Note: Descriptions are shown in the official language in which they were submitted.
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TAGGED MICROPARTICLE COMPOSITIONS AND METHODS
This invention relates to methods and compositions for detecting and/or
measuring
multiple analytes in a sample using microparticle-bound binding pairs with
releasable
molecular tags.
BACKGROUND OF THE INVENTION
The development of several powerful technologies for genome-wide and
proteome-wide expression measurements has created an opportunity to study and
understand the coordinated activities of large sets of, if not all, an
organism's genes in
response to a wide variety of conditions and stimuli, e.g. DeRisi et al,
Science, 278: 680-
686 (1997); Wodicka et al, Nature Biotechnology, 15: 1359-1367 (1997);
Velculescu et al,
Cell, 243-251 (1997); Brenner et al, Nature Biotechnology, 18: 630-634 (2000);
McDonald et al, Disease Markers, 18: 99-105 (2002); Patterson, Bioinformatics,
18 (Suppl
2): 5181 (2002). Studies using these technologies have shown that reduced
subsets of
genes appear to be co-regulated to perform particular functions and that
subsets of
expressed genes and proteins can be used to classify cells phenotypically,
e.g. Shiffman
and Porter, Current Opinion in Biotechnology, 11: 598-601 (2000); Afshari et
al, Nature,
403: 503-511 (2000); Golub et al, Science, 286: 531-537 (1999); van't Veer et
al, Nature,
415: 530-536 (2002); and the like.
An area of interest in drug development is the expression profiles of genes
and
proteins involved with the metabolism or toxic effects of xenobiotic
compounds. Several
studies have shown that sets of several tens of genes can serve as indicators
of compound
toxicity, e.g. Thomas et al, Molecular Pharmacology, 60: 1189-1194 (2001);
Waring et al,
Toxicology Letters, 120: 359-368 (2001); Longueville et al, Biochem.
Pharmacology, 64:
137-149 (2002); and the like. Similarly, in the area of cancer diagnostics and
prognosis,
the differential expression of sets of a few tens of genes or proteins has
been shown
frequently to have strong correlations with the progression and prognosis of a
cancer.
Accordingly, there is an interest in technologies that provide convenient and
accurate measurements of multiple expressed genes in a single assay, either at
the
messenger RNA level or the protein level, or both. Current approaches to such
measurements include multiplexed polymerase chain reaction (PCR), spotted and
synthesized DNA microarrays, color-coded microbeads, and single-analyte
assays, such as
enzyme-linked immunosorbant assays (ELISAs) or Taqman-based PCR, used with
robotics apparatus, e.g. Longueville et al (cited above); Elnifro et al,
Clinical
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Microbiology Reviews, 13: 559-570 (2000); Chen et al, Genome Research, 10: 549-
557
(2000); and the like. Unfortunately, none of the approaches provides a
completely
satisfactory solution for the desired measurements for several reasons
including difficulty
in automating, reagent usage, sensitivity, consistency of results, and so on,
e.g. Elnifro et
al (cited above); Hess et al, Trends in Biotechnology, 19: 463-468 (2001);
King and Sinha,
JAMA, 286: 2280-2288 (2001).
In view of the above, the availability of a convenient and cost effective
technique
for measuring the presence or absence or quantities of multiple analytes, such
as gene
expression products, in a single assay reaction would advance the art in many
fields where
such measurements are becoming increasingly important, including life science
research,
medical research and diagnostics, drug discovery, genetic identification,
animal and plant
science, and the like.
Summary of the Invention
The present invention is directed to methods and compositions for determining
the
presence and/or amount of one or more target analytes in a sample using
microparticles
derivatized with a plurality of releasable molecular tags that have distinct
separation
characteristics.
In one aspect, the invention includes a composition comprising a mixture of
more
than one microparticle, each microparticle in the mixture having molecular
tags attached
by cleavable linkages such that different molecular tags are attached to
different
microparticles, the molecular tags being selected from a plurality of
molecular tags such
that each molecular tag of the plurality has one or more physical and/or
optical
characteristics distinct from those of the other molecular tags of the
plurality so that each
molecular tag forms a distinguishable peak upon cleavage and separation based
on such
one or more physical and/or optical characteristics.
In another aspect, the invention includes a composition comprising: (i) a
mixture
of more than one microparticle, each microparticle having a surface with
binding moieties
and molecular tags attached, each binding moiety being specific for a
predetermined
analyte and the molecular tags being attached by cleavable linkages, such that
different
pairs of molecular tags and binding moieties are attached to different
microparticles, the
molecular tags being selected from a plurality of molecular tags such that
each molecular
tag of the plurality has one or more physical and/or optical characteristics
distinct from
those of the other molecular tags of the plurality so that each molecular tag
forms a
distinguishable peak upon cleavage and separation based on such one or more
physical
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and/or optical characteristics; and (ii) a second binding composition
comprising at least
one binding moiety specific for each of the predetermined analytes, each such
binding
moiety having a sensitizer for generating an active species capable of
cleaving the
cleavable linkage.
In yet another aspect, the invention provides methods of using the above
compositons to determine the presence or absence or quantities of multiple
target analytes
in a sample. Such methods comprise the steps of (i) combining with a sample a
mixture of
microparticles and a second binding composition such that in the presence of a
target
analyte a complex is formed between the target analyte and at least one first
binding
moiety and at least one second binding moiety specific therefor, and such that
the
sensitizer of the second binding moiety causes the generation of an active
species and the
cleavage of one or more cleavable linkages to release one or more molecular
tags from at
least one microparticle; and (ii) separating and identifying the released
molecular tags by
the one or more physical and/or optical characteristics to determine the
target analytes in
the sample.
In another aspect, the present invention includes kits for performing the
methods of
the invention, such kits comprising a mixture of microparticles for detecting
or measuring
the quantities of each of one or more target analytes. Such kits further
comprise a
cleavage agent and appropriate buffers for cleaving the cleavable linkages
between
molecular tags and binding moieties that form stable complexes with a target
analyte.
Such kit futher comprise separation standards for aiding in making
quantitative
measurements of the separated molecular tags.
In another aspect, compositions of the invention may be used as identifying
agents,
or taggants, for monitoring or tracking materials or products including
natural resources
such as animals, plants, oil, minerals, and water; chemicals such as drugs,
solvents,
petroleum products, and explosives; commercial by-products including
pollutants such as
radioactive or other hazardous waste; and articles of manufacture such as
guns,
typewriters, automobiles and automobile parts. In this aspect, a composition
of the
invention comprising a plurality of microparticles each coated with a
different kind of
releaseable molecular tag is incorporated or added to a material to be tracked
and/or
identified.
The present invention provides a detection and signal generation means with
several advantages for multiplexed measurements of target analytes, including
but not
limited to (1) the detection and/or measurement of molecular tags that are
separated from
the assay mixture provide greatly reduced background and a significant gain in
sensitivity;
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(2) the use of tags that are specially designed for ease of separation thereby
providing
convenient multiplexing capability; (3) providing greater sensitivity by
attaching analyte
binding moieties to solid phase supports, and (4) providing increased numbers
of
molecular tags released per analyte detection event by use of microparticle
supports.
Brief Description of the Drawings
Figure 1 illustrates a method of using the compositions of the invention to
detect
multiple analytes.
Figure 2 illustrates one exemplary synthetic approach starting with
commercially
available 6-carboxy fluorescein, where the phenolic hydroxyl groups are
protected using
an anhydride. Upon standard extractive workup, a 95% yield of product is
obtained. This
material is phosphitylated to generate the phosphoramidite monomer.
Figure 3 illustrates the use of a symmetrical bis-amino alcohol linker as the
amino
alcohol with the second amine then coupled with a multitude of carboxylic acid
derivatives.
Figure 4 shows the structure of several benzoic acid derivatives that can
serve as
mobility modifiers.
Figure 5 illustrates the use of an alternative strategy that uses 5-
aminofluorescein
as starting material and the same series of steps to convert it to its
protected
phosphoramidite monomer.
Figure 6 illustrates several amino alcohols and diacid dichlorides that can be
assembled into mobility modifiers in the synthesis of molecular tags.
Figures 7 A-F illustrate oxidation-labile linkages and their respective
cleavage
reactions mediated by singlet oxygen.
Figures 8 A-B illustrate the general methodology for conjugation of an e-tag
moiety to an antibody to form an e-tag probe, and the reaction of the
resulting probe with
singlet oxygen to produce a sulfinic acid moiety as the released molecular
tag.
Figures 9A-J show the structures of e-tag moieties that have been designed and
synthesized. (Prol is commercially available from Molecular Probes, Inc.)
Figures 10 A-I illustrate the chemistries of synthesis of the e-tag moieties
illustrated in Figure 9.
Definitions
"Analyte" means a substance, compound, or component in a sample whose
presence or absence is to be detected or whose quantity is to be measured.
Analytes
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include but are not limited to peptides, proteins, polynucleotides,
polypeptides,
oligonucleotides, organic molecules, haptens, epitopes, parts of biological
cells,
posttranslational modifications of proteins, receptors, complex sugars,
vitamins,
hormones, and the like. There may be more than one analyte associated with a
single
molecular entity, e.g. different phosphorylation sites on the same protein.
"Antibody" means an immunoglobulin that specifically binds to, and is thereby
defined as complementary with, a particular spatial and polar organization of
another
molecule. The antibody can be monoclonal or polyclonal and can be prepared by
techniques that are well known in the art such as immunization of a host and
collection of
sera (polyclonal) or by preparing continuous hybrid cell lines and collecting
the secreted
protein (monoclonal), or by cloning and expressing nucleotide sequences or
mutagenized
versions thereof coding at least for the amino acid sequences required for
specific binding
of natural antibodies. Antibodies may include a complete immunoglobulin or
fragment
thereof, which immunoglobulins include the various classes and isotypes, such
as IgA,
IgD, IgE, IgGl, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include
Fab,
Fv and F(ab)2, Fab', and the like. In addition, aggregates, polymers, and
conjugates of
immunoglobulins or their fragments can be used where appropriate so long as
binding
affinity for a particular polypeptide is maintained.
"Antibody binding composition" means a molecule or a complex of molecules that
comprise one or more antibodies and derives its binding specificity from an
antibody.
Antibody binding compositions include, but are not limited to, antibody pairs
in which a
first antibody binds specifically to a target molecule and a second antibody
binds
specifically to a constant region of the first antibody; a biotinylated
antibody that binds
specifically to a target molecule and streptavidin derivatized with moieties
such as
molecular tags or photosensitizers; antibodies specific for a target molecule
and
conjugated to a polymer, such as dextran, which, in turn, is derivatized with
moieties such
as molecular tags or photosensitizers; antibodies specific for a target
molecule and
conjugated to a bead, or microbead, or other solid phase support, which, in
turn, is
derivatized with moieties such as molecular tags or photosensitizers, or
polymers
containing the latter.
"Capillary-sized" in reference to a separation column means a capillary tube
or
channel in a plate or microfluidics device, where the diameter or largest
dimension of the
separation column is between about 25-500 microns, allowing efficient heat
dissipation
throughout the separation medium, with consequently low thermal convection
within the
medium.
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"Chromatography" or "chromatographic separation" as used herein means or
refers
to a method of analysis in which the flow of a mobile phase, usually a liquid,
containing a
mixture of compounds, e.g. including analytes, promotes the separation of such
compounds by a differential distribution between the mobile phase and a
stationary phase,
usually a solid.
A "separation profile" in reference to the separation of molecular tags means
a
chart, graph, curve, bar graph, or other representation of signal intensity
data versus time,
or other variable related to time, that provides a readout, or measure, of the
number of
molecular tags of each type produced in an assay. A separation profile may be
an
electropherogram, a chromatogram, an electrochromatogram, or like graphical
representations of data depending on the separation technique employed. A
"peak" or a
"band" or a "zone" in reference to a separation profile means a region where a
separated
compound is concentrated. There may be multiple separation profiles for a
single assay if,
for example, different molecular tags have different fluorescent labels having
distinct
emission spectra and data is collected and recorded at multiple wavelengths.
"Microparticles" as used herein means solid phase particulate supports to
which
molecular tags can be covalently attached and/or to which binding moieties,
such as
antibodies, can be covalently, or in some embodiments, non-covalently
attached. Such
particulate supports are small-sized to provide large surface area-to-volume
ratios and are
monodisperse under assay conditions. Microparticles may vary widely in size,
shape, and
composition. In one aspect, microparticles are uniformly sized microspheres
having a
diameter in the range of from a few tens of manometers to several tens of
micrometers, e.g.
in the range of from 20 nm to 50 gm, or from 0.5 gm to 25 gm, or from 1 gm to
10 gm.
Typically, diameters of populations of uniformly sized microparticles have a
coeffient of
variation of less than ten percent. In one aspect, microparticles are are
mechanically rigid
and substantially non-swellable under assay conditions. In another aspect,
microparticles
are non-porous. Microparticles may be made from a variety of materials
including
polymers, such as polystyrene, polymethylacrylate, glycidal methacrylate,
nylon, or the
like, or minerals, such as silica, alumina, or the like. In another aspect,
microparticles
include colloidal particles, dentrimers, liposomes, and other non-rigid
particle-like
supports, e.g. Hermanson, Bioconjugate Techniques (Academic Press, New York,
1996);
Frechet, Science, 263: 1710-1705 (1994); Klajnert et al, Acta. Biochim. Pol.,
48: 199-208
(2001); Singh et al, Clinical Chemistry, 40: 1845-1849 (1994); and the like.
Microparticles further include magnetic microbeads, e.g. DynadeadsTm, as
disclosed in
U.S. patents 4,186,120; 4,530,956; 4,563,510; and 4,654,267.
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"Specific" or "specificity" in reference to the binding of one molecule to
another
molecule, such as a probe for a target polynucleotide, means the recognition,
contact, and
formation of a stable complex between the two molecules, together with
substantially less
recognition, contact, or complex formation of that molecule with other
molecules. In one
aspect, "specific" in reference to the binding of a first molecule to a second
molecule
means that to the extent the first molecule recognizes and forms a complex
with another
molecules in a reaction or sample, it forms the largest number of the
complexes with the
second molecule. Preferably, this largest number is at least fifty percent.
Generally,
molecules involved in a specific binding event have areas on their surfaces or
in cavities
giving rise to specific recognition between the molecules binding to each
other. Examples
of specific binding include antibody-antigen interactions, enzyme-substrate
interactions,
formation of duplexes or triplexes among polynucleotides and/or
oligonucleotides,
receptor-ligand interactions, and the like. As used herein, "contact" in
reference to
specificity or specific binding means two molecules are close enough that weak
noncovalent chemical interactions, such as Van der Waal forces, hydrogen
bonding, ionic
and hydrophobic interactions, and the like, dominate the interaction of the
molecules. As
used herein, "stable complex" in reference to two or more molecules means that
such
molecules form noncovalently linked aggregates, e.g. by specific binding, that
under assay
conditions are thermodynamically more favorable than a non-aggregated state.
As used herein, the term "spectrally resolvable" in reference to a plurality
of
fluorescent labels means that the fluorescent emission bands of the labels are
sufficiently
distinct, i.e. sufficiently non-overlapping, that molecular tags to which the
respective
labels are attached can be distinguished on the basis of the fluorescent
signal generated by
the respective labels by standard photodetection systems, e.g. employing a
system of band
pass filters and photomultiplier tubes, or the like, as exemplified by the
systems described
in U.S. Pat. Nos. 4,230,558; 4,811,218, or the like, or in Wheeless et al,
pgs. 21-76, in
Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New York,
1985).
"Oligonucleotide" as used herein means linear oligomers of natural or modified
nucleosidic monomers linked by phosphodiester bonds or analogs thereof.
Oligonucleotides include deoxyribonucleosides, ribonucleosides, anomeric forms
thereof, peptide nucleic acids (PNAs), and the like, capable of specifically
binding to a
target polynucleotide by way of a regular pattern of monomer-to-monomer
interactions,
such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse
Hoogsteen types of base pairing, or the like. Usually monomers are linked by
phosphodiester bonds or analogs thereof to form oligonucleotides ranging in
size from a
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few monomeric units, e.g. 3-4, to several tens of monomeric units, e.g. 40-60.
Whenever an oligonucleotide is represented by a sequence of letters, such as
"ATGCCTG," it will be understood that the nucleotides are in 5'0 3' order from
left to
right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G"
denotes
deoxyguanosine, "T" denotes deoxythymidine, and "U" denotes the
ribonucleoside,
uridine, unless otherwise noted. Usually oligonucleotides of the invention
comprise the
four natural deoxynucleotides; however, they may also comprise ribonucleosides
or non-
natural nucleotide analogs. It is clear to those skilled in the art when
oligonucleotides
having natural or non-natural nucleotides may be employed in the invention.
For
example, where processing by an enzyme is called for, usually oligonucleotides
consisting of natural nucleotides are required. Likewise, where an enzyme has
specific
oligonucleotide or polynucleotide substrate requirements for activity, e.g.
single stranded
DNA, RNA/DNA duplex, or the like, then selection of appropriate composition
for the
oligonucleotide or polynucleotide substrates is well within the knowledge of
one of
ordinary skill, especially with guidance from treatises, such as Sambrook et
al,
Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York,
1989),
and like references.
"Perfectly matched" in reference to a duplex means that the poly- or
oligonucleotide strands making up the duplex form a double stranded structure
with one
another such that every nucleotide in each strand undergoes Watson-Crick
basepairing
with a nucleotide in the other strand. The term also comprehends the pairing
of
nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine
bases, and
the like, that may be employed. In reference to a triplex, the term means that
the triplex
consists of a perfectly matched duplex and a third strand in which every
nucleotide
undergoes Hoogsteen or reverse Hoogsteen association with a basepair of the
perfectly
matched duplex. Conversely, a "mismatch" in a duplex between a tag and an
oligonucleotide means that a pair or triplet of nucleotides in the duplex or
triplex fails to
undergo Watson-Crick and/or Hoogsteen and/or reverse Hoogsteen bonding. As
used
herein, "stable duplex" between complementary oligonucleotides or
polynucleotides
means that a significant fraction of such compounds are in duplex or double
stranded
form with one another as opposed to single stranded form. Preferably, such
significant
fraction is at least ten percent of the strand in lower concentration, and
more preferably,
thirty percent.
As used herein, "nucleoside" includes the natural nucleosides, including 2'-
deoxy
and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA
Replication, 2nd
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Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleosides
includes
synthetic nucleosides having modified base moieties and/or modified sugar
moieties, e.g.
described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman
and
Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the only
proviso that
they are capable of specific hybridization. Such analogs include synthetic
nucleosides
designed to enhance binding properties, reduce complexity, increase
specificity, and the
like.
A probe is "capable of hybridizing" to a nucleic acid sequence if at least one
region
of the probe shares substantial sequence identity with at least one region of
the
complement of the nucleic acid sequence. "Substantial sequence identity" is a
sequence
identity of at least about 80%, preferably at least about 85%, more preferably
at least about
90%, and most preferably 100%. It should be noted that for the purpose of
determining
sequence identity of a DNA sequence and a RNA sequence, U and T are considered
the
same nucleotide. For example, a probe comprising the sequence ATCAGC is
capable of
hybridizing to a target RNA sequence comprising the sequence GCUGAU.
"Normal phase" in reference to chromatographic separation means that
separation
operates on the basis of hydrophilicity and lipophilicity by using a polar
stationary phase
and a less polar mobile phase. Thus hydrophobic compounds elute more quickly
than do
hydrophilic compounds. Exemplary groups on a solid phase for normal phase
chromatography are amine (-NH2) and hydroxyl (-OH) groups.
"Reverse phase" in reference to chromatographic separation means that
separation
operates on the basis of hydrophilicity and lipophilicity. The stationary
phase usually
consists of silica based packings with n-alkyl chains or phenyl groups
covalently bound.
For example, C-8 signifies an octyl chain and C-18 an octadecyl ligand in the
matrix. The
more hydrophobic the matrix on each ligand, the greater is the tendancy of the
column to
retain hydrophobic moieties. Thus hydrophilic compounds elute more quickly
than do
hydrophobic compounds.
"Ion-exchange" in reference to chromatographic separation means that
separation
operates on the basis of selective exchange of ions in the sample with
counterions in the
stationary phase. Ion exchange is performed with columns containing charge-
bearing
functional groups attached to a polymer matrix. The functional ions are
permanently
bonded to the column and each has a counterion attached. The sample is
retained by
replacing the counterions of the stationary phase with its own ions. The
sample is eluted
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from the column by changing the properties of the mobile phase do that the
mobile phase
will now displace the sample ions from the stationary phase, (ie. changing the
pH).
As used herein, the term "Tin" is used in reference to the "melting
temperature."
The melting temperature is the temperature at which a population of double-
stranded
nucleic acid molecules becomes half dissociated into single strands. Several
equations for
calculating the Tm of nucleic acids are well known in the art. As indicated by
standard
references, a simple estimate of the T,, value may be calculated by the
equation. Tm =
81.5 + 0.4 1 (% G + C), when a nucleic acid is in aqueous solution at I M
NaC1(see e.g.,
Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid
Hybridization
(1985). Other references (e.g., Allawi, H.T. & SantaLucia, J., Jr.,
Biochemistry 36, 10581-
94 (1997)) include alternative methods of computation which take structural
and
environmental, as well as sequence characteristics into account for the
calculation of Tm.
The term "sample" in the present specification and claims is used in a broad
sense.
On the one hand it is meant to include a specimen or culture (e.g.,
microbiological
cultures). On the other hand, it is meant to include both biological and
environmental
samples. A sample may include a specimen of synthetic origin. Biological
samples may
be animal, including human, fluid, solid (e.g., stool) or tissue, as well as
liquid and solid
food and feed products and ingredients such as dairy items, vegetables, meat
and meat by-
products, and waste. Biological samples may include materials taken from a
patient
including, but not limited to cultures, blood, saliva, cerebral spinal fluid,
pleural fluid,
milk, lymph, sputum, semen, needle aspirates, and the like. Biological samples
may be
obtained from all of the various families of domestic animals, as well as
feral or wild
animals, including, but not limited to, such animals as ungulates, bear, fish,
rodents, etc.
Environmental samples include environmental material such as surface matter,
soil, water
and industrial samples, as well as samples obtained from food and dairy
processing
instruments, apparatus, equipment, utensils, disposable and non-disposable
items. These
examples are not to be construed as limiting the sample types applicable to
the present
invention.
The term "isothermal" in reference to assay conditions means a uniform or
constant temperature at which the cleavage of the binding compound in
accordance with
the present invention is carried out. The temperature is chosen so that the
duplex formed
by hybridizing the probes to a polynucleotide with a target polynucleotide
sequence is in
equilibrium with the free or unhybridized probes and free or unhybridized
target
polynucleotide sequence, a condition that is otherwise referred to herein as
"reversibly
hybridizing" the probe with a polynucleotide. Normally, at least 1%,
preferably 20 to 80%,
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usually less than 95% of the polynucleotide is hybridized to the probe under
the isothermal
conditions. Accordingly, under isothermal conditions there are molecules of
polynucleotide that are hybridized with the probes, or portions thereof, and
are in dynamic
equilibrium with molecules that are not hybridized with the probes. Some
fluctuation of
the temperature may occur and still achieve the benefits of the present
invention. The
fluctuation generally is not necessary for carrying out the methods of the
present invention
and usually offer no substantial improvement. Accordingly, the term
"isothermal" includes
the use of a fluctuating temperature, particularly random or uncontrolled
fluctuations in
temperature, but specifically excludes the type of fluctuation in temperature
referred to as
thermal cycling, which is employed in some known amplification procedures,
e.g.,
polymerase chain reaction.
As used herein, the term "kit" refers to any delivery system for delivering
materials. In the context of reaction assays, such delivery systems include
systems that
allow for the storage, transport, or delivery of reaction reagents (e.g.,
probes, enzymes, etc.
in the appropriate containers) and/or supporting materials (e.g., buffers,
written
instructions for performing the assay etc.) from one location to another. For
example, kits
include one or more enclosures (e.g., boxes) containing the relevant reaction
reagents
and/or supporting materials. Such contents may be delivered to the intended
recipient
together or separately. For example, a first container may contain an enzyme
for use in an
assay, while a second container contains probes.
"Polypeptide" refers to a class of compounds composed of amino acid residues
chemically bonded together by amide linkages with elimination of water between
the
carboxy group of one amino acid and the amino group of another amino acid. A
polypeptide is a polymer of amino acid residues, which may contain a large
number of
such residues. Peptides are similar to polypeptides, except that, generally,
they are
comprised of a lesser number of amino acids. Peptides are sometimes referred
to as
oligopeptides. There is no clear-cut distinction between polypeptides and
peptides. For
convenience, in this disclosure and claims, the term "polypeptide" will be
used to refer
generally to peptides and polypeptides. The amino acid residues may be natural
or
synthetic.
"Protein" refers to a polypeptide, usually synthesized by a biological cell,
folded
into a defined three-dimensional structure. Proteins are generally from about
5,000 to
about 5,000,000 or more in molecular weight, more usually from about 5,000 to
about
1,000,000 molecular weight, and may include posttranslational modifications,
such
acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of
flavin,
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covalent attachment of a heme moiety, covalent attachment of a nucleotide or
nucleotide
derivative, covalent attachment of a lipid or lipid derivative, covalent
attachment of
phosphotidylinositol, cross-linking, cyclization, disulfide bond formation,
demethylation,
formation of covalent cross-links, formation of cystine, formation of
pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation,
iodination, methylation, myristoylation, oxidation, phosphorylation,
prenylation,
racemization, selenoylation, sulfation, and ubiquitination, e.g. Wold, F.,
Post-translational
Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Post-
translational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983.
Proteins
include, by way of illustration and not limitation, cytokines or interleukins,
enzymes such
as, e.g., kinases, proteases, galactosidases and so forth, protamines,
histones, albumins,
immunoglobulins, scleroproteins, phosphoproteins, mucoproteins,
chromoproteins,
lipoproteins, nucleoproteins, glycoproteins, T-cell receptors, proteoglycans,
unclassified
proteins, e.g., somatotropin, prolactin, insulin, pepsin, proteins found in
human plasma,
blood clotting factors, blood typing factors, protein hormones, cancer
antigens, tissue
specific antigens, peptide hormones, nutritional markers, tissue specific
antigens, and
synthetic peptides.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the present invention is directed to methods and compositions
for
determining the presence and/or amount of one or more analytes in a sample by
releasing
microparticle-bound molecular tags as the result of a binding reaction. The
binding
reaction is between analytes and binding moieties operationally associated
with the
microparticle-bound molecular tags. Usually, a binding moiety is operationally
associated
with a particular species of molecular tag by being attached to the same
microparticle. In
one embodiment, such a binding moiety binds to an analyte together with a
second binding
moiety having a sensitizer attached. Such a binding event brings molecular
tags on the
microparticle into the effective proximity of the sensitizer. When a target
analyte is a
polynucleotide, both binding moieties comprise oligonucleotides specific for
such
polynucleotide. When a target analyte is a protein, or other analyte having a
molecular
weight greater than about 4-6000 daltons, the binding moieties may both be
antibody
binding compositions. When a target analyte is a surface membrane receptor of
a
biological cell, the binding moiety attached to the microparticle is a ligand
or candidate
ligand for the receptor. In the later embodiment, a sensitizer may be a lipid-
photosensitizer conjugate disposed in the same membrane as the receptor.
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Methods using compositions of the invention may be operated in either a
homogeneous or a heterogeneous format. One embodiment of a homogeneous format
is
illustrated in Figure 1. Mixture (100) of microparticles derivatized with
molecular tags
("T1", "T2", "T3") (101) and first binding moieties (`B1", "B2", `B3") (103)
are combined
(106) with target analytes ("Al", "A2", "A3") (102) and second binding
composition
("Bk'-PS") (104) comprising conjugates of first binding moieties (105) and
photosensitizers ("PS") (107). Assay conditions are selected so that complexes
(108) form
among the first and second binding moieties and target analytes. After an
incubation
period to allow such complexes to form, the assay mixture is subjected to
illumination
(110) by light that excites photosensitizers (107) so that they react with
molecular oxygen
to produce singlet oxygen (111) that, in turn, reacts with cleavable linkages
("L") (113) to
release molecular tag (101). Such incubation period can vary from a few
minutes, e.g. 15
min, to several hours, e.g. 2 hours, 4 hours, 8 hours, to overnight, depending
on the nature
of the binding moieties and analytes. Released molecular tags are then
separated from the
assay mixture and from one another (114) to produce separation profile (116)
from which
the presence, absence, and/or quantities of target analytes are determined.
A heterogeneous format may be desirable whenever sensitizers are either long-
lived molecular entities or act throughout the entire reaction mixture rather
than in a
limited region, e.g. light when the cleavable linkages are photo-cleavable,
acid when the
cleavable linkages are acid labile, etc. In such embodiments, after formation,
microparticles containing complexes (108) are separated from microparticles
lacking such
complexes, after which they treated as described above.
As described more fully below, target analytes are determined by separation
and
identification of the released molecular tags. A wide variey of separation
techniques may
be employed that can distinguish molecules based on one or more physical,
chemical, or
optical differences among molecules being separated including but not limited
to
electrophoretic mobility, molecular weight, shape, solubility, pKa,
hydrophobicity, charge,
charge/mass ratio, polarity, or the like. In one aspect, molecular tags in a
plurality differ
in electrophoretic mobility and optical detection characteristics and are
separated by
electrophoresis. In another aspect, molecular tags in a plurality differ in
molecular weight,
shape, solubility, pKa, hydrophobicity, charge, polarity, and are separated by
normal phase
or reverse phase HPLC, ion exchange HPLC, capillary electrochromatography, or
like
technique.
Another aspect of the present invention is providing sets of molecular tags
that
may be separated into distinct bands or peaks by the separation technique
employed after
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they are released from microparticles. Molecular tags within a set may be
chemically
diverse; however, for convenience, sets of molecular tags are usually
chemically related.
For example, they may all be peptides, or they may consist of different
combinations of
the same basic building blocks or monomers, or they may be synthesized using
the same
basic scaffold with different substituent groups for imparting different
separation
characteristics, as described more fully below. The number of molecular tags
in a
plurality may vary depending on several factors including the mode of
separation
employed, the labels used on the molecular tags for detection, the sensitivity
of the
binding moieties, the efficiency with which the cleavable linkages are
cleaved, and the
like. In one aspect, the number of molecular tags in a plurality ranges from 2
to several
hundred, e.g. 200. In other aspects, the size of the plurality may be in the
range of from 5
to 100, and more usually, in the range of from 5 to 50, or in the range of
from 5 to 30, or in
the range of from 5 to 20.
Microparticle Supports for Molecular Tags and Binding Moieties
In one aspect, compositions of the invention comprise mixtures of
microparticles
having molecular tags covalently attached by way of cleavable linkages.
Usually, each
microparticle has only one kind of molecular tag attached, so that different
molecular
tags are attached to different microparticles; however, in some embodiments,
two or
more different kinds of molecular tag may be attached to the same
microparticle.
Compositions of the invention also comprise mixtures of microparticles having
both
molecular tags covalently attached by cleavage linkages and binding moieties
attached
either covalently or non-covalently. As above, usually when both molecular
tags and
binding moieties are attached to microparticles, only pairs of a single kind
of molecular
tag and a single kind of binding moiety are attached to the same
microparticle; thus,
different molecular tag-binding moiety pairs are attached to different
microparticles.
The ratio of molecular tags to binding moieties on a microparticle is readily
varied by
one of ordinary skill in the art and depends on the requirements of a
particular assay.
Other embodiments of the invention include combinations of multiple molecular
tags
and/or multiple binding moieties on single microparticles. Making compositions
of the
invention based on such embodiments are design choices available to one of
ordinary
skill in the art depending on the requirements of a particular assay.
Microparticles for use with the invention may be derivatized with many
different
functional groups that permit the covalent attachment of molecular tags and/or
binding
moieties. Such functional groups include but are not limited to amino,
carboxyl,
hydroxyl, hydrazide, chloromethyl, silanol, and the like. Molecular tags are
attached by
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reacting a functional group on the microparticles with a complementary
functionality on
a precursor of the molecular tag. For example, carboxyl-modified
microparticles may be
coupled to an amino complementary functionality of a molecular tag via a water
soluble
carbodiimide cross-linking agent, amino-modified microparticles may be coupled
to an
amino complementary functionality of a molecular tag via a glutaraldehyde
cross-linking
agent, hydroxyl-modified microparticles may be coupled to an amino
complementary
functionality of a molecular tag via cyanogens bromide, and the like.
Extensive
guidance can be found in the literature for covalently linking molecular tags
and binding
moieties, such as antibodies, to microparticles, e.g. Bangs Labortories
(Fishers, IN)
Technical Note 205 (30 March 2002); Hermanson, Bioconjugate Techniques,
(Academic
Press, New York, 1996), and the like.
In one aspect, carboxyl-derivatized microparticles (many sizes and varieties
available from Bangs Laboratories, Fishers, IN) are treated with a polymeric
carrier
molecules, such as polylysine, aminodextran, or the like, to create an amino
derivatized
surface for reacting with complementary functionalities of molecular tags
and/or binding
moieties. Preferably, carboxyl-derivatized microparticles are treated with
aminodextran
to prepare their surfaces for reaction with amine-reactive groups on molecular
tags
and/or binding moieties, e.g. as disclosed by Pollner, U.S. patent 6,346,384;
and Patel,
International patent publication WO 01/90399.
In one aspect of the invention, molecular tags and antibodies are attached to
amino derivatized microparticles, e.g. hydroxypropylaminodextran coated
microspheres,
as described below. Preferably, attachment is done in a two step process. In
the first
stage, a mixture of NHS-ester of a molecular tag and an NHS-ester cross-
linking agent
are reacted with the free amines on the microparticle. The proportion of each
compound
in the mixture depends on several factors including the proportion of
molecular tag to
antibody that is desired on the surface of the microparticle, relative
reaction rates,
efficiency of the reaction of the second stage of the process, and the like.
Numerous
suitable cross linking agents may be used including but not limited to
succinimidyl 6-
((iodoacetyl)amino)hexanoate (SIAX), succinimidyl 6-[6-(((iodoacetyl)amino)-
hexanoyl)amino]hexanoate (SIAXX), N-succinimidyl 3-(2-pyridyldithio)propionate
(SPDP), and like compounds well known in the art, e.g. Hermanson (cited
above);
Haugland, Handbook of Fluorescent Probes and Research Products, Ninth Edition
(Molecular Probes, Eugene, OR, 2002). In the second stage, an appropriately
derivatized antibody binding moiety is reacted with the other functionality of
the cross-
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linking agent, such as the iodoacetyl group of SIAX. By way of example, for
such a
reaction, an amino group of an antibody binding moiety may be converted to a
sulfhydryl group by treatment with N-succinimidyl-S-acetylthioacetate (SATA),
e.g.
Hermanson (cited above).
In another aspect of the invention, molecular tags and binding moieties are
attached to microparticles by providing biotinylated forms of molecular tags
and binding
moieties and avidinated microparticles. A mixture of biotinylated molecular
tags and
biotinylated binding moieties, such as antibodies, can then be combined with
the
avidinated microparticle for attachment. Binding moieties such as antibodies,
fragments
thereof, or other proteins, are readily biotinylated using commercial
reagents, e.g. NHS-
esters of biotin (Pierce Chemical Co.).
In yet another aspect, molecular tags and oligonucleotides are attached to
amino
derivatized microparticles, e.g. hydroxypropylaminodextran coated
microspheres, as
described in Pollner (cited above); Patel (cited above); or Beaudet et al,
Genome
Research, 11:600-608(2001).
Once microparticles are separately derivatized by a plurality of different
molecular tags and/or binding moieties they are pooled to produce the
compositions of
the invention. Usually, each different kind of microparticle is present in a
composition in
the same proportion; however, proportions may be varied as a design choice so
that one
or a subset of particular microparticles are present in greater or lower
proportion
depending on the desirability or requirements for a particular embodiment or
assay.
Molecular Tags and Cleavable Linkages
In one embodiment, molecular tags are cleaved from a microparticle by reaction
of
a cleavable linkage with an active species, such as singlet oxygen, generated
by a
cleavage-inducing moiety, e.g. Singh et al, International patent publication
WO 01/83502.
A cleavable linkage can be virtually any chemical linking group that may be
cleaved under
conditions that do not degrade the structure or affect detection
characteristics of the
released molecular tag. Whenever compositions of the invention are used in a
homogeneous assay format, the cleavable linkage holding a molecular tag to a
microparticle is cleaved by a cleavage agent that acts over a short distance
so that only
cleavable linkages in its immediate proximity are cleaved. Typically, such an
agent must
be activated by making a physical or chemical change to the reaction mixture
so that the
agent produces an short lived active species that diffuses to a cleavable
linkage to effect
cleavage. In a homogeneous format, the cleavage agent is preferably attached
to a binding
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agent, such as an antibody, that targets the cleavage agent to a particular
site prior to
activation, e.g. on an analyte, in the proximity of the binding compound.
In a non-homogeneous, or heterogeneous format, stable complexes between
binding compounds and analytes are separated from unbound binding compounds.
Thus,
a wider selection of cleavable linkages and cleavage agents are available for
use with the
invention. Cleavable linkages may not only include linkages that are labile to
reaction
with a locally acting reactive species, such as singlet oxygen, but also
include linkages that
are labile to agents that operate throughout a reaction mixture, such as a
base cleaving all
base-labile linkages, general illumination by light of an appropriate
wavelength cleaving
all photocleavable linkages, and so on. Additional linkages cleavable by
agents that act
generally throughout a reaction mixture include linkages cleavable by
reduction, linkages
cleaved by oxidation, acid-labile linkages, peptide linkages cleavable by
specific
proteases, and the like. References describing many such linkages include
Greene and
Wuts, Protective Groups in Organic Synthesis, Second Edition (John Wiley &
Sons, New
York, 1991); Hermanson, Bioconjugate Techniques (Academic Press, New York,
1996);
and Still et al, U.S. patent 5,565,324.
An aspect of the invention includes providing mixtures of pluralities of
different
microparticles, wherein each different microparticle has one or more molecular
tags
attached through cleavable linkages. In another aspect, such microparticles
have attached
pairs of molecular tags and binding moieties. A binding moiety is a compound
that is
capable of forming a stable complex with an analyte under assay conditions.
The nature
of the binding moiety, cleavable linkage, and molecular tag may vary widely. A
binding
moiety may be an antibody binding composition, an antibody, a peptide, a
peptide or non-
peptide ligand for a cell surface receptor, an oligonucleotide, an
oligonucleotide analog,
such as a peptide nucleic acid, a lectin, or any other molecular entity that
is capable of
specific binding or complex formation with an analyte of interest and that can
be attached
to a microparticle. In one aspect, a molecular tag attached to a microparticle
can be
represented by the formula:
P-(L-E)k
wherein P is a microparticle; L is a cleavable linkage; and E is a molecular
tag.
Preferably, in homogeneous assays for non-polynucleotide analytes, cleavable
linkage, L,
is an oxidation-labile linkage, and more preferably, it is a linkage that may
be cleaved by
singlet oxygen. The moiety "-(L-E)k" indicates that a single microparticle has
multiple
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molecular tags attached via cleavable linkages. In one aspect, k is an integer
greater than
several hundred, e.g. 100 to 500, or k is greater than several hundred to as
many as several
thousand, e.g. 500 to 5000. Within a composition of the invention, each of the
plurality
of different types of microparticle has a different molecular tag, E.
Cleavable linkages,
e.g. oxidation-labile linkages, and molecular tags, E, are attached to P by
way of
conventional chemistries.
When L is oxidation labile, L is preferably a thioether or its selenium
analog; or an
olefin, which contains carbon-carbon double bonds, wherein cleavage of a
double bond to
an oxo group, releases the molecular tag, E. Illustrative olefins include
vinyl sulfides,
vinyl ethers, enamines, imines substituted at the carbon atoms with an a-
methine (CH, a
carbon atom having at least one hydrogen atom), where the vinyl group may be
in a ring,
the heteroatom may be in a ring, or substituted on the cyclic olefinic carbon
atom, and
there will be at least one and up to four heteroatoms bonded to the olefinic
carbon atoms.
The resulting dioxetane may decompose spontaneously, by heating above ambient
temperature, usually below about 75 C, by reaction with acid or base, or by
photo-
activation in the absence or presence of a photosensitizer. Such reactions are
described in
the following exemplary references: Adam and Liu, J. Amer. Chem. Soc. 94, 1206-
1209,
1972, Ando, et al., J.C.S. Chem. Comm. 1972, 477-8, Ando, et al., Tetrahedron
29, 1507-
13, 1973, Ando, et al., J. Amer. Chem. Soc. 96, 6766-8, 1974, Ando and Migita,
ibid. 97,
5028-9, 1975, Wasserman and Terao, Tetra. Lett. 21, 1735-38, 1975, Ando and
Watanabe,
ibid. 47, 4127-30, 1975, Zaklika, et al., Photochemistry and Photobiology 30,
35-44, 1979,
and Adam, et al., Tetra. Lett. 36, 7853-4, 1995. See also, U.S. Patent no.
5,756,726.
The formation of dioxetanes is obtained by the reaction of singlet oxygen with
an
activated olefin substituted with an molecular tag at one carbon atom and the
binding
moiety at the other carbon atom of the olefin. See, for example, U.S. Patent
No.
5,807,675. These cleavable linkages may be depicted by the following formula:
-W-(X).C a = Cp(Y)(Z)-
wherein:
W may be a bond, a heteroatom, e.g., 0, S, N, P, M (intending a metal that
forms a
stable covalent bond), or a functionality, such as carbonyl, imino, etc., and
may be bonded
to X or C,,;
at least one X will be aliphatic, aromatic, alicyclic or heterocyclic and
bonded to C a
through a hetero atom, e.g., N, 0, or S and the other X may be the same or
different and
may in addition be hydrogen, aliphatic, aromatic, alicyclic or heterocyclic,
usually being
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aromatic or aromatic heterocyclic wherein one X may be taken together with Y
to form a
ring, usually a heterocyclic ring, with the carbon atoms to which they are
attached,
generally when other than hydrogen being from about 1 to 20, usually 1 to 12,
more
usually 1 to 8 carbon atoms and one X will have 0 to 6, usually 0 to 4
heteroatoms, while
the other X will have at least one heteroatom and up to 6 heteroatoms, usually
1 to 4
heteroatoms;
Y will come within the definition of X, usually being bonded to Cp through a
heteroatom and as indicated may be taken together with X to form a
heterocyclic ring;
Z will usually be aromatic, including heterocyclic aromatic, of from about 4
to 12,
usually 4 to 10 carbon atoms and 0 to 4 heteroatoms, as described above, being
bonded
directly to Cp or through a heteroatom, as described above;
n is 1 or 2, depending upon whether the molecular tag is bonded to Ca or X;
wherein one of Y and Z will have a functionality for binding to the binding
moiety,
or be bound to the binding moiety, e.g. by serving as, or including a linkage
group, to a
binding moiety, T.
Preferably, W, X, Y, and Z are selected so that upon cleavage molecular tag,
E, is
within the size limits described below.
Illustrative cleavable linkages include S(molecular tag)-3-thiolacrylic acid,
N(molecular tag), N-methyl 4-amino-4-butenoic acid, 3-hydroxyacrolein, N-(4-
carboxyphenyl)-2-(molecular tag)-imidazole, oxazole, and thiazole.
Also of interest are N-alkyl acridinyl derivatives, substituted at the 9
position with
a divalent group of the formula:
- (CO) X' (A) -
wherein:
X1 is a heteroatom selected from the group consisting of 0, S, N, and Se,
usually
one of the first three; and
A is a chain of at least 2 carbon atoms and usually not more than 6 carbon
atoms
substituted with an molecular tag, where preferably the other valences of A
are satisfied by
hydrogen, although the chain may be substituted with other groups, such as
alkyl, aryl,
heterocyclic groups, etc., A generally being not more than 10 carbon atoms.
Also of interest are heterocyclic compounds, such as diheterocyclopentadienes,
as
exemplified by substituted imidazoles, thiazoles, oxazoles, etc., where the
rings will
usually be substituted with at least one aromatic group and in some instances
hydrolysis
will be necessary to release the molecular tag.
Also of interest are tellurium (Te) derivatives, where the Te is bonded to an
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ethylene group having a hydrogen atom R to the Te atom, wherein the ethylene
group is
part of an alicyclic or heterocyclic ring, that may have an oxo group,
preferably fused to
an aromatic ring and the other valence of the Te is bonded to the molecular
tag. The rings
may be coumarin, benzoxazine, tetralin, etc.
Several preferred cleavable linkages and their cleavage products are
illustrated in
Figures 6 A-F. The thiazole cleavable linkage, "-CH2-thiazole-(CH2)n C(=O)-NH-
protein," shown in Fig. 6A, results in an molecular tag with the moiety "-CH2-
C(=O)-NH-
CHO." Preferably, n is in the range of from 1 to 12, and more preferably, from
1 to 6.
The oxazole cleavable linkage, "-CH2-oxazole-(CH2)õC(=O)-NH-protein," shown in
Fig.
6B, results in an molecular tag with the moiety "-CH2-C(=O)O-CHO." An olefin
cleavable linkage (Fig. 6C) is shown in connection with the binding compound
embodiment "P-L-M-D," described above and with D being a fluorescein dye. The
olefin
cleavable linkage may be employed in other embodiments also. Cleavage of the
illustrated olefin linkage results in an molecular tag of the form: "R-(C=O)-M-
D," where
"R" may be any substituent within the general description of the molecular
tags, E,
provided above. Preferably, R is an electron-donating group, e.g. Ullman et
al, U.S. patent
6,251,581; Smith and March, March's Advanced Organic Chemistry: Reactions,
Mechanisms, and Structure, 5th Edition (Wiley-Interscience, New York, 2001);
and the
like. More preferably, R is an electron-donating group having from 1-8 carbon
atoms and
from 0 to 4 heteroatoms selected from the group consisting of 0, S, and N. In
further
preference, R is -N(Q)2, -OQ, p-[C6H4N(Q)2], furanyl, n-alkylpyrrolyl, 2-
indolyl, or the
like, where Q is alkyl or aryl. In further reference to the olefin cleavable
linkage of Fig.
6C, substituents "X" and "R" are equivalent to substituents "X" and "Y" of the
above
formula describing cleavable linkage, L. In particular, X in Fig. 6C is
preferably
morpholino, -OR', or -SR", where R' and R" are aliphatic, aromatic, alicyclic
or
heterocyclic having from 1 to 8 carbon atoms and 0 to 4 heteroatoms selected
from the
group consisting of 0, S. and N. A preferred thioether cleavable linkage is
illustrated in
Fig. 6D having the form "-(CH2)2-S-CH(C6H5)C(=O)NH-(CH2),, NH-," wherein n is
in the
range of from 2 to 12, and more preferably, in the range of from 2 to 6.
Thioether
cleavable linkages of the type shown in Fig. 6D may be attache to binding
moieties, T, and
molecular tags, E, by way of precursor compounds shown in Figures 6E and 6F.
To attach
to an amino group of a binding moiety, T, the terminal hydroxyl is converted
to an NHS
ester by conventional chemistry. After reaction with the amino group and
attachment, the
Fmoc protection group is removed to produce a free amine which is then reacted
with an
NHS ester of the molecular tag, such as compounds produced by the schemes of
Figures 1,
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2, and 4, with the exception that thelast reaction step is the addition of an
NHS ester,
instead of a phosphoramidite group.
Molecular tag, E, is a water soluble organic compound that is stable with
respect to
the active species, especially singlet oxygen, and that includes a detection
or reporter
group. Otherwise, E may vary widely in size and structure. In one aspect, E
has a
molecular weight in the range of from about 100 to about 2500 daltons, more
preferably,
from about 100 to about 1500 daltons. Preferred structures of E are described
more fully
below. The detection group may generate an electrochemical, fluorescent, or
chromogenic
signal. Preferably, the detection group generates a fluorescent signal.
Molecular tags within a plurality of a composition each have either a unique
chromatographic separation characteristics and/or a unique optical property
with respect to
the other members of the same plurality. In one aspect, the chromatographic
separation
characteristic is retention time in the column used for separation. In another
aspect, the
optical property is a fluorescence property, such as emission spectrum,
fluorescence
lifetime, fluorescence intensity at a given wavelength or band of wavelengths,
or the like.
Preferably, the fluorescence property is fluorescence intensity. For example,
each
molecular tag of a plurality may have the same fluorescent emission
properties, but each
will differ from one another by virtue of a unique retention time in the
column of choice.
On the other hand, or two or more of the molecular tags of a plurality may
have identical
retention times, but they will have unique fluorescent properties, e.g.
spectrally resolvable
emission spectra, so that all the members of the plurality are distinguishable
by the
combination of molecular separation and fluorescence measurement.
In one aspect, molecular tag, E, is (M, D), where M is a mobility-modifying
moiety
and D is a detection moiety. The notation "(M, D)" is used to indicate that
the ordering of
the M and D moieties may be such that either moiety can be adjacent to the
cleavable
linkage, L. That is, "P-L-(M, D)" designates binding compound of either of two
forms: "P-
L-M-D" or "P-L-D-M."
Detection moiety, D, may be a fluorescent label or dye, a chromogenic label or
dye,
an electrochemical label, or the like. Preferably, D is a fluorescent dye.
Exemplary
fluorescent dyes for use with the invention include water-soluble rhodamine
dyes,
fluoresceins, 4,7-dichlorofluoresceins, benzoxanthene dyes, and energy
transfer dyes,
disclosed in the following references: Handbook of Molecular Probes and
Research
Reagents, 8th ed., (Molecular Probes, Eugene, 2002); Lee et al, U.S. patent
6,191,278; Lee
et al, U.S. patent 6,372,907; Menchen et al, U.S. patent 6,096,723; Lee et al,
U.S. patent
5,945,526; Lee et al, Nucleic Acids Research, 25: 2816-2822 (1997); Hobb, Jr.,
U.S. patent
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4,997,928; Khanna et al., U.S. patent 4,318,846; Reynolds, U.S. patent
3,932,415; Eckert et
al, U.S. patent 2,153,059; Eckert et al, U.S. patent 2,242,572; Taing et al,
International
patent publication WO 02/30944; and the like. Further specific exemplary
fluorescent dyes
include 5- and 6-carboxyrhodamine 6G; 5- and 6-carboxy-X-rhodamine, 5- and 6-
carboxytetramethylrhodamine, 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-
dichlorofluorescein, 2',7'-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein,
2',7'-
dimethoxy-4',5'-dichloro-5- and 6-carboxyfluorescein, 2',7'-dimethoxy-4',5'-
dichloro-5- and
6-carboxy-4,7-dichlorofluorescein, 1',2',7',8'-dibenzo-5- and 6-carboxy-4,7-
dichlorofluorescein, 1',2',7',8'-dibenzo-4',5'-dichloro-5- and 6-carboxy-4,7-
dichlorofluorescein, 2',7'-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein,
and 2',4',5',7'-
tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein. Most preferably, D is a
fluorescein or
a fluorescein derivative.
The size and composition of mobility-modifying moiety, M, can vary from a bond
to about 100 atoms in a chain, usually not more than about 60 atoms, more
usually not
more than about 30 atoms, where the atoms are carbon, oxygen, nitrogen,
phosphorous,
boron and sulfur. Generally, when other than a bond, the mobility-modifying
moiety has
from about 0 to about 40, more usually from about 0 to about 30 heteroatoms,
which in
addition to the heteroatoms indicated above may include halogen or other
heteroatom.
The total number of atoms other than hydrogen is generally fewer than about
200 atoms,
usually fewer than about 100 atoms. Where acid groups are present, depending
upon the
pH of the medium in which the mobility-modifying moiety is present, various
cations may
be associated with the acid group. The acids may be organic or inorganic,
including
carboxyl, thionocarboxyl, thiocarboxyl, hydroxamic, phosphate, phosphite,
phosphonate,
phosphinate, sulfonate, sulfinate, boronic, nitric, nitrous, etc. For positive
charges,
substituents include amino (includes ammonium), phosphonium, sulfonium,
oxonium,
etc., where substituents are generally aliphatic of from about 1 - 6 carbon
atoms, the total
number of carbon atoms per heteroatom, usually be less than about 12, usually
less than
about 9. The side chains include amines, ammonium salts, hydroxyl groups,
including
phenolic groups, carboxyl groups, esters, amides, phosphates, heterocycles. M
may be a
homo-oligomer or a hetero-oligomer, having different monomers of the same or
different
chemical characteristics, e.g., nucleotides and amino acids.
In another aspect, (M,D) moieties are constructed from chemical scaffolds used
in
the generation of combinatorial libraries. For example, the following
references describe
scaffold compound useful in generating diverse mobility modifying moieties:
peptoids
(PCT Publication No WO 91/19735, Dec. 26, 1991), encoded peptides (PCT
Publication
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WO 03/042699 PCT/US02/35907
WO 93/20242, Oct. 14 1993), random bio-oligomers (PCT Publication WO 92/00091,
Jan.
9, 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomeres such as
hydantoins,
benzodiazepines and dipeptides (Hobbs DeWitt, S. et al., Proc. Nat. Acad. Sci.
U.S.A. 90:
6909-6913 (1993), vinylogous polypeptides (Hagihara et al. J.Amer. Chem. Soc.
114:
6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding
(Hirschmann, R. et al., J.Amer. Chem. Soc. 114: 9217-9218 (1992)), analogous
organic
syntheses of small compound libraries (Chen, C. et al. J.Amer. Chem. Soc. 116:
2661(1994)), oligocarbamates (Cho, C. Y. et al. Science 261: 1303(1993)),
peptidyl
phosphonates (Campbell, D. A. et al., J. Org. Chem. 59:658(1994)); Cheng et
al, U.S.
patent 6,245,937; Heizmann et al, "Xanthines as a scaffold for molecular
diversity," Mol.
Divers. 2: 171-174 (1997); Pavia et al, Bioorg. Med. Chem., 4: 659-666 (1996);
Ostresh et
al, U.S. patent 5,856,107; Gordon, E. M. et al., J. Med. Chem. 37: 1385
(1994); and the
like. Preferably, in this aspect, D is a substituent on a scaffold and M is
the rest of the
scaffold.
In yet another aspect, (M, D) moieties are constructed from one or more of the
same or different common or commercially available linking, cross-linking, and
labeling
reagents that permit facile assembly, especially using a commercial DNA or
peptide
synthesizer for all or part of the synthesis. In this aspect, (M, D) moieties
are made up of
subunits usually connected by phosphodiester and amide bonds. Exemplary,
precusors
include, but are not limited to, dimethoxytrityl (DMT)-protected hexaethylene
glycol
phosphoramidite, 6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-
diisopropyl)-
phosphoramidite, 12-(4-Monomethoxytritylamino)dodecyl-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramidite, 2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-
cyanoethyl), N,N-diisopropyl)-phosphoramidite, (S-Trityl-6-mercaptohexyl)- (2-
cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 5'-Fluorescein phosphoramidite,
5'-
Hexachloro-Fluorescein Phosphoramidite, 5'-Tetrachloro-Fluorescein
Phosphoramidite, 9-
0-Dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-
phosphoramidite, 3(4,4'Dimethoxytrityloxy)propyl-l-[(2-cyanoethyl)-(N,N-
diisopropyl)]-
phosphoramidite, 5'-O-Dimethoxytrityl-l',2'-Dideoxyribose-3'-[(2-cyanoethyl)-
(N,N-
diisopropyl)]-phosphoramidite, 18-0 Dimethoxytritylhexaethyleneglycol,1-[(2-
cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 12-(4,4'-
Dimethoxytrityloxy)dodecyl-l-
[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 1,3-bis-[5-(4,4'-
dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-
phosphoramidite, 1-[5-(4,4'-dimethoxytrityloxy)pentylamido]-3-[5-
fluorenomethoxycarbonyloxy
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pentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Tris-
2,2,2-
[3-(4,4'-dimethoxytrityloxy)propyloxymethyl] ethyl- [(2-cyanoethyl)-(N,N-
diisopropyl)]-
phosphoramidite, succinimidyl trans-4-(maleimidylmethyl) cyclohexane-l-
carboxylate
(SMCC), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl
acetylthioacetate, Texas Red-X-succinimidyl ester, 5- and 6-
carboxytetramethylrhodamine
succinimidyl ester, bis-(4-carboxypiperidinyl)sulfonerhodamine di(succinimidyl
ester), 5-
and 6-((N-(5-aminopentyl)aminocarbonyl)tetramethylrhodamine, succinimidyl 4-(p-
maleimidophenyl)butyrate (SMPB); N-y-maleimidobutyryl-oxysuccinimide ester
(GMBS); p-nitrophenyl iodoacetate (NPIA); 4-(4-N-maleimidophenyl)butyric acid
hydrazide (MPBH); and like reagents. The above reagents are commercially
available,
e.g. from Glen Research (Sterling, VA), Molecular Probes (Eugene, OR), Pierce
Chemical, and like reagent providers. Use of the above reagents in
conventional synthetic
schemes is well known in the art, e.g. Hermanson, Bioconjugate Techniques
(Academic
Press, New York, 1996). In particular, M may be constructed from the following
reagents:
dimethoxytrityl (DMT)-protected hexaethylene glycol phosphoramidite, 6-(4-
Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite,
12-
(4-Monomethoxytritylamino)dodecyl-(2-cyanoethyl)-(N,N-diisopropyl)-
phosphoramidite,
2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl), N,N-diisopropyl)-
phosphoramidite, (S-Trityl-6-mercaptohexyl)- (2-cyanoethyl)-(N,N-diisopropyl)-
phosphoramidite, 9-0-Dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-
(N,N-
diisopropyl)]-phosphoramidite, 3(4,4Dimethoxytrityloxy)propyl-l-[(2-
cyanoethyl)-(N,N-
diisopropyl)]-phosphoramidite, 5'-O-Dimethoxytrityl-l',2'-Dideoxyribose-3'-[(2-
cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 18-0
Dimethoxytritylhexaethyleneglycol, l-[(2-cyanoethyl)-(N,N-diisopropyl)]-
phosphoramidite, 12-(4,4'-Dimethoxytrityloxy)dodecyl-l-[(2-cyanoethyl)-(N,N-
diisopropyl)]-phosphoramidite, 1,3-bis-[5-'(4,4'-
dimethoxytrityloxy)pentylamido]propyl-2-
[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 1-[5-(4,4'-
dimethoxytrityloxy)pentylamido]-3-[5-fluorenomethoxycarbonyloxy
pentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Tris-
2,2,2-
[3-(4,4'-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-
diisopropyl)]-
phosphoramidite, succinimidyl trans-4-(maleimidylmethyl) cyclohexane-l-
carboxylate
(SMCC), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl
acetylthioacetate, succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB); N-y-
maleimidobutyryl-oxysuccinimide ester (GMBS); p-nitrophenyl iodoacetate
(NPIA); and
4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH).
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M may also comprise polymer chains prepared by known polymer subunit
synthesis methods. Methods of forming selected-length polyethylene oxide-
containing
chains are well known, e.g. Grossman et al, U.S. patent 5,777,096. It can be
appreciated
that these methods, which involve coupling of defined-size, multi-subunit
polymer units to
one another, directly or via linking groups, are applicable to a wide variety
of polymers,
such as polyethers (e.g., polyethylene oxide and polypropylene oxide),
polyesters (e.g.,
polyglycolic acid, polylactic acid), polypeptides, oligosaccharides,
polyurethanes,
polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block
copolymers
thereof, including polymers composed of units of multiple subunits linked by
charged or
uncharged linking groups. In addition to homopolymers, the polymer chains used
in
accordance with the invention include selected-length copolymers, e.g.,
copolymers of
polyethylene oxide units alternating with polypropylene units. As another
example,
polypeptides of selected lengths and amino acid composition (i.e., containing
naturally
occurring or man-made amino acid residues), as homopolymers or mixed polymers.
In another aspect, after release, molecular tag, E, is defined by the formula:
A-M-D
wherein:
A is -C(=O)R, where R is aliphatic, aromatic, alicyclic or heterocyclic having
from
1 to 8 carbon atoms and 0 to 4 heteroatoms selected from the group consisting
of 0, S. and
N; -CH2-C(=O)-NH-CHO; -SO2H; -CH2-C(=O)O-CHO; -C(=O)NH-(CH2)n-NH-
C(=O)C(=O)-(C6H5), where n is in the range of from 2 to 12;
D is a fluorescent dye; and
M is as described above, with the proviso that the total molecular weight of A-
M-D
be within the range of from about 100 to about 2500 daltons.
In another aspect, D is a fluorescein and the total molecular weight of A-M-D
is in
the range of from about 100 to about 1500 daltons.
In another aspect, M may be synthesized from smaller molecules that have
functional groups that provide for linking of the molecules to one another,
usually in a
linear chain. Such functional groups include carboxylic acids, amines, and
hydroxy- or
thiol- groups. In accordance with the present invention the charge-imparting
moiety may
have one or more side groups pending from the core chain. The side groups have
a
functionality to provide for linking to a label or to another molecule of the
charge-
imparting moiety. Common functionalities resulting from the reaction of the
functional
groups employed are exemplified by forming a covalent bond between the
molecules to be
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conjugated. Such functionalities are disulfide, amide, thioamide, dithiol,
ether, urea,
thiourea, guanidine, azo, thioether, carboxylate and esters and amides
containing sulfur
and phosphorus such as, e.g., sulfonate, phosphate esters, sulfonamides,
thioesters, etc.,
and the like.
Cleavage-Inducing Moieties Producing Active Species
A cleavage-inducing moiety is a group that produces an active species that is
capable of cleaving a cleavable linkage, preferably by oxidation. Preferably,
the active
species is a chemical species that exhibits short-lived activity so that its
cleavage-inducing
effects are only in the proximity of the site of its generation. Either the
active species is
inherently short lived, so that it will not create significant background
because beyond the
proximity of its creation, or a scavenger is employed that efficiently
scavenges the active
species, so that it is not available to react with cleavable linkages beyond a
short distance
from the site of its generation. Illustrative active species include singlet
oxygen, hydrogen
peroxide, NADH, and hydroxyl radicals, phenoxy radical, superoxide, and the
like.
Illustrative quenchers for active species that cause oxidation include
polyenes,
carotenoids, vitamin E, vitamin C, amino acid-pyrrole N-conjugates of
tyrosine, histidine,
and glutathione, and the like, e.g. Beutner et al, Meth. Enzymol., 319: 226-
241 (2000).
An important consideration for the cleavage-inducing moiety and the cleavable
linkage is that they not be so far removed from one another when bound to a
target protein
that the active species generated by the sensitizer diffuses and loses its
activity before it
can interact with the cleavable linkage. Accordingly, a cleavable linkage
preferably are
within 1000 nm, preferably 20-100 nm of a bound cleavage-inducing moiety. This
effective range of a cleavage-inducing moiety is referred to herein as its
"effective
proximity."
Generators of active species include enzymes, such as oxidases, such as
glucose
oxidase, xanthene oxidase, D-amino acid oxidase, NADH-FMN oxidoreductase,
galactose
oxidase, glyceryl phosphate oxidase, sarcosine oxidase, choline oxidase and
alcohol
oxidase, that produce hydrogen peroxide, horse radish peroxidase, that
produces hydroxyl
radical, various dehydrogenases that produce NADH or NADPH, urease that
produces
ammonia to create a high local pH.
A sensitizer is a compound that can be induced to generate a reactive
intermediate,
or species, usually singlet oxygen. Preferably, a sensitizer used in
accordance with the
invention is a photosensitizer. Other sensitizers included within the scope of
the invention
are compounds that on excitation by heat, light, ionizing radiation, or
chemical activation
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will release a molecule of singlet oxygen. The best known members of this
class of
compounds include the endoperoxides such as 1,4-biscarboxyethyl-1,4-
naphthalene
endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-
tetraphenyl
naphthalene 5,12-endoperoxide. Heating or direct absorption of light by these
compounds
releases singlet oxygen. Further sensitizers are disclosed in the following
references: Di
Mascio et al, FEBS Lett., 355: 287 (1994)(peroxidases and oxygenases);
Kanofsky, J.Biol.
Chem. 258: 5991-5993 (1983)(lactoperoxidase); Pierlot et al, Meth. Enzymol.,
319: 3-20
(2000)(thermal lysis of endoperoxides); and the like.
Attachment of a binding agent to the cleavage-inducing moiety may be direct or
indirect, covalent or non-covalent and can be accomplished by well-known
techniques,
commonly available in the literature. See, for example, "Immobilized Enzymes,"
Ichiro
Chibata, Halsted Press, New York (1978); Cuatrecasas, J. Biol. Chem., 245:3059
(1970).
A wide variety of functional groups are available or can be incorporated.
Functional
groups include carboxylic acids, aldehydes, amino groups, cyano groups,
ethylene groups,
hydroxyl groups, mercapto groups, and the like. The manner of linking a wide
variety of
compounds is well known and is amply illustrated in the literature (see
above). The length
of a linking group to a binding agent may vary widely, depending upon the
nature of the
compound being linked, the effect of the distance on the specific binding
properties and
the like.
It may be desirable to have multiple cleavage-inducing moieties attached to a
binding agent to increase, for example, the number of active species
generated. This can
be accomplished with a polyfunctional material, normally polymeric, having a
plurality of
functional groups, e.g., hydroxy, amino, mercapto, carboxy, ethylenic,
aldehyde, etc., as
sites for linking. Alternatively a support may be used. The support can have
any of a
number of shapes, such as particle including bead, film, membrane, tube, well,
strip, rod,
and the like. For supports in which photosensitizer is incorporated, the
surface of the
support is, preferably, hydrophilic or capable of being rendered hydrophilic
and the body
of the support is, preferably, hydrophobic. The support may be suspendable in
the
medium in which it is employed. Examples of suspendable supports, by way of
illustration and not limitation, are polymeric materials such as latex, lipid
bilayers, oil
droplets, cells and hydrogels. Other support compositions include glass,
metals, polymers,
such as nitrocellulose, cellulose acetate, poly(vinyl chloride),
polyacrylamide,
polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene,
polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate),
etc.; either
used by themselves or in conjunction with other materials. Attachment of
binding agents
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to the support may be direct or indirect, covalent or non-covalent and can be
accomplished
by well-known techniques, commonly available in the literature as discussed
above. See,
for example, "Immobilized Enzymes," Ichiro Chibata, supra. The surface of the
support
will usually be polyfunctional or be capable of being polyfunctionalized or be
capable of
binding to a target-binding moiety, or the like, through covalent or specific
or non-specific
non-covalent interactions.
The cleavage-inducing moiety may be associated with the support by being
covalently or non-covalently attached to the surface of the support or
incorporated into the
body of the support. Linking to the surface may be accomplished as discussed
above. The
cleavage-inducing moiety may be incorporated into the body of the support
either during
or after the preparation of the support. In general, the cleavage-inducing
moiety is
associated with the support in an amount necessary to achieve the necessary
amount of
active species. Generally, the amount of cleavage-inducing moiety is
determined
empirically.
Photosensitizers as Cleavage-Inducing Moieties
As mentioned above, the preferred cleavage-inducing moiety in accordance with
the present invention is a photosensitizer that produces singlet oxygen. As
used herein,
"photosensitizer" refers to a light-adsorbing molecule that when activated by
light
converts molecular oxygen into singlet oxygen. Photosensitizers may be
attached directly
or indirectly, via covalent or non-covalent linkages, to the binding agent of
a class-specific
reagent. Guidance for constructiing of such compositions, particularly for
antibodies as
binding agents, available in the literature, e.g. in the fields of
photodynamic therapy,
immunodiagnostics, and the like. The following are exemplary references:
Ullman, et al.,
'Proc. Natl. Acad. Sci. USA 91, 5426-5430 (1994); Strong et al, Ann. New York
Acad.
Sci., 745: 297-320 (1994); Yarmush et al, Crit. Rev. Therapeutic Drug Carrier
Syst., 10:
197-252 (1993); Pease et al, U.S. patent 5,709,994; Ullman et al, U.S. patent
5,340,716;
Ullman et al, U.S. patent 6,251,581; McCapra, U.S. patent 5,516,636; and the
like.
Likewise, there is guidance in the literature regarding the properties and
selection
of photosensitizers suitable for use in the present invention. The following
are exemplary
references: Wasserman and R.W. Murray. Singlet Oxygen. (Academic Press, New
York,
1979); Baumstark, Singlet Oxygen, Vol. 2 (CRC Press Inc., Boca Raton, FL
1983); and
Turro, Modern Molecular Photochemistry (University Science Books, 1991).
The photosensitizers are sensitizers for generation of singlet oxygen by
excitation
with light. The photosensitizers include dyes and aromatic compounds, and are
usually
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compounds comprised of covalently bonded atoms, usually with multiple
conjugated
double or triple bonds. The compounds typically absorb light in the wavelength
range of
about 200 to about 1,100 rum, usually, about 300 to about 1,000 nm,
preferably, about 450
to about 950 nm, with an extinction coefficient at its absorbance maximum
greater than
about 500 M-1 cm 1, preferably, about 5,000 M-1 cm 1, more preferably, about
50,000 M-1
cm 1, at the excitation wavelength. The lifetime of an excited state produced
following
absorption of light in the absence of oxygen will usually be at least about
100
nanoseconds, preferably, at least about 1 millisecond. In general, the
lifetime must be
sufficiently long to permit cleavage of a linkage in a reagent in accordance
with the
present invention. Such a reagent is normally present at concentrations as
discussed
below. The photosensitizer excited state usually has a different spin quantum
number (S)
than its ground state and is usually a triplet (S=1) when the ground state, as
is usually the
case, is a singlet (S=0). Preferably, the photosensitizer has a high
intersystem crossing
yield. That is, photoexcitation of a photosensitizer usually produces a
triplet state with an
efficiency of at least about 10%, desirably at least about 40%, preferably
greater than
about 80%.
Photosensitizers chosen are relatively photostable and, preferably, do not
react
efficiently with singlet oxygen. Several structural features are present in
most useful
photosensitizers. Most photosensitizers have at least one and frequently three
or more
conjugated double or triple bonds held in a rigid, frequently aromatic
structure. They will
frequently contain at least one group that accelerates intersystem crossing
such as a
carbonyl or imine group or a heavy atom selected from rows 3-6 of the periodic
table,
especially iodine or bromine, or they may have extended aromatic structures.
A large variety of light sources are available to photo-activate
photosensitizers to
generate singlet oxygen. Both polychromatic and monchromatic sources may be
used as
long as the source is sufficiently intense to produce enough singlet oxygen in
a practical
time duration. The length of the irradiation is dependent on the nature of the
photosensitizer, the nature of the cleavable linkage, the power of the source
of irradiation,
and its distance from the sample, and so forth. In general, the period for
irradiation may
be less than about a microsecond to as long as about 10 minutes, usually in
the range of
about one millisecond to about 60 seconds. The intensity and length of
irradiation should
be sufficient to excite at least about 0.1% of the photosensitizer molecules,
usually at least
about 30% of the photosensitizer molecules and preferably, substantially all
of the
photosensitizer molecules. Exemplary light sources include, by way of
illustration and not
limitation, lasers such as, e.g., helium-neon lasers, argon lasers, YAG
lasers, He/Cd lasers,
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and ruby lasers; photodiodes; mercury, sodium and xenon vapor lamps;
incandescent
lamps such as, e.g., tungsten and tungsten/halogen; flashlamps; and the like.
Examples of photosensitizers that may be utilized in the present invention are
those
that have the above properties and are enumerated in the following references:
Turro,
Modem Molecular Photochemistry (cited above); Singh and Ullman,U.S. patent
5,536,834; Li et al, U.S. patent 5,763,602; Ullman, et al., Proc. Natl. Acad.
Sci. USA 91,
5426-5430 (1994); Strong et al, Ann. New York Acad. Sci., 745: 297-320 (1994);
Martin
et al, Methods Enzymol., 186: 635-645 (1990);Yarmush et al, Crit. Rev.
Therapeutic Drug
Carrier Syst., 10: 197-252 (1993); Pease et al, U.S. patent 5,709,994; Ullman
et al, U.S.
patent 5,340,716; Ullman et al, U.S. patent 6,251,581; McCapra, U.S. patent
5,516,636;
Wohrle, Chimia, 45: 307-310 (1991); Thetford, European patent publ. 0484027;
Sessler et
al, SPIE, 1426: 318-329 (1991); Madison et al, Brain Research, 522: 90-98
(1990); Polo et
al, Inorganica Chimica Acta, 192: 1-3 (1992); Demas et al, J. Macromol. Sci.,
A25: 1189-
1214 (1988); and the like. Exemplary photosensitizers are listed in Table lb.
Table lb
Exemplary Photosensitizers
Hypocrellin A Tetraphenylporphyrin
Hypocrellin B Halogenated derivatives of rhodamine
dyes
Hypericin metallo-Porphyrins
Halogenated derivatives of fluorescein Phthalocyanines
dyes
Rose bengal Naphthalocyanines
Merocyanine 540 Texaphyrin-type macrocycles
Methylene blue Hematophorphyrin
9-Thioxanthone 9,10-Dibromoanthracene
Chlorophylls Benzophenone
Phenaleone Chlorin e6
Protoporphyrin Perylene
Benzo o in A monacid Benzo o in B monacid
In certain embodiments the photosensitizer moiety comprises a support, as
discussed above with respect to the cleavage-inducing moiety. The
photosensitizer may
be associated with the support by being covalently or non-covalently attached
to the
surface of the support or incorporated into the body of the support as
discussed above. In
general, the photosensitizer is associated with the support in an amount
necessary to
achieve the necessary amount of singlet oxygen. Generally, the amount of
photosensitizer
is determined empirically. Photosensitizers used as the photosensitizer are
preferably
relatively non-polar to assure dissolution into a lipophilic member when the
photosensitizer is incorporated in, for example, a latex particle to form
photosensitizer
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beads, e.g. as disclosed by Pease et al., U.S. patent 5,709,994. For example,
the
photosensitizer rose bengal is covalently attached to 0.5 micron latex beads
by means of
chloromethyl groups on the latex to provide an ester linking group, as
described in J.
Amer. Chem. Soc., 97: 3741 (1975).
In one aspect of the invention, a class-specific reagent comprises a first
binding
agent that is an antibody and a cleavage-inducing moiety that is a
photosensitizer, such
that the photosensitizer is covalently linked to the antibody, e.g. using well
know
techniques as disclosed in Strong et al (cited above); Yarmush et al (cited
above); or the
like. Alternatively, a class-specific reagent comprises a solid phase support,
e.g. a bead, to
which a photosensitizer is covalently or non-covalently attached and an
antibody is
attached, preferably convalently, either directly or by way of a
functionalized polymer,
such as amino-dextran, or the like.
Conjugation of sensitizer molecules to assay reagents: Sensitizer molecules
can be
conjugated to an antibody, antigen, avidin, biotin, mononucleotides,
polynucleotides, small
molecules, large molecules and others by various methods and configurations.
For example,
an activated (NHS ester, aldehyde, sulfonyl chloride, etc) sensitizer (Rose
Bengal,
phthalocyanine, etc.) can be reacted with reactive amino-group containing
moieties
(antibody, avidin or other proteins, H2N-LC-Biotin, aminodextran, amino-group
containing
other small and large molecules). The formed conjugates can be used directly
(for example
the antibody-sensitizer conjugate, Biotin-LC-sensitizer, etc.) in various
assays. Also, the
formed conjugates can be further coupled with antibody (for example,
aminodextran-
sensitizer conjugate containing 20 - 200 sensitizers and 200 - 500 amino-
groups can be
coupled to periodate oxidized antibody molecules to generate the antibody-
dextran-
sensitizer conjugate) or with the antibody and a particle. For example,
aminodextran-
sensitizer conjugate containing 20 - 200 sensitizers and 200 - 500 amino-
groups can be
coupled to carboxylated polystyrene beads by EDC coupling chemistry to form
the
sensitizer-aminodextran-particle conjugate. Methods for incorporation of a
sensitizer into a
particle are given in, e.g., U.S. Pat. No. 5,340,716. Then the Na-periodate
oxidized antibody
molecules can be reacted with the amino-groups of the aminodextran molecule,
in presence
of sodium cyanoborohydride, to generate the antibody-dextran-sensitizer-
particle conjugate).
It should be noted that instead of an antibody molecule, avidin or other
molecules can be
used.
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Separation of Released Molecular Tags
As mentioned above, molecular tags are designed for separation by a separation
technique that can distinguish molecular tags based on one or more physical,
chemical,
and/or optical characteristics. Preferably, such separation technique is
capable of
providing quantitative information as well as qualitative information about
the presence or
absence of molecular tags (and therefore, corresponding analytes). In one
aspect, a liquid
phase separation technique is employed so that a solution, e.g. buffer
solution, reaction
solvent, or the like, containing a mixture of molecular tags is processed to
bring about
separation of individual kinds of molecular tags. Usually, such separation is
accompanied
by the differential movement of molecular tags from such a starting mixture
along a path
until discernable peaks or bands form that correspond to regions of increased
concentration of the respective molecular tags. Such a path may be defined by
a fluid
flow, electric field, magnetic field, or the like. The selection of a
particular separation
technique depends on several factors including the expense and convenience of
using the
technique, the resolving power of the technique given the chemical nature of
the molecular
tags, the number of molecular tags to be separated, the type of detection mode
employed,
and the like. Preferably, molecular tags are electrophoretically or
chromatographically
separated.
A. Electrophoretic Separation
Methods for electrophoresis of are well known and there is abundant guidance
for
one of ordinary skill in the art to make design choices for forming and
separating
particular pluralities of molecular tags. The following are exemplary
references on
electrophoresis: Krylov et al, Anal. Chem., 72: 111R-128R (2000); P.D.
Grossman and
J.C. Colburn, Capillary Electrophoresis: Theory and Practice, Academic Press,
Inc., NY
(1992); U.S. Patents 5,374,527; 5,624,800; 5,552,028; ABI PRISM 377 DNA
Sequencer
User's Manual, Rev. A, January 1995, Chapter 2 (Applied Biosystems, Foster
City, CA);
and the like. In one aspect, molecular tags are separated by capillary
electrophoresis.
Design choices within the purview of those of ordinary skill include but are
not limited to
selection of instrumentation from several commercially available models,
selection of
operating conditions including separation media type and concentration, pH,
desired
separation time, temperature, voltage, capillary type and dimensions,
detection mode, the
number of molecular tags to be separated, and the like.
In one aspect of the invention, during or after electrophoretic separation,
the
molecular tags are detected or identified by recording fluorescence signals
and migration
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times (or migration distances) of the separated compounds, or by constructing
a chart of
relative fluorescent and order of migration of the molecular tags (e.g., as an
electropherogram). To perform such detection, the molecular tags can be
illuminated by
standard means, e.g. a high intensity mercury vapor lamp, a laser, or the
like. Typically,
the molecular tags are illuminated by laser light generated by a He-Ne gas
laser or a solid-
state diode laser. The fluorescence signals can then be detected by a light-
sensitive
detector, e. g., a photomultiplier tube, a charged-coupled device, or the
like. Exemplary
electrophoresis detection systems are described elsewhere, e.g., U.S. Patent
Nos.
5,543,026; 5,274,240; 4,879,012; 5,091,652; 6,142,162; or the like. In another
aspect,
molecular tags may be detected electrochemically detected, e.g. as described
in U.S.
Patent No. 6,045,676.
Electrophoretic separation involves the migration and separation of molecules
in an electric field based on differences in mobility. Various forms of
electrophoretic
separation include, by way of example and not limitation, free zone
electrophoresis, gel
electrophoresis, isoelectric focusing, isotachophoresis, capillary
electrochromatography, and micellar electrokinetic chromatography. Capillary
electrophoresis involves electroseparation, preferably by electrokinetic flow,
including
electrophoretic, dielectrophoretic and/or electroosmotic flow, conducted in a
tube or
channel of from about 1 to about 200 micrometers, usually, from about 10 to
about 100
micrometers cross-sectional dimensions. The capillary may be a long
independent
capillary tube or a channel in a wafer or film comprised of silicon, quartz,
glass or
plastic.
In capillary electroseparation, an aliquot of the reaction mixture containing
the
molecular tags is subjected to electroseparation by introducing the aliquot
into an
electroseparation channel that may be part of, or linked to, a capillary
device in which
the amplification and other reactions are performed. An electric potential is
then
applied to the electrically conductive medium contained within the channel to
effectuate migration of the components within the combination. Generally, the
electric
potential applied is sufficient to achieve electroseparation of the desired
components
according to practices well known in the art. One skilled in the art will be
capable of
determining the suitable electric potentials for a given set of reagents used
in the
present invention and/or the nature of the cleaved labels, the nature of the
reaction
medium and so forth. The parameters for the electroseparation including those
for the
medium and the electric potential are usually optimized to achieve maximum
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separation of the desired components. This may be achieved empirically and is
well
within the purview of the skilled artisan.
Detection may be by any of the known methods associated with the analysis of
capillary electrophoresis columns including the methods shown in U.S. Patent
Nos.
5,560,811 (column 11, lines 19-30), 4,675,300, 4,274,240 and 5,324,401. Those
skilled in
the electrophoresis arts will recognize a wide range of electric potentials or
field strengths
may be used, for example, fields of 10 to 1000 V/cm are used with about 200 to
about 600
V/cm being more typical. The upper voltage limit for commercial systems is
about 30 kV,
with a capillary length of about 40 to about 60 cm, giving a maximum field of
about 600
V/cm. For DNA, typically the capillary is coated to reduce electroosmotic
flow, and the
injection end of the capillary is maintained as a negative potential.
For ease of detection, the entire apparatus may be fabricated from a plastic
material
that is optically transparent, which generally allows light of wavelengths
ranging from
about 180 to about 1500 nm, usually about 220 to about 800 nm, more usually
about 450
to about 700 nm, to have low transmission losses. Suitable materials include
fused silica,
plastics, quartz, glass, and so forth.
B. Chromatographic Separation
In one aspect of the invention, pluralities of molecular tags are designed for
separation by chromatography based on one or more physical characteristics
that include
but are not limited to molecular weight, shape, solubility, pKa,
hydrophobicity, charge,
polarity, or the like. A chromatographic separation technique is selected
based on
parameters such as column type, solid phase, mobile phase, and the like,
followed by
selection of a plurality of molecular tags that may be separated to form
distinct peaks or
bands in a single operation. Several factors determine which HPLC technique is
selected
for use in the invention, including the number of molecular tags to be
detected (i.e. the size
of the plurality), the estimated quantities of each molecular tag that will be
generated in
the assays, the availability and ease of synthesizing molecular tags that are
candidates for a
set to be used in multiplexed assays, the detection modality employed, and the
availability,
robustness, cost, and ease of operation of HPLC instrumentation, columns, and
solvents.
Generally, columns and techniques are favored that are suitable for analyzing
limited
amounts of sample and that provide the highest resolution separations.
Guidance for
making such selections can be found in the literature, e.g. Snyder et al,
Practical HPLC
Method Development, (John Wiley & Sons, New York, 1988); Millner, "High
Resolution
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Chromatography: A Practical Approach", Oxford University Press, New York
(1999),
Chi-San Wu, "Column Handbook for Size Exclusion Chromatography", Academic
Press,
San Diego (1999), and Oliver, "HPLC of Macromolecules: A Practical Approach,
Oxford
University Press", Oxford, England (1989). In particular, procedures are
available for
systematic development and optimization of chromatographic separations given
conditions, such as column type, solid phase, and the like, e.g. Haber et al,
J. Chromatogr.
Sci., 38: 386-392 (2000); Outinen et al, Eur. J. Pharm. Sci., 6: 197-205
(1998); Lewis et al,
J. Chromatogr., 592: 183-195 and 197- 208 (1992); and the like.
In one aspect, initial selections of molecular tag candidates are governed by
the
physiochemical properties of molecules typically separated by the selected
column and
stationary phase. The initial selections are then improved empirically by
following
conventional optimization procedure, as described in the above reference, and
by
substituting more suitable candidate molecular tags for the separation
objectives of a
particular embodiment. In one aspect, separation objectives of the invention
include (i)
separation of the molecular tags of a plurality into distinguishable peaks or
bands in a
separation time of less than 60 minutes, and more preferably in less than 40
minutes, and
still more preferably in a range of between 10 to 40 minutes, (ii) the
formation of peaks or
bands such that any pair has a resolution of at least 1.0, more preferably at
least 1.25, and
still more preferably, at least 1.50, (iii) column pressure during separation
of less than 150
bar, (iv) separation temperature in the range of from 25 C to 90 C, preferably
in the range
of from 35 C to 80 C, and (v) the plurality of distinguishable peaks is in the
range of from
5 to 30 and all of the peaks in the same chromatogram. As used herein,
"resolution" in
reference to two peaks or bands is the distance between the two peak or band
centers
divided by the average base width of the peaks, e.g. Snyder et al (cited
above).
A chromatographic method is used to separate molecular tags based on their
chromatographic properties. A chromatographic property can be, for example, a
retention
time of a molecular tag on a specific chromatographic medium under defined
conditions,
or a specific condition under which a molecular tag is eluted from a specific
chromatographic medium. A chromatographic property of a molecular tag can also
be an
order of elution, or pattern of elution, of a molecular tag contained in a
group or set of
molecular tags being chromatographically separated using a specific
chromatographic
medium under defined conditions. A chromatographic property of a molecular tag
is
determined by the physical properties of the molecular tag and its
interactions with a
chromatographic medium and mobile phase. Defined conditions for chromatography
include particular mobile phase solutions, column geometry, including column
diameter
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and length, pH, flow rate, pressure and temperature of column operation, and
other
parameters that can be varied to obtain the desired separation of molecular
tags. A
molecular tag, or chromatographic property of a molecular tag, can be detected
using a
variety of chromatography methods.
Although standard liquid chromatography methods can be used to separate
molecular tags, high pressure (or performance) liquid chromatography (HPLC)
provides
the advantages of high resolution, increased speed of analysis, greater
reproducibility, and
ease of automation of instrument operation and data analysis. HPLC methods
also allow
separation of molecular tags based on a variety of physiochemical properties.
Molecular
tags having similar properties can be used together in the same experiment
since HPLC
can be used to differentiate between closely related tags. The high degree of
resolution
achieved using HPLC methods allows the use of large sets of tagged probes
because the
resulting molecular tags can be distinguished from each other. The ability to
detect large
sets of tagged probes is an advantage when performing multiplexed detection of
target
nucleic acids and target analytes. As used herein, "HPLC" refers to a liquid
phase
chromatographic separation that (i) employs a rigid cylindrical separation
column having a
length of up to 300 mm and an inside diameter of up to 5 mm, (ii) has a solid
phase
comprising rigid spherical particles (e.g. silica, alumina, or the like)
having the same
diameter of up to 5 m packed into the separation column, (iii) takes place at
a
temperature in the range of from 35 C to 80 C and at column pressure up to 150
bars, and
(iv) employs a flow rate in the range of from 1 pL/min to 4 mUmin. Solid phase
particles
for use in HPLC are further characterized in (i) having a narrow size
distribution about the
mean particle diameter, with substantially all particle diameters being within
10% of the
mean, (ii) having the same pore size in the range of from 70 to 300 angstroms,
(iii) having
a surface area in the range of from 50 to 250 m2/g, and (iv) having a bonding
phase density
(i.e. the number of retention ligands per unit area) in the range of from 1 to
5 per nm2.
Sets of molecular tags detected in a single experiment generally are a group
of
chemically related molecules that differ by mass, charge, mass-charge ratio,
detectable tag,
such as differing fluorophores or isotopic labels, or other unique
characteristic. Therefore,
both the chemical nature of the molecular tag and the particular differences
among
molecular tags in a group of molecular tags can be considered when selecting a
suitable
chromatographic medium for separating molecular tags in a sample.
Reverse phase chromatography is a type of chromatography in which the
chemically bonded phase is hydrophobic (nonpolar) than the mobile phase. This
is
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"reversed" from normal phase chromatography, in which the stationary phase is
hydrophilic (polar), and the starting mobile phase is more nonpolar than the
stationary
phase. Mobile phase gradients that increase in concentration of an organic
modifier
(usually acetonitrile or methanol) are commonly used in reverse phase HPLC.
These
gradients elute solute molecules in order of increasing hydrophobicity.
Exemplary mobile
phases for use with the invention to separate water soluble molecular tags
include but are
not limited to water, nitromethane, methanol, dimethyl sulfoxide,
dimethylformamide,
acetonitrile, acetic acid, methoxyethanol, benzyl alcohol, acetone, and the
like. The
mobile phases may be used isocratically or they may be combined and delivered
to a
column in continuously varying proportions. In the latter case, usually two
solvents are
combined in proportions that vary linearly over time, i.e. gradient delivery.
Various mobile phase additives can be used to provide different selectivity to
improve separation of molecular tags. For example, ion pairing reagents may be
used in
reverse phase HPLC methods. Exemplary ion pairing reagents include
trifluoroacetic acid
(TFA), which is an anionic ion-pairing reagent, and tetrabutylammonium
phosphate,
which is a cationic ion pairing reagent.
Reverse phase HPLC can be used to separate a variety of types of molecular
tags,
including organic molecules, oligonucleotides, peptides and polypeptides.
Reversed phase
HPLC is particularly useful for separating peptide or polypeptide molecular
tags that are
closely related to each other. Exemplary reversed phase chromatography media
for
separating molecular tags include particles, e.g. silica or alumina, having
bonded to their
surfaces retention ligands, such as phenyl groups, cyano groups, or aliphatic
groups
selected from the group including C8 through C18 . Preferably, the particles
have a pore
size in the range of from 80 to 300 angstroms.
Exemplary reversed phase chromatography media for separating molecular tags
that are peptides, include particles having aliphatic retention ligands in the
range of from
C8 to C18 bonded to their surfaces and having a pore size of between 60 and 80
angstroms.
Commercial preparations useful for separating molecular tags include, for
example, Apex
WP Octadecyl C18, Octyl C8, Butyl C4 and Phenyl, Aquaprep RP-3000 C4 and C8,
Bakerbond WP Octadecyl C18, Octyl C8, Butyl C4 and Diphenyl.
Prior to separation by HPLC, a sample can be fractionated or subjected to a
pre-
separation step, for example, to remove particulate matter or molecules other
than reporter
tags. In addition to standard biochemical methods for fractionating samples,
such as
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centrifugation, precipitation, filtration and extraction, a variety of HPLC
pre-columns or
guard columns can be used for this purpose.
Separated molecular tags can be detected using a variety of analytical
methods,
including detection of intrinsic properties of molecular tags, such as
absorbance,
fluorescence or electrochemical properties, as well as detection of a
detection group or
moiety attached to a molecular tag. Although not required, a variety of
detection groups
or moieties can be attached to molecular tags to facilitate detection after
chromatographic
separation.
Detection methods for use with liquid chromatography are well known,
commercially available, and adaptable to automated and high-throughput
sampling. The
detection method selected for analysis of molecular tags will depend upon
whether the
molecular tags contain a detectable group or moiety, the type of detectable
group used,
and the physicochemical properties of the molecular tag and detectable group,
if used.
Detection methods based on fluorescence, electrolytic conductivity, refractive
index, and
evaporative light scattering can be used to detect various types of molecular
tags.
A variety of optical detectors can be used to detect a molecular tag separated
by
liquid chromatography. Methods for detecting nucleic acids, polypeptides,
peptides, and
other macromolecules and small molecules using ultraviolet (UV)/visible
spectroscopic
detectors are well known, making UV/visible detection the most widely used
detection
method for HPLC analysis. Infrared spectrophotometers also can be used to
detect
macromolecules and small molecules when used with a mobile phase that is a
transparent
polar liquid.
Variable wavelength and diode-array detectors represent two commercially
available types of UV/visible spectrophotometers. A useful feature of some
variable
wavelength UV detectors is the ability to perform spectroscopic scanning and
precise
absorbance readings at a variety of wavelengths while the peak is passing
through the
flowcell. Diode array technology provides the additional advantage of allowing
absorbance measurements at two or more wavelengths, which permits the
calculation of
ratios of such absorbance measurements. Such absorbance rationing at multiple
wavelengths is particularly helpful in determining whether a peak represents
one or more
than one molecular tag.
Fluorescence detectors can also be used to detect fluorescent molecular tags,
such
as those containing a fluorescent detection group and those that are
intrinsically
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fluorescent. Typically, fluorescence sensitivity is relatively high, providing
an advantage
over other spectroscopic detection methods when molecular tags contain a
fluorophore.
Although molecular tags can have detectable intrinsic fluorescence, when a
molecular tag
contains a suitable fluorescent detection group, it can be possible to detect
a single
molecular tag in a sample.
Electrochemical detection methods are also useful for detecting molecular tags
separated by HPLC. Electrochemical detection is based on the measurement of
current
resulting from oxidation or reduction reaction of the molecular tags at a
suitable electrode.
Since the level of current is directly proportional to molecular tag
concentration,
electrochemical detection can be used quantitatively, if desired.
Mass spectrometry methods also can be used to detect molecular tags separated
by
HPLC. Mass spectrometers can resolve ions with small mass differences and
measure the
mass of ions with a high degree of accuracy and sensitivity. Mass spectrometry
methods
are well known in the art (see Burlingame et al. Anal. Chem. 70:647R-716R
(1998);
Linter and Sherman, Protein Sequencing and Identification Using Tandem Mass
Spectrometry Wiley-Interscience, New York (2000)).
Analysis of data obtained using any detection method, such as spectral
deconvolution and quantitative analysis can be manual or computer-assisted,
and can be
performed using automated methods. A variety of computer programs can be used
to
determine peak integration, peak area, height and retention time. Such
computer programs
can be used for convenience to determine the presence of a molecular tag
qualitatively or
quantitatively. Computer programs for use with HPLC and corresponding
detectors are
well known to those skilled in the art and generally are provided with
commercially
available HPLC and detector systems.
The particular molecular tags contained in a sample can be determined, for
example, by comparison with a database of known chromatographic properties of
reference molecular tags, or by algorithmic methods such as chromatographic
pattern
matching, which allows the identification of components in a sample without
the need to
integrate the peaks individually. The identities of molecular tags in a sample
can be
determined by a combination of methods when large numbers of molecular tags
are
simultaneously identified, if desired.
A variety of commercially available systems are well-suited for high
throughput
analysis of molecular tags. Those skilled in the art can determine appropriate
equipment,
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such as automated sample preparation systems and autoinjection systems, useful
for
automating HPLC analysis of molecular tags. Automated methods can be used for
high-
throughput analysis of molecular tags, for example, when a large number of
samples are
being processes or for multiplexed application of the methods of the invention
for
detecting target analytes. An exemplary HPLC instrumentation system suitable
for use
with the present invention is the Agilent 1100 Series HPLC system (Agilent
Technologies,
Palo Alto, CA).
Those skilled in the art will be aware of quality control measures useful for
obtaining reliable analysis of molecular tags, particular when analysis is
performed in a
high-throughput format. Such quality control measures include the use of
external and
internal reference standards, analysis of chromatograph peak shape, assessment
of
instrument performance, validation of the experimental method, for example, by
determining a range of linearity, recovery of sample, solution stability of
sample, and
accuracy of measurement.
In another aspect of the invention, molecular tags are separated by capillary
electrochromatography (CEC). In CEC, the liquid phase is driven by
electroosmotic flow
through a capillary-sized column, e.g. with inside diameters in the range of
from 30 to 100
gm. CEC is disclosed in Svec, Adv. Biochem. Eng. Biotechnol. 76: 1-47 (2002);
Vanhoenacker et al, Electrophoresis, 22: 4064-4103 (2001); and like
references. CEC
column may used the same solid phase materials as used in conventional reverse
phase
HPLC and additionally may use so-called "monolithic" non-particular packings.
In some
forms of CEC, pressure as well as electroosmosis drives a sample-containing
solvent
through a column.
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Synthesis of Molecular Tags and Binding Cpmounds
The chemistry for performing the types of syntheses to form the charge-
imparting
moiety or mobility modifier as a peptide chain is well known in the art. See,
for example,
Marglin, et al., Ann. Rev. Biochem. (1970) 39:841-866. In general, such
syntheses
involve blocking, with an appropriate protecting group, those functional
groups that are
not to be involved in the reaction. The free functional groups are then
reacted to form the
desired linkages. The peptide can be produced on a resin as in the Merrifield
synthesis
(Merrifield, J. Am. Chem. Soc. (1980) 85:2149-2154 and Houghten et al., Int.
J. Pep. Prot.
Res. (1980) 16:311-320. The peptide is then removed from the resin according
to known
techniques.
A summary of the many techniques available for the synthesis of peptides may
be
found in J. M. Stewart, et al., "Solid Phase Peptide Synthesis, W. H. Freeman
Co, San
Francisco (1969); and J. Meienhofer, "Hormonal Proteins and Peptides", (1973),
vol. 2, p.
46, Academic Press (New York), for solid phase peptide synthesis; and E.
Schroder, et al.,
"The Peptides", vol. 1, Academic Press (New York), 1965 for solution
synthesis.
In general, these methods comprise the sequential addition of one or more
amino
acids, or suitably protected amino acids, to a growing peptide chain.
Normally, a suitable
protecting group protects either the amino or carboxyl group of the first
amino acid. The
protected or derivatized amino acid can then be either attached to an inert
solid support or
utilized in solution by adding the next amino acid in the sequence having the
complementary (amino or carboxyl) group suitably protected, under conditions
suitable for
forming the amide linkage. The protecting group is then removed from this
newly added
amino acid residue and the next amino acid (suitably protected) is then added,
and so
forth. After all the desired amino acids have been linked in the proper
sequence, any
remaining protecting groups (and any solid support) are removed sequentially
or
concurrently, to afford the final peptide. The protecting groups are removed,
as desired,
according to known methods depending on the particular protecting group
utilized. For
example, the protecting group may be removed by reduction with hydrogen and
palladium
on charcoal, sodium in liquid ammonia, etc.; hydrolysis with trifluoroacetic
acid,
hydrofluoric acid, and the like.
For synthesis of binding compounds employing phosphoramidite, or related,
chemistry many guides are available in the literature: Handbook of Molecular
Probes and
Research Products, 8tt, edition (Molecular Probes, Inc., Eugene, OR, 2002);
Beaucage and
Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al, U.S. patent 4,980,460;
Koster et al,
U.S. patent 4,725,677; Caruthers et al, U.S. patents 4,415,732; 4,458,066; and
4,973,679;
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and the like. Many of these chemistries allow components of the binding
compound to be
conveniently synthesized on an automated DNA synthesizer, e.g. an Applied
Biosystems,
Inc. (Foster City, California) model 392 or 394 DNA/RNA Synthesizer, or the
like.
Synthesis of molecular tag reagents comprising nucleotides as part of the
mobility-
modifying moiety can be easily and effectively achieved via assembly on a
solid phase
support using standard phosphoramidite chemistries. The resulting mobility
modifying
moiety may be linked to the label and/or polypeptide-binding moiety as
discussed above.
Exemplary Synthetic Approaches for Molecular Tags
One exemplary synthetic approach is outlined in Figure 1. Starting with
commercially available 6-carboxy fluorescein, the phenolic hydroxyl groups are
protected
using an anhydride. Isobutyric anhydride in pyridine was employed but other
variants are
equally suitable. It is important to note the significance of choosing an
ester functionality
as the protecting group. This species remains intact throughout the
phosphoramidite
monomer synthesis as well as during oligonucleotide construction. These groups
are not
removed until the synthesized oligonucleotide is deprotected using ammonia.
After
protection the crude material is then activated in situ via formation of an N-
hydroxysuccinimide ester (NHS-ester) using DCC as a coupling agent. The DCU by
product is filtered away and an amino alcohol is added. Many amino alcohols
are
commercially available some of which are derived from reduction of amino
acids. When
the amino alcohol is of the form "H2N-(CH2)n OH," n is in the range of from 2
to 12, and
more preferably, from 2 to 6. Only the amine is reactive enough to displace N-
hydroxysuccinimide. Upon standard extractive workup, a 95% yield of product is
obtained. This material is phosphitylated to generate the phosphoramidite
monomer. For
the synthesis of additional molecular tags, a symmetrical bis-amino alcohol
linker is used
as the amino alcohol (Figure 2). As such, the second amine is then coupled
with a
multitude of carboxylic acid derivatives (exemplified by several possible
benzoic acid
derivatives shown in Figure 3 prior to the phosphitylation reaction.
Alternatively, molecular tags may be made by an alternative strategy that uses
5-
aminofluorescein as starting material (Figure 4). Addition of 5-
aminofluorescein to a
great excess of a diacid dichloride in a large volume of solvent allows for
the predominant
formation of the monoacylated product over dimer formation. The phenolic
groups are
not reactive under these conditions. Aqueous workup converts the terminal acid
chloride
to a carboxylic acid. This product is analogous to 6-carboxyfluorescein, and
using the
same series of steps is converted to its protected phosphoramidite monomer.
There are
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many commercially available diacid dichlorides and diacids, which can be
converted to
diacid dichlorides using SOC12 or acetyl chloride. There are many commercial
diacid
dichlorides and amino alcohols (Figure 5). These synthetic approaches are
ideally suited
for combinatorial chemistry.
The molecular tags constructed with the schemes of Figures 1, 2, and 4 are
further
reacted either before or after phosphitylation to attach a cleavable linkage,
e.g. using
chemistry as described below.
The molecular tag may be assembled having an appropriate functionality at one
end for linking to the polypeptide-binding moieties. A variety of
functionalities can be
employed. Thus, the functionalities normally present in a peptide, such as
carboxy,
amino, hydroxy and thiol may be the targets of a reactive functionality for
forming a
covalent bond. The molecular tag is linked in accordance with the chemistry of
the
linking group and the availability of functionalities on the polypeptide-
binding moiety.
For example, as discussed above for antibodies, and fragments thereof such as
Fab'
fragments, specific for a polypeptide, a thiol group will be available for
using an active
olefin, e.g., maleimide, for thioether formation. Where lysines are available,
one may use
activated esters capable of reacting in water, such as nitrophenyl esters or
pentafluorophenyl esters, or mixed anhydrides as with carbodiimide and half-
ester
carbonic acid. There is ample chemistry for conjugation in the literature, so
that for each
specific situation, there is ample precedent in the literature for the
conjugation.
In an illustrative synthesis a diol is employed. Examples of such diols
include an
alkylene diol, polyalkylene diol, with alkylene of from 2 to 3 carbon atoms,
alkylene
amine or poly(alkylene amine) diol, where the alkylenes are of from 2 to 3
carbon atoms
and the nitrogens are substituted, for example, with blocking groups or alkyl
groups of
from 1- 6 carbon atoms, where one diol is blocked with a conventional
protecting group,
such as a dimethyltrityl group. This group can serve as the mass-modifying
region and
with the amino groups as the charge-modifying region as well. If desired, the
mass
modifier can be assembled by using building blocks that are joined through
phosphoramidite chemistry. In this way the charge modifier can be interspersed
between
the mass modifier. For example, a series of polyethylene oxide molecules
having 1, 2, 3, n
units may be prepared. To introduce a number of negative charges, a small
polyethylene
oxide unit may be employed. The mass and charge-modifying region may be built
up by
having a plurality of the polyethylene oxide units joined by phosphate units.
Alternatively, by employing a large spacer, fewer phosphate groups would be
present, so
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that without large mass differences, large differences in mass-to- charge
ratios may be
realized.
The chemistry that is employed is the conventional chemistry used in
oligonucleotide synthesis, where building blocks other than nucleotides are
used, but the
reaction is the conventional phosphoramidite chemistry and the blocking group
is the
conventional dimethoxytrityl group. Of course, other chemistries compatible
with
automated synthesizers can also be used. However, it is desirable to minimize
the
complexity of the process.
As mentioned above, in one embodiment the hub nucleus is a hydrophilic
polymer,
generally, an addition or condensation polymer with multiple functionality to
permit the
attachment of multiple moieties. One class of polymers that is useful for the
reagents of
the present invention comprises the polysaccharide polymers such as dextrans,
sepharose,
polyribose, polyxylose, and the like. For example, the hub may be dextran to
which
multiple molecular tags may be attached in a cleavable manner consistent with
the present
invention. A few of the aldehyde moieties of the dextran remain and may be
used to
attach the dextran molecules to amine groups on an oligonucleotide by
reductive
amination. In another example using dextran as the hub nucleus, the dextran
may be
capped with succinic anhydride and the resulting material may be linked to
amine-
containing oligonucleotides by means of amide formation.
Besides the nature of the linker and mobility-modifying moiety, as already
indicated, diversity can be achieved by the chemical and optical
characteristics of the
fluorescer, the use of energy transfer complexes, variation in the chemical
nature of the
linker, which affects mobility, such as folding, interaction with the solvent
and ions in the
solvent, and the like. As already suggested, in one embodiment the linker is
an oligomer,
where the linker may be synthesized on a support or produced by cloning or
expression in
an appropriate host. Conveniently, polypeptides can be produced where there is
only one
cysteine or serine/threonine/tyrosine, aspartic/glutamic acid, or
lysine/arginine/histidine,
other than an end group, so that there is a unique functionality, which may be
differentially
functionalized. By using protective groups, one can distinguish a side-chain
functionality
from a terminal amino acid functionality. Also, by appropriate design, one may
provide
for preferential reaction between the same functionalities present at
different sites on the
linking group. Whether one uses synthesis or cloning for preparation of
oligopeptides,
will to a substantial degree depend on the length of the linker.
Methods of Using Binding Compositions of the Invention
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In one aspect, the invention provides a method for detecting or measuring one
or
more target analytes from biological sources. Conventional methodologies are
employed
to prepare samples for analysis. For example, for protein analytes guidance in
sample
preparation can be found in Scopes, Protein Purification, chapter 2 (Springer-
Verlag, New
York), where a range of procedures are disclosed for preparing protein
extracts from
different sources. Preparative techniques include mild cell lysis by osmotic
disruption of
cellular membranes, to enzymatic digestion of connective tissue followed by
osmotic-
based lysis, to mechanical homogenization, to ultrasonication.
For sources containing target polynucleotides, guidance for sample preparation
techniques can be found in standard treatises, such as Sambrook et al,
Molecular Cloning,
Second Edition (Cold Spring Harbor Laboratory Press, New York, 1989); Innis et
al,
editors, PCR Protocols (Academic Press, New York, 1990); Berger and Kimmel,
"Guide
to Molecular Cloning Techniques," Vol. 152, Methods in Enzymology (Academic
Press,
New York, 1987); or the like. For mammalian tissue culture cells, or like
sources, samples
of target RNA may be prepared by conventional cell lysis techniques (e.g. 0.14
M NaCl,
1.5 mM MgC12, 10 mM Tris-Cl (pH 8.6), 0.5% Nonidet P-40, 1 mM dithiothreitol,
1000
units/mL placential RNAase inhibitor or 20 mM vanadyl-ribonucleoside
complexes).
In carrying out the assays, the components, i.e., the sample, composition of
microparticles, and in some embodiments a cleavage-inducing moiety, are
combined in an
assay medium in any order, usually simultaneously. Alternatively, one or more
of the
reagents may be combined with one or more of the remaining agents to form a
subcombination. The subcombination can then be subjected to incubation. Then,
the
remaining reagents or subcombination thereof may be combined and the mixture
incubated. The amounts of the reagents are usually determined empirically. The
components are combined under binding conditions, usually in an aqueous
medium,
generally at a pH in the range of about 5 to about 10, with buffer at a
concentration in the
range of about 10 to about 200 mM. These conditions are conventional, where
conventional buffers may be used, such as phosphate, carbonate, HEPES, MOPS,
Tris,
borate, etc., as well as other conventional additives, such as salts,
stabilizers, organic
solvents, etc. The aqueous medium may be solely water or may include from 0.01
to 80 or
more volume percent of a co-solvent.
The combined reagents are incubated for a time and at a temperature that
permit a
substantial number of binding events to occur. The time for incubation after
combination
of the reagents varies depending on the (i) nature and expected concentration
of the
analyte being detected, (ii) the mechanism by which the binding compounds for
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complexes with analytes, and (iii) the affinities of the specific reagents
employed.
Moderate temperatures are normally employed for the incubation and usually
constant
temperature. Incubation temperatures will normally range from about 5 to 99
C, usually
from about 15 to 85 C, more usually 35 to 75 C.
Generally, the concentrations of the various agents involved with an assay of
the
invention will vary with the concentration range of the individual analytes in
the samples
to be analyzed, generally being in the range of about 10 nM to about 10 mM.
Buffers will
ordinarily be employed at a concentration in the range of about 10 to about
200 mM. The
concentration of each analyte will generally be in the range of about 1 pM to
about 100
M, more usually in the range of about 100 pM to about 10 M. In specific
situations the
concentrations may be higher or lower, depending on the nature of the analyte,
the affinity
of the binding compounds, the efficiency of release of the molecular tags, the
sensitivity
with which the molecular tags are detected, and the number of analytes to be
determined
in the assay, as well as other considerations.
In heterogeneous assays it is required that the unbound labeled reagent be
separable from the bound labeled reagent. This can be achieved in a variety of
ways, each
requiring a reagent bound to a solid support that distinguishes between the
complex of
labeled reagent and polypeptide. The solid support may be a vessel wall, e.g.,
microtiter
well plate well, capillary, plate, slide, beads, including magnetic beads,
liposomes, or the
like. The primary characteristics of the solid support is that it permits
segregation of the
bound labeled specific binding member from unbound probe and that the support
does not
interfere with the formation of the binding complex, nor the other operations
of the
determination.
The solid support may have the complex directly or indirectly bound to the
support. For directly bound, one may have the binding compound or second
binding
compound covalently or non-covalently bound to the support. The surface may be
activated with various functionalities that will form covalent bonds with the
second
binding compound. These groups may include imino halides, activated carboxyl
groups,
e.g., mixed anhydrides or acyl halides, amino groups, (X-halo or
pseudohaloketones, etc. A
specific binding member bound to the surface of the support may be used to
bind a
member of the complex.
In some embodiments, where components of the assay mixture interfere with a
chromatographic analysis, the molecular tags may be required to be separated
from the
assay mixture prior to chromatographic analysis, or certain components of the
assay
mixture, e.g. binding moieties with unreleased molecular tags, may be required
to be
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excluded from the chromatographic analysis. Depending on the nature of the
molecular
tags and the components of the assay mixture, one may sequester or adsorb or
exclude
such binding moieties by using guard column, and the like. Alternatively, one
may have a
capture ligand attached to binding compounds for the purpose of removing such
interfering components in the mixture.
An additional degree of flexibility can be conferred on an assay by the stage
at
which the molecular tags are labeled. A molecular tag may contain a
functionality
allowing it to bind to a label after reaction with the sample is complete. In
this
embodiment, a molecular tag comprising a functionality for binding to a
detectable label is
combined with a sample. After a binding reaction takes place and molecular
tags are
released, additional reagents are combined in a sample vessel with the
products of the first
reaction, which react with the released molecular tags to add a detectable
label.
For quantitation, one may choose to use controls, which provide a signal in
relation
to the amount of the target that is present or is introduced. A control to
allow conversion
of relative fluorescent signals into absolute quantities is accomplished by
addition of a
known quantity of a fluorophore to each sample before separation of the
molecular tags.
Any fluorophore that does not interfere with detection of the molecular tag
signals can be
used for normalizing the fluorescent signal. Such standards preferably have
separation
properties that are different from those of any of the molecular tags in the
sample, and
could have the same or a different emission wavelength. Exemplary fluorescent
molecules
for standards include ROX, FAM, and fluorescein and derivatives thereof.
One example of an assay in accordance with the present invention involves the
detection of the phosphorylation of a polypeptide. The sample comprises
cellular material
and the post-translational modification is the phosphorylation of a particular
polypeptide,
referred to as a target polypeptide. The sample is combined with a second
binding
compound comprising a photosensitizer linked to a metal affinity agent to
which is bound
a metal ion. If the phosphorylated target polypeptide is present, the
phosphate group binds
to the metal-metal affinity agent complex. A binding composition is combined
with the
above reaction mixture. The binding composition comprises an antibody for the
target
polypeptide, to which is cleavably linked one or more molecular tags. The
cleavable
linkage comprises a moiety that is cleavable by singlet oxygen. After addition
of the
binding composition and an appropriate incubation period, the reaction mixture
is
irradiated with light to excite the photosensitizer, which generates singlet
oxygen. The
cleavable moiety is cleaved by the singlet oxygen because the cleavable moiety
is in close
proximity to the photosensitizer and the active species, namely, singlet
oxygen, retains
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sufficient activity to cleave the cleavable moiety and release a molecular
tag. Binding
compounds that do not become bound to target polypeptide because the target
polypeptide
is not present, or excess binding compound, or binding compound that binds to
a
polypeptide that is not phosphorylated, does not yield cleaved molecular tags
because the
activity of the singlet oxygen is very short-lived and the cleavable moiety in
any binding
compound that is not bound to the second binding compound by virtue of the
presence of
phosphorylated target polypeptide does not yield cleaved molecular tags. The
released
molecular tag is separated on the basis of its different mobility and detected
on the basis of
the detection moiety that remains attached to the mobility modifying moiety of
the
molecular tag. The presence and/or amount of the released molecular tag
indicates the
presence and/or amount of the target polypeptide.
The present invention finds particular use in multiplexed assays for target
polypeptides. An example of an assay in accordance with this aspect of the
present
invention involves the detection of the phosphorylation of multiple
polypeptides. The
sample comprises cellular material and the post-translational modification is
the
phosphorylation of several polypeptides, referred to as target polypeptides.
The sample is
combined with a second binding compound comprising a photosensitizer linked to
a metal
affinity agent to which is bound a metal ion. The second binding compound is a
class-
specific reagent in that it binds to any phosphate group present in the
reaction mixture. If
the phosphorylated target polypeptides are present, the phosphate group binds
to the
metal-metal affinity agent complex. A plurality of binding compounds is
combined with
the above reaction mixture. Each of the binding compounds comprises an
antibody for a
particular target polypeptide, to which is cleavably linked an molecular tag
that is unique
for the particular target polypeptide. The cleavable link comprises a moiety
that is
cleavable by singlet oxygen. After addition of the binding compounds and an
appropriate
incubation period, the reaction mixture is irradiated with light to excite the
photosensitizer,
which generates singlet oxygen. The cleavable moiety is cleaved by the singlet
oxygen
because the cleavable moiety is in close proximity to the photosensitizer and
the active
species, namely, singlet oxygen, retains sufficient activity to cleave the
cleavable moiety
and release molecular tags from all binding compounds that are bound to a
target
polypeptide bound to the class-specific reagent. Again, binding compounds,
which do not"
become bound to target polypeptides bound to the class-specific reagent, do
not yield
cleaved molecular tags for the reasons given above. The released molecular
tags are
separated on the basis of their differences in mobility and detected on the
basis of the
detection moiety that remains attached to the mobility modifying moiety of the
molecular
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tag. The presence and/or amount of each of the released molecular tags
indicate the
presence and/or amount of each of the respective target polypeptides. In this
fashion
various cellular pathways may be studied on a real time basis. Protein
phosphorylation
and de-phosphorylation reactions may be studied to develop more information
about
metabolic regulation and signal transduction pathways. The above method may be
repeated at various times during the cell cycle to follow the progression of
the cell.
Another application of the present invention is to detect multiple
phosphorylations
of a target polypeptide. For example, it is desirable to know whether a
polypeptide has
been mono-phosphorylated, bis-phosphorylated or even higher multiples of
phosphorylation. An example of an assay in accordance with this aspect of the
present
invention involves the detection of the degree of phosphorylation of a target
polypeptide.
The sample, which comprises cellular material, is combined with a second
binding
compound comprising a multiple photosensitizer molecules linked to a hub
molecule to
which multiple molecules of a metal affinity agent with bound metal are also
linked. By
appropriate titration of the class-specific reagent, the level of
phosphorylation of the target
polypeptide can be determined. If the phosphorylated target polypeptides are
present, the
phosphate group binds to the metal-metal affinity agent complex. An binding
compound is
combined with the above reaction mixture. The binding compound comprises an
antibody
for the particular target polypeptide, to which is cleavably linked an
molecular tag that is
unique for the particular target polypeptide. The cleavable link comprises a
moiety that is
cleavable by singlet oxygen. After addition of the binding compound and an
appropriate
incubation period, the reaction mixture is irradiated with light to excite the
photosensitizer,
which generates singlet oxygen. The cleavable moiety is cleaved by the singlet
oxygen
because the cleavable moiety is in close proximity to the photosensitizer. The
active
species, namely, singlet oxygen, retains sufficient activity to cleave the
cleavable moiety
and release molecular tags from the binding compound that is bound to a target
polypeptide bound to the class-specific reagent. Again, binding compounds,
which do not
become bound to target polypeptides bound to the class-specific reagent, do
not yield
cleaved molecular tags for the reasons given above. The released molecular tag
is
separated on the basis of differences in mobility and detected on the basis of
the detection
moiety that remains attached to the mobility modifying moiety of the molecular
tag. The
presence and/or amount of the released molecular tag may be correlated with
the amount
of class-specific reagent added to determine the level of phosphorylation of
the target
polypeptide.
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The present invention may be employed to determine the site or sites of
phosphorylation on a target polypeptide. In an example of an assay in
accordance with this
aspect of the present invention, the sample, which comprises cellular
material, is combined
with a second binding compound comprising a chemical protease linked to a
metal affinity
agent to which is bound a metal ion. If the phosphorylated target polypeptide
is present,
the phosphate group binds to the metal-metal affinity agent complex. The
chemical
protease is activated by irradiation with light and site specific cleavage
takes place on the
target polypeptide whose phosphate group is bound to the metal affinity-metal
complex.
On the other hand, one or more binding compounds may be combined with the
above
reaction mixture to provide a detection moiety for the unique moieties. Each
binding
compound comprises an antibody for a cleaved moiety, to which is attached the
detection
moiety. The molecular tag and is separated on the basis of its different
mobility and
detected on the basis of the detection moiety that is attached. The presence
of the
molecular tag is indicative of the site of phosphorylation of the target
polypeptide.
Taggant Use
Compositions of the invention may be used to label and track materials, such
as
liquids, wherein compositions of microparticles are mixed with the material or
liquid to be
identified or tracked, referred to herein as a "tagged material" or "tagged
liquid." The
origin or distribution of the tagged liquid or material can be determined by
isolating a
sample containing microparticles. The molecular tags of the microparticles are
released,
separated, and identified. Identity may determined by the presence or absence
of a
plurality of molecular tags (i.e. a "bar code"), or the presence, absence, or
quantity of a
plurality of molecular tags. For example, in the former case, if 15 different
kinds of
microparticles were employed in a composition, over 32,000 (=215) liquids or
materials
could be uniquely labeled. Guidance for the types of liquids and materials
suitable for
tagging, amounts to use for tagging, and method of extracting microparticles
from a
sample of such liquid or material is found in Slater et al., U.S. Patent
5,643,728. In one
aspect, liquids are labeled such that there are from 1 to 100 microparticles
of each kind per
mL of liquid, or from 10 to 1000 microparticles of each kind per mL of liquid.
In another
aspect, microparticles may be separated from the liquid by conventional
techniques, such as
flow cytometry. After such separation, molecular tags are cleaved, separated,
and identified,
thereby providing the identity of the liquid from which they were sampled.
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Kits for Use of Microparticle Compositions
As a matter of convenience, predetermined amounts of reagents employed in
the present invention can be provided in a kit in packaged combination. One
exemplary kit for polypeptide analysis can comprise in packaged combination a
microparticle composition of the invention and a second binding composition of
the
invention. The kit can further comprise standards for separating released
molecular
tags under a pretermined separation technique. In another embodiment,
microparticles having pairs of molecular tags and binding moieties attached
may be
packaged separately.
The kits will include microparticle compositions having from 2 to 50, and
more usually from 5 to 30, different kinds of microparticles each with a
different
molecular tag attached, such that the released molecular tags form
distinguishable
peaks upon separation using a specified separation protocol.
The kit may further comprise a device for conducting chromatography or
electrophoresis as well as reagents that may be necessary to activate the
cleavage-inducing
moiety of the cleavage-inducing reagent. The kit can further include various
buffered
media, some of which may contain one or more of the above reagents.
The relative amounts of the various reagents in the kits can be varied widely
to
provide for concentrations of the reagents necessary to achieve the objects of
the present
invention. Under appropriate circumstances one or more of the reagents in the
kit can be
provided as a dry powder, usually lyophilized, including excipients, which on
dissolution
will provide for a reagent solution having the appropriate concentrations for
performing a
method or assay in accordance with the present invention. Each reagent can be
packaged
in separate containers or some reagents can be combined in one container where
cross-reactivity and shelf life permit. The kits may also include a written
description of a
method in accordance with the present invention as described above.
EXAMPLES
The invention is demonstrated further by the following syntheses and
illustrative
examples. Parts and percentages are by weight unless otherwise indicated.
Temperatures
are in degrees Centigrade ( C) unless otherwise specified. The following
preparations and
examples illustrate the invention but are not intended to limit its scope.
Unless otherwise
indicated, peptides used in the following examples were prepared by synthesis
using an
automated synthesizer and were purified by gel electrophoresis or HPLC.
The following abbreviations have the meanings set forth below:
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Tris HCl - Tris(hydroxymethyl)aminomethane-HC1 (a lOx solution) from
BioWhittaker,
Walkersville, MD
TLC - thin layer chromatography
BSA - bovine serum albumin, e.g. available from Sigma Chemical Company (St.
Louis,
MO), or like reagent supplier.
EDTA - ethylene diamine tetra-acetate from Sigma Chemical Company
FAM - carboxyfluorescein
EMCS - N-c-maleimidocaproyloxy-succinimide ester
EDC - l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
NHS - N-hydroxysuccinimide
DCC - 1,3-dicylcohexylcarbodiimide
DMF - dimethylformamide
Fmoc - N-(9-fluorenylmethoxycarbonyl)-
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Example 1
Conjugation of photosensitizer molecules to assay reagents
Photosensitizer molecules are conjugated to a metal affinity agent, a boronic
acid
containing agent, a hub molecule, and the like by various conventional methods
and
configurations. For example, an activated (NHS ester, aldehyde, sulfonyl
chloride, etc)
photosensitizer (Rose Bengal, phthalocyanine, etc.) can be reacted with
reactive amino-
group containing moieties (aminodextran, amino-group containing agents (with
appropriate protection of metal binding sites), other small and large
molecules). The
formed conjugates can be used directly (for example the antibody-
photosensitizer
conjugate, Biotin-LC-photosensitizer, etc.) in various assays. Also, the
formed conjugates
can be further coupled with antibody (for example, aminodextran-
photosensitizer
conjugate containing 20 - 200 photosensitizers and 200 - 500 amino-groups can
be
coupled to periodate oxidized antibody molecules to generate the antibody-
dextran-
sensitizer conjugate) or with the antibody and a particle. For example,
aminodextran-
sensitizer conjugate containing 20 - 200 photosensitizers and 200 - 500 amino-
groups can
be coupled to carboxylated polystyrene beads by EDC coupling chemistry to form
the
photosensitizer-aminodextran-particle conjugate. Methods for incorporation of
a
photosensitizer into a particle are given in, e.g., U.S. Pat. No. 5,340,716.
Then the Na-
periodate oxidized antibody molecules can be reacted with the amino-groups of
the
aminodextran molecule, in presence of sodium cyanoborohydride, to generate the
antibody-dextran-photosensitizer-particle conjugate, referred to herein as a
"photosensitizer bead." It should be noted that instead of an antibody
molecule, avidin or
other molecules can be used also.
Example 2
Preparation of Aminodextran Derivatized Microspheres
Aminodextran is prepared as described in Pollner, U.S. patent 6,346,384.
Briefly,
hydroxypropylaminodextran (1NH2 /16 glucose) is prepared by dissolving Dextran
T-500
(Pharmacia, Uppsala, Sweden) (100 g) in 500 mL of H2O in a 3-neck round-bottom
flask
equipped with mechanical stirrer and dropping funnel. To the above solution is
added 45 g
sodium hydroxide, 50 mg EDTA, 50 mg NaBH4, 50 mg hydroquinone and 200 g N-(2,3-
epoxypropyl) phthalimide. The mixture is heated and stirred in a 90 C water
bath for 2 hr.
A small aliquot is precipitated three times from methanol and analyzed by NMR.
Appearance of a peak at 7.3-7.66 indicates incorporation of phthalimide. The
main
reaction mixture is precipitated by addition to 3.5 L of methanol and the
solid is collected.
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The phthalimide protecting group is removed by dissolving the product above in
500 mL
of 0.1 M acetate buffer, adding 50 mL of 35% hydrazine and adjusting the pH to
3.5. The
mixture is heated at 80 C. for 1 hr, the pH is readjusted to 3.2, and the
mixture is heated
for an additional one-half hour. An aliquot is precipitated three times in
methanol. The
reaction mixture is neutralized to pH 8 and stored at room temperature. The
product is
purified by tangential flow filtration using a 50,000 molecular weight cut-off
filter,
washing with about 8 L water, 0.5 L of 0. 1M HCI, 0.5 L of 0.01 M NaOH, and
finally 3 L
of water. The product solution is concentrated by filtration to 700 mL and
then is
lyophilized. Determination of reactive amines using trinitrobenzenesulfonate
indicates
about 1 amine per 16 glucose residues.
A solution of hydroxypropylaminodextran (synthesized as described above) is
prepared at 2 mg/mL in 50 mM MES (pH 6). One hundred fifty (150) mg carboxyl-
modified microspheres (Bangs Laboratories, Fishers, IN) in 7.5 mL water is
added
dropwise to 7.5 mL of the hydroxypropylaminodextran solution while vortexing.
One
hundred eighty eight (188) L of EDAC solution (80 mg/mL) in water is added to
the
coating mixture while vortexing. The mixture is incubated overnight at room
temperature
in the dark. The mixture is diluted with 12 mL water and centrifuged. The
supernatant is
discarded and the bead pellet is suspended in 40 mL water by sonication. The
beads are
washed 3 times with water (40 mL per wash) by repeated centrifugation and
suspension by
sonication. The final pellet is suspended in 5 mL water.
Example 3
Conjugation and Release of a Molecular Tag
Figure 7A-B summarize the methodology for conjugation of molecular tag
precursor to an antibody or other binding moiety with a free amino group, and
the reaction
of the resulting conjugate with singlet oxygen to produce a sulfinic acid
moiety as the
released molecular tag. Figure 8 A-J shows several molecular tag reagents,
most of which
utilize 5- or 6-carboxyfluorescein (FAM) as starting material.
Example 4
Preparation of Pro2, Pro4, and Pro6 through Pro13
The scheme outlined in Figure 9A shows a five-step procedure for the
preparation
of the carboxyfluorescein-derived molecular tag precursors, namely, Pro2,
Pro4, Pro6,
Pro7, Pro8, Pro9, ProlO, Proll, Prol2, and Prol3. The first step involves the
reaction of a
5- or 6-FAM with N-hydroxysuccinimide (NHS) and 1,3-dicylcohexylcarbodiimide
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(DCC) in DMF to give the corresponding ester, which was then treated with a
variety of
diamines to yield the desired amide, compound 1. Treatment of compound 1 with
N-
succinimidyl iodoacetate provided the expected iodoacetamide derivative, which
was not
isolated but was further reacted with 3-mercaptopropionic acid in the presence
of
triethylamine. Finally, the resulting (3-thioacid (compound 2) was converted,
as described
above, to its NHS ester. The various e-tag moieties were synthesized starting
with 5- or 6-
FAM, and one of various diamines. The diamine is given H2N A X A NH2 in the
first
reaction of Figure 9A. The regioisomer of FAM and the chemical entity of "X"
within the
diamine are indicated in the table below for each of the molecular tag
precursors
synthesized. Clearly, the diamine, X, can have a wide range of additional
forms, as
described above in the discussion of the mobility modifier moiety.
Precursor FAM X
Prot 5-FAM C(CH3)2
Pro4 5-FAM no carbon
Pro6 5-FAM (CH2)8
Pro? 5-FAM CH2OCH2CH2OCH2
Pro8 5-FAM CH2CH2OCH2CH2OCH2CH2OCH2CH2
Pro9 5-FAM 1,4-phenyl
ProlO 6-FAM C(CH3)2
Proll 6-FAM no carbon
Pro12 6-FAM CH2OCH2CH2OCH2
Pro13 6-FAM CH2CH2OCH2CH2OCH2CH2OCH2CH2
Synthesis of compound 1
To a stirred solution of 5- or 6-carboxyfluorescein (0.5 mmol) in dry DMF (5
mL)
were added N-hydroxysuccinimide (1.1 equiv.) and 1,3-dicylcohexylcarbodiimide
(1.1
equiv.). After about 10 minutes, a white solid (dicyclohexylurea) started
forming. The
reaction mixture was stirred under nitrogen at room temperature overnight. TLC
(9:1
CH2C12-MeOH) indicated complete disappearance of the starting material.
The supernatant from the above mixture was added dropwise to a stirred
solution
of diamine (2-5 equiv.) in DMF (10 mL). As evident from TLC (40:9:1 CH2C12-
MeOH-
H20), the reaction was complete instantaneously. The solvent was removed under
reduced pressure. Flash chromatography of the resulting residue on Iatrobeads
silica
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provided the desired amine (compound 1) in 58-89% yield. The 1H NMR (300 MHz,
DMSO-d6) of compound 1 was in agreement with the assigned structure.
Synthesis of compound 2
To the amine (compound 1) (0.3 mmol) were sequentially added dry DMF (10 mL)
and N-succinimidyl iodoacetate (1.1 equiv.). The resulting mixture was stirred
at room
temperature until a clear solution was obtained. TLC (40:9:1 CH2C12-MeOH-H20)
revealed completion of the reaction.
The above reaction solution was then treated with triethylamine (1.2 equiv.)
and 3-
mercaptopropionic acid (3.2 equiv.). The mixture was stirred at room
temperature
overnight. Removal of the solvent under reduced pressure followed by flash
chromatography afforded the (3-thioacid (compound 2) in 62-91% yield. The
structure of
compound 2 was assigned on the basis of its 1NMR (300 MHz, DMSO-d6).
Synthesis of Pro2, Pro4, and Pro6 through Prol3
To a stirred solution of the (3-thioacid (compound 2) (0.05 mmol) in dry DMF
(2
mL) were added N-hydroxysuccinimide (1.5 equiv.) and 1,3-
dicylcohexylcarbodiimide
(1.5 equiv.). The mixture was stirred at room temperature under nitrogen for
24-48 h
(until all of the starting material had reacted). The reaction mixture was
concentrated
under reduced pressure and then purified by flash chromatography to give the
target
molecule in 41-92% yield.
Preparation of Prol
The compounds of this reaction are shown in Figure 9B. To a stirred solution
of 5-
iodoacetamidofluorescein (compound 4) (24 mg, 0.047 mmol) in dry DMF (2 mL)
were
added triethylamine (8 L, 0.057 mmol) and 3-mercaptopropionic acid (5 L,
0.057
mmol). The resulting solution was stirred at room temperature for 1.5 h. TLC
(40:9:1
CH2C12-MeOH-H20) indicated completion of the reaction. Subsequently, N-
hydroxysuccinimide (9 mg, 0.078 mmol) and 1,3-dicylcohexylcarbodiimide (18 mg,
0.087
mmol) were added. The reaction mixture was stirred at room temperature under
nitrogen
for 19 h at which time TLC showed complete disappearance of the starting
material.
Removal of the solvent under reduced pressure and subsequent flash
chromatography
using 25:1 and 15:1 CH2C12-MeOH as eluant afforded Prol (23 mg, 83%).
Preparation of Pro3
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The compounds of this reaction are shown in Figure 9C. To a stirred solution
of 6-
iodoacetamidofluorescein (compound 5) (26 mg, 0.050 mmol) in dry DMF (2 mL)
were
added triethylamine (8 L, 0.057 mmol) and 3-mercaptopropionic acid (5 L,
0.057
mmol). The resulting solution was stirred at room temperature for 1.5 h. TLC
(40:9:1
CH2C12-MeOH-H20) indicated completion of the reaction. Subsequently, N-
hydroxysuccinimide (11 mg, 0.096 mmol) and 1,3-dicylcohexylcarbodiimide (18
mg,
0.087 mmol) were added. The reaction mixture was stirred at room temperature
under
nitrogen for 19 h at which time TLC showed complete disappearance of the
starting
material. Removal of the solvent under reduced pressure and subsequent flash
chromatography using 30:1 and 20:1 CH2C12-MeOH as eluant provided Pro3 (18 mg,
61%).
Preparation of ProS
The compounds of this reaction are shown in Figure 9D.
Synthesis of compound 7
To a stirred solution of 5-(bromomethyl)fluorescein (compound 6) (40 mg, 0.095
mmol) in dry DMF (5 mL) were added triethylamine (15 L, 0.108 mmol) and 3-
mercaptopropionic acid (10 L, 0.115 mmol). The resulting solution was stirred
at room
temperature for 2 days. TLC (40:9:1 CH2C12-MeOH-H20) indicated completion of
the
reaction. The reaction solution was evaporated under reduced pressure.
Finally, flash
chromatography employing 30:1 and 25:1 CH2C12-MeOH as eluant provided the J3-
thioacid (compound 7) (28 mg, 66%).
Synthesis of ProS
To a solution of the acid (compound 7) (27 mg, 0.060 mmol) in dry DMF (2 mL)
were added N-hydroxysuccinimide (11 mg, 0.096 mmol) and 1,3-
dicylcohexylcarbodiimide (20 mg, 0.097 mmol). The reaction mixture was stirred
at room
temperature under nitrogen for 2 days at which time TLC (9:1 CH2C12-MeOH)
showed
complete disappearance of the starting material. Removal of the solvent under
reduced
pressure and subsequent flash chromatography with 30:1 CH2C12-MeOH afforded
ProS
(24 mg, 73%).
Preparation of Pro14
The compounds of this reaction are shown in Figure 9E.
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Synthesis of compound 9
To 5-aminoacetamidofluorescein (compound 8) (49 mg, 0.121 mmol) were
sequentially added dry DMF (4 rL) and N-succinimidyl iodoacetate (52 mg,
0.184). A
clear solution resulted and TLC (40:9:1 CH2C12-MeOH-H20) indicated complete
disappearance of the starting material.
The above reaction solution was then treated with triethylamine (30 L, 0.215
mmol) and 3-mercaptopropionic acid (30 L, 0.344 mmol). The resulting mixture
was
stirred for 2 h. Removal of the solvent under reduced pressure followed by
flash
chromatography using 20:1 and 15:1 CH2C12-MeOH as eluant gave the j3-thioacid
(compound 9) (41 mg, 62%). The structural assignment was made on the basis of
1NMR
(300 MHz, DMSO-d6).
Synthesis of Pro14
To a stirred solution of compound 9 (22 mg, 0.04 mmol) in dry DMF (2 mL) were
added N-hydroxysuccinimide (9 mg, 0.078 mmol) and 1,3-dicylcohexylcarbodiimide
(16
mg, 0.078 mmol). The resulting solution was stirred at room temperature under
nitrogen
for about 24 h. The reaction mixture was concentrated under reduced pressure
and the
residue purified by flash chromatography using 30:1 and 20:1 CH2C12-MeOH as
eluant to
give Pro14 (18 mg, 70%).
Synthesis of Prol5, Pro20, Pro22, and Pro28
The synthesis schemes for producing NHS esters of molecular tags Pro 15,
Pro20,
Pro22, and Pro28 are shown in Figures 16 F-I, respectively. All of the reagent
and
reaction conditions are conventional in the art and proceed similarly as the
reactions
described above.
Example 5
Attachment of Molecular Tags and Antibodies to
Aminodextran Derivatized Microspheres
In this example, Pro28-NHS and the heterobifunctional cross-linking agent SIAX
are reacted with aminodextran-coated microspheres to give microspheres having
molecular tags and sulfhydryl-reactive iodoacetyl moieties attached.
Separately,
antibodies having free sulfhydyl groups are prepared using the
heterobifunctional cross-
linking agent succinimidyl acetylthioacetate (SATA) in a conventional reaction
that
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converts free amine groups of the antibodies to free sulfhydryls. In a second
step, the
SATA-modified antibodies are reacted with the microspheres to produce the
desired
composition.
Sixty five (65) mg of hydroxypropylaminodextran-coated microspheres (prepared
as described above) are suspended in 5 mL 50 mM MOPS pH 7. A 10% (w/v)
solution
(SAIX:Pro28 in 1:2 molar ratio)("tag solution") is prepared in DMSO and 77 L
was
added to the microsphere suspension while vortexing. The mixture is incubated
at room
temperature for an additional 90 minutes in the dark and then a second 77 tL
aliquot of
tag solution is added and the mixture is incubated for an additional 60
minutes. The
suspension is centrifuged and the supernatant is discarded. The microsphere
pellet is
suspended in 6 mL water by sonication and the centrifugation repeated. The
pellet is
suspended in 6.5 mL water and stored at 4 C.
SATA-modified antibody is prepared as described in Hermanson (cited above),
pgs. 467-469, and is purified by gel filtration on a Sephadex G-25 column, or
like column.
In preparation for antibody coupling the microspheres are centrifuged, the
supernatant is
discarded and 1.34 mL coupling buffer is added to the pellet. Coupling buffer
consists of
the following mixture: 900 L 0.2 M borate, 2 mM EDTA pH 9 and 333 L of 0.4 M
borate pH 9.45 and 1000 L of 2 M sodium sulfate. The mixture is degassed and
saturated
with argon and then 9 L of 10% Tween 20 detergent is added. The antibody
solution is
added to the pelleted microspheres in coupling buffer and the mixture is
sonicated to
suspend the microspheres. The suspension is incubated at 37 C for 23 hr.
Residual iodo
groups of the iodoaminodextran coat are capped by reaction with mercaptoacetic
acid.
After centrifugation, the pelleted microspheres are suspended by sonication in
5 mL of 10
mM mercaptoacetic acid in 0.4 M borate pH 9.45 and the mixture is incubated at
37 C.
for 1 hr. After several washes, the derivatived microspheres may be combined
with
similarly prepared microspheres having different molecular tags and antibodies
to form a
composition of the invention.
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