pH 5.5, with a linear gradient of 0-70~ methanol
in 30 min. The bimane-labeled Tp~s)T eluted at 21 min, while
the product TpT eluted at 16 min.
At low pH values (3-7) less than 5~ of the triester
was hydrolyzed after a 20 h incubation as determined by
integration of the corresponding HPLC peaks. ~see Figure 2).
Upon incubation with Tris-HCl at pH 8 for 20 h, 11% o the
triester was hydrolyzed. At pH 9, a 20 h incubation resulted
in 40~ of the hydrolysis product. The triester was
completely hydrolyzed within 15 h at pH 10 and within 1 h at
pII 11 (see Figure 2). EIPLC analysis confirmed that ;
hydrolysis occurred by cleavage of the P-S bond and formation
of TpT as expected.
To further characterize the reaction of '~
monobromobimane with a phosphorothioate diester, the reaction
was performed with an oligodeoxynucleotide which at ambient
temperature exists largely in the double-stranded form. The
reaction of the octamer d[GpCp(s)CpCpGpGpGpC] with a 10-fold
excess of monobromobimane was performed in either distilled
water or Tris-HCl pH 7, at ambient temperature. The HPLC ;
analysis ater a 5-h incubation (Figure 3) showed the
3 starting material (14.88 min), a monobromobimane hydrolysis
product (15.3 min), a product peak ~17.75 min), and
" ,
~ 30-
,
monobromobimane (25.21 min~. The starting material was
1 completely consumed within 23 h. With a 5-fold excess of
moIlobromobimane, the reaction was complete within 48 h. The
reaction proceeded equally well with either 1_he Rp or the Sp
diastereoisomer. A control reaction containing an
oligodeoxynucleotide with only phosphodiesters failed to show
any conversion to a labeled product.
4~ P N~IR Studies
The 31p ~MR studies were done at l21.5 MHz using a
varian m~ltinuclear FT-NMR. Positi~e chemical shift values
10 are reported in parts per million ~ppm) downfield from the -
external standard of aqueous 85% phosphoric acid. NMR
analysis was done on a sample containing 1.2 umol of
Tp(s)Tp(s)Tp(s)T (3.5 umol of phosphorothioate diesters) and
20 mM Na2EDTA. The sample was adjusted to a volume of 250 uL
with D2O. After NMR analysis of the tetramer, 10 umol of
monobromobimane (a 3-fold excess with respect to the
diesters) in 100 uL of acetonitrile was added to the NM~ tu~e
with a final volume of 350 uL. The sample was allowed to
react for 2.5 h at ambient temperature in the dark. NMR
analysis was then repeated.
5) ~adioisotopic Labeling (32p End Labeling)
A reaction mixture in a final volume of 200 uL
containing 40.1 uM 15-mer (1 A260 unit), 40.7 uM ATP, 10 mM
MgC12, 10 mM dithiothreitol, 5 ug/mL bovine serum albumin, 40
mM Tris-HCl, pH 8.7, 0.127 uM (0.152 mCi) [~ -3 P]ATP, and 10
units of T4 polynucleotide kinase was incubated at 37C for
18 h. After the addition of the reaction mixture to the
Sep-pak cartidge (prewashed with 20 mL of methanol and 20 mL
of distilled water), it was washed with 10 mL of 1~ aqueous
3 methanol to elute the unincorporated ATP and buffer salts.
The oligodeoxynucleotide was eluted with 10 mL of 50~ aqueous
. : ........ ... .. . . . . . ..
,, , . . ~ . . . .
-31-
methanol. The solution containing the DNA fragment was
1 evaporated to dryness and redissolved in 0.4 ~I distilled
water. Isolated yields ranged from 60 to 80%.
The 21-mer, 23.3 uM ~1 A260 unit), was end labeled
in an analogous manner but could not be eluted with aqueous
methanol. In this case, the Sep-pak cartidge was prewashed
with acetonitrile and distilled water. The unincorporated
ATP and salts were then eluted with 1% aqueous acetonitrile
while the oligodeoxynucleotide was eluted with 50~ aqueous
acetonitrile. Isolated yields also ranged from 60 to 80~.
6) Post-Assay Labeling
Gel electrophoreseis was performed on 20 x 20 x ; ;
0.04 cm or 34 x 42 x 0.04 cm gels of 20~ acrylamide, 2% ~
bis(acrylamide) [or 6% acrylamide and 0.6t~ bis(acrylamide)l, ;
50 mM Na2EDTA, and 13 mM sodium persulfate. Post-assay
labeling was performed both in the presence and in the
absence of 7 M urea. The DNA was fixed in the gel by soaking
it in 10~ aqueous acetic acid for 5 min. The gel was then ;~
transferred to a 4 mM solution of monobromobimane in 50~
aqueous acetonitrile and allowed to react overnight (18 h~ in
the dark. The gel ~as destained by shaking in 50~ aqueous
acetonitrile for 1 h. The short destaining appeared
necessary because of minor reactions with the gel components
and monobromobimane. Following a brief treatment (5 min) in ;- -
60 or 75~ aqueous simethylformamide, the DNA was viewed on a
standard long-wavelenth ultraviolet transilluminator (~max =
366 nm). In some cases for internal standardization, the
fluorescent bands of DNA were cut out of the gel and -~
lyophilized before determination of the amount of DNA present
in the gel via scintillation counting.
`
''
;. .:
~
~32-
The effect of solvents on fluorescent intensity was
l also investigated. ~fter post~assay labeling and destaining,
the gels were treated with one of the following: 75% aqueous
mixtures of methanol, ethanol, butanol, dimethylformamide, or
concentrated glycerol. The gels were viewed using a long
ultraviolet wavelength light transilluminator.
7) Fluorescent Studies
The fluorescense (excitation 385 nm, emission
465 nm) of varying solutions o~ bimane-labeled Tp (s) T in 5 mM
K~l2P04, p~ 4.5, was measured by using a fluorescence
spectrophotometer, and a standard curve of fluorescence vs.
phosphorothioate diester concentratio~ was fitted to the data
employing a linear least-squares analysis.
After post-assay fluorescent labeling (see above)
with monobromobimane, the 5,_32p end-labeled 15-mer was
electroeluted for 2 h from a 20~ polyacrylamide gel into
dialysis tubing containing 0.5x TBE buffer. The solution was
evaporated to dryness, redissolved in 1 mL of distilled
water, and desalted using a column of Sephadex G-10. The DNA
fragment was collected, evaporated to dryness, and
redissolved in 3 mL of 5 mM KH2P04, pH 4.5. The fluorescence
of the solution was measured and the concent:ration of the
15-mer determined by scintillation counting. The
fluorescence as a function of concentration of the
phosphorothioate diesters was plotted on the standard
bimane-labeled Tp(s)T curve.
In similar fashion, the 5,_32p end-labeled 21-mer
was electroeluted for 24 h from the polyacrylamide gel after
post-assay labeling. The solution was evaporated to dryness
and redissolved in 0.5 mL of distilled water. In this case,
3 the solution containing the 21-mer was~adjusted to 10 mM
MgC12 and 2 M ammonium acetate, 1 volume of ice-cold
:" ' ' ~ ' " : `
: . .
-33-
acetonitrile was added, and the solution was kept at -70C
l for 18 h. The salt precipitated out of solution while
essentially all of the DNA remained in the supernatant. The
solubility of the labeled 21-mer in acetonitrile is largely a -~
result of the increased hydrophobicity conferred upon the
oligonucleotide due to the presence of the ~lmane residues.
The supernatent was decanted, evaporated to dryness, and
dissolved in 3 mL of 5 mM KH2PO4, pH 4.5. The fluorescence
and radioactivity were measured and compared with the
standard curve.
8) DNA Polymerase and Restriction Endonuclease Reactions
M13 mpl8 DNA was converted to the replicative form
(RF) as follows. The template DNA t2.5 ug) and universal
primer ~0.1 ug) were annealed in 25 uL of buffer containing
100 mM NaCl, 20 mM MgC12, and 100 mM Tris-HCl, pH 8.0, by
heating the mixture to 56C for 15 min followed by slow
cooling to ambient temperature. The final 50-uL reaction
mixture containing d~TP, dGTP, dCTP, dTTP (500 uM each), ATP
(1 mM), DNA polymerase 1 (Escherichia coli, 10 units), and
T4 DNA ligase (8 units) was incubated overniyht at 16~C.
Substitution of the appropriate dNTP ~S derivative(s) for the
corresponding dNTP(s) essentially as described (Taylor,
et al., Nucleic Acids Res., 13: 8749-8764, 1985) allowed the
enzymatic incoporation of phosphorothioate diesters in place
of phosphodiesters. In some cases for internal
standaxdization, ~-[35S]-dATP (1.15 Ci/mmol) was employed in
the elongation reaction.
Restriction digests with AvaI and HpaII were
performed as follows. The AvaI reaction mixture contained RF
M13mpl9 DNA, 100 mM NaCl, 20 mM MgCl2, and lO0 mM Tris-HCl,
3 pH 8Ø The HpaII reaction mixture contained RF Ml3mpl8 DNA,
3 mM KCl, 5 mM MgCl2, 100 ug/mL BSA, and 5 mM Tris-HCl,
'' "'
- 35 ~
.'~.'.~'; .~.
-34-
p~l 7.4. The reactions were initiated by the addition of the
1 enzyme and incubated at 37C for 2 h. The reaction mixture
was loaded OlltO 6~ acrylamide, 0.6~ bis(acrylamide) gels (20
x 20 x 0.04 cm or 34 x 42 x 0.04 cm) containing 3 mM Na2EDTA,
7 M urea, and 50 mM Tris-borate, p~l 8.3. Fluorescent
labeling proceeded as described above.
9) Detection of Nucleic Acids
The 5'-3 P end-labeled 21-mer was viewed on a
transilluminator ( ~ max = 366 nm) after gel analysis and
post-assay labeling. The bluish green bands were excised
from the gel and lyophilized, and the amount of DNA present
was determined by scintillation counting. The amount of the
oligodeoxynucleotide visible as a result of the bimane
fluorescence has decreased such that 500 fmol (500 x 10 15
mol) of the DNA fragments could be observed.
Longer DNA fragments containing phosphorothioates
can be prepared by enzymatic synthesis if the dNTP substrates
are substituted by the ~ -thio derivatives (Taylor et al.,
Nucleic ~cids Res., 13: 8749-8764, 1985). In order to
generate fragments of defined length, an oligonucleotide
primer was extended using an M13mpl8 or M13mpl9 template and
the resulting material was hydrolyzed with a restriction
endonuclease. It was possible to prepare M13 RF D~A
containing phosphorothioates at each position. Cleavage of
the elongated DNA with HpaII produced fragments which
migrated in the 6% polyacrylamide gel and could be visualized
Dy ?ost-assay fluorescent labeling (Fiaure 4A). A similar
experiment with the AvaI restriction endonuclease produced a
444 nucleotide fragment which could be visualized by
post-assay covalent labeling ~Figure 4B). Some high
3 molecular weight DWA could also be observed in this gel at
the edge of the sample well ~Figure 4B). With the 444-mer,
, ... .. . . ..
: . . : : : .,
/ - ~
-35-
:
the bands were excised, and the amount of DNA was determined
l by scintillation counting. Approximately 40 fmol (40 x 10
mol) of the 444-mer (containing a maximum of 104
bimane-labeled phosphorothioate diesters) could be visualized
in this e~periment.
10) Synthesis of oli~odeoxynucleot_des containing
a single phosphorothioate diester -
Two oligonucleotides were synthesized for covalent
attachment of a variety of reporter groups, including spin
labels, fluorophores and drug derivatives. A
dodecadeoxynucleotide and an eicosodeoxynucleotide were
chemically synthesized by the phosphoramidite method
described in Example 1 and altering the oxidation step at the
appropriate cycle, resulting in two phosphorus diastereomers
(Rp and Sp). It is possible to prepare the oligonucleotide
such that it contains a pure phosphorus diastereoisomer as
described lConnolly et al., Biochemistry 23: 3443-3453,
1984: Taylor et al., 1985].
Specifically, the dodecamer has the sequence
d[CGCA(s)AAAA~GCGl and the eicosomer has the sequence
dl CGTACTAGTT~s)AACTAGTACG].
~dditionally Tp(s)T was reacted ~ith a number of -
fluorophores or reporter groups containing a variety of
functional groups. Three functionalities, ~-bromo- d ,~un-
saturated carbonyls, iodo (or bromo) acetamides, and
aziridinyl sulfonamides, were observed to effectively label
phosphorothioate diestérs and produce the corresponding-
phosphorothioate triester carrying the desired reporter
group.
': :
, ' '~.. :
'
-36-
11) Phosphorothioate triester
1 oli~ xynucleotldes carryinq various reporter groups
Oligodeoxynucleotides of Example 10 containing a
single covalently bound reporter group (Fig. 5) were obtained
by incubation of the phosphorothioate-containing DNA fragment
with the reporter group of choice in aqueous or largely
aqueous solutions at pH values from 5 to 8. These reactions
were performed at 25 to 50C and usually proceeded with
yields greater than 85% after 24 h at 50C. Resolution of
the reaction mi~ture and isolation of the triester product
10 was accomplished by using HPLC (4.6 X 250 mm Hypersil-QDS
with 0.02 ~I KH3PO~ pH 5.5 and a metha~ol gradient).
Modification of the phosphorothioate was observed to be more
efficient for the single-stranded dodecamer than the
self-complementary eicosomer. This difference in reactivity
was partially overcome when the reaction mi~ture was heated
at 50~C. IJ1 the absence of the phosphorothioate diester,
control reactions using native oligodeoxynucleotides dicl not
result in any significant labeling.
al Attachment of a PROXYL spin label:
The reaction to produce the compound in Fig.
5a was conducted as described above using the following
specific conditions: 10 mM 3-~2-iodoacetamido)PROXYL, 0.15
mM dodecamer, pH 8.0 (phosphate) at 50C in a solution
containing 4% DMP. Similar conditions were employed to label
the eicosomer.
b) Attachment~of-a ~-1065 drug analogue: a
derivative of the dihydropyrroloindole subunit:
The reaction to produce the compound in Fig.
5b was conducted as described above using the following
3 specific conditions: 5 mM dihydropyrroloindole deriva~ive,
., ,
~5
_. .
~37-
0.07 mM dodecamer, p~ 8.0 ~Tris) at 50C in a solution
1 containing 60% DMF. This xeaction required 48 h at 50C or
80 h at 25C at which timç it was 70-80~ complete. Similar
conditions were employed to label the eicosomer.
c) Attachment of a sulfonamide-linked dansyl
fluorophore:
The reaction to produce the compound in FigO
5c was conducted as described above using the following
specific conditions: 12 mM N-dansylaziridine, 0.34 mM
dodecamer, pl~ 8.0 (phosphate) at 2SC in a solution
containing 50~ acetonitrile. Similar condiitons were
employed to label the eicosomer.
At 50C, HPLC analysis of the dansylaziridine
reaction indicated the presence of minor products, suggesting ~-
some nonspecific reaction with the DNA. Labeling conducted
at 25C (pH 8.0) proceeded more slowly, but did not indicate
the presence of any species other than the desired product
and starting materials. ~lowever, the possibility of some ;;
nonspecific modification of the DNA even at 25C can not be
excluded.
d) Attachment of an N-linked dansyl fluorophore:
The reaction to produce the compound in Fig. '~ ~
5d was conducted as aescribed above using the following ~ -
specific conditions: 10 mM 1,5-I-AEDANS, 0.80 mM dodecamer, ~ -
pH 6.0 lphosphate) at 50C in a solution containing 25~ DMF.
Similar conditions were employed to label the eicosomer.
12) StabilitY and properties of phosphorothioate
triesters from examples 10 and 11
The unlabeled dodecamer helix, dlCGCA(s)AAAAAGCG~
dlCGGTTTTTTGCG], exhibited a Tm of 55C, and this was
3 indistinguishable from the Tm values obtained for the PROXYL-
labeled (a in Figure 5) or drug-labeled (b in Figure 5)
' ' ' '.~'' .'' ~
~
:
-38- .
helices. The T value for the self-complementary eicosomer,
1 dlCGTACTAGTT(s)AACT~GTACG]2 with two labels was also largely
unchanged (68.5C) in comparison to the unlabele~ fragment
(Tm = 67C)-
The hydrolytic stabili-ty of the phosophorothioate
triesters is an important practical consideration for the
value of such derivatives in many studies. ~ydrolysis of the
triesters proceeded by desulfurization (monitored by HPLC and
confirmed by comparison with authentic standards). No
detectable cleavage of the oligodeoxynucleotide at the point
of attachment was observed. This agrees with the results of
ethylated or hydroxyethylated derivatives, which result in
primarily desulfurization and only very minor amounts of
chain cleavage.
Less than 5~ of the Tp(s~T triester carrying the
PROXYL spin label was hydrolyzed after 24 h at pH 7. At pH 8
this increased to 28%, and at pH 10 the triester was
completely hydrolyzed within 11 h. With longer fragments,
the hydrolytic stability of the triester increased [the
labeled dodecamer was hydrolyzed < 1~, 30~, and 99~ at p~
values 7, 8, and 10, respectively; the values for the
eicosomer were <1%, 2%, and 63%(24 h)]. The triester
prepared from a ~-bromo- ~, fi-unsaturated carbonyl (b in
Figure 5) exhibited stability similar to that of the
PROXYL-labeled derivatives while that resulting from reaction
with the aziridinyl sulfonamide (c in Figure 5) was more
stable lthe Tp(s)T-labeled triester was hydrolyzed <1~ (pH
7), 5~(pH 8), and 34% (pH 10) after 24 h at ambient
temperature].
It is noteworthy that the triester produced from
3 1,5-I-AEDANS and Tp(s)T was significantly less stable than
the PROXYL-labeled derivative although the triesters ~ormea
: -39- :
' .
both resulted from iodoacetamides. The ~EDANS-labeled dimer
l eY~hibited l9~i (p~ 7) and 88~i (p~ 8) hydrolysis (24 h); it was
completely hydrolyzed within 2 h at pH lO. However, the
AED~NS-labeled dodecamer (d in Figure 5) exhibited only
1~, 49%, and 99% hydrolysis at the same respective pH values
~24 h).
An additional dodecamer was labeled with the
bromoacetamideo derivative i. Although the three `
acetamido-linked adducts are similar in structure, that
prepared from i proved to be more stable than either a or d
(~igure 1) (only 13~i of the triester formed from i was
hydroly2ed after 24 h at pH 8.0).
Derivative i:
CH~OC~ ~c~
'
~..
: .
3
:'. :' .. .' ..
: .
... . , , .... , .. ~ . ., ,.. , . . ` . .... ~ . . ... .. .. .. .
