U.S. patent application number 11/592042 was filed with the patent office on 2007-03-08 for fluorescence polarization assay.
Invention is credited to I. Lawrence Greenfield, Michael M. A. Sekar.
Application Number | 20070054308 11/592042 |
Document ID | / |
Family ID | 31495914 |
Filed Date | 2007-03-08 |
United States Patent
Application |
20070054308 |
Kind Code |
A1 |
Sekar; Michael M. A. ; et
al. |
March 8, 2007 |
Fluorescence polarization assay
Abstract
The present invention relates to methods for detecting the
presence of one or more analytes of interest in a sample by
measuring changes in fluorescence anisotropy as a result of binding
of the analytes to specific aptamers. The aptamers are immobilized
on a solid support and may be in the form of an array.
Inventors: |
Sekar; Michael M. A.; (Santa
Clara, CA) ; Greenfield; I. Lawrence; (San Mateo,
CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
31495914 |
Appl. No.: |
11/592042 |
Filed: |
November 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10628879 |
Jul 28, 2003 |
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11592042 |
Nov 1, 2006 |
|
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60401021 |
Aug 2, 2002 |
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Current U.S.
Class: |
435/6.19 ;
436/518 |
Current CPC
Class: |
C12Q 1/6837 20130101;
G01N 33/54313 20130101; C12Q 1/6837 20130101; G01N 33/582 20130101;
C12Q 2561/119 20130101; C12Q 2525/205 20130101 |
Class at
Publication: |
435/006 ;
436/518 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/543 20060101 G01N033/543 |
Claims
1. A method for detecting an analyte in a sample comprising: (a)
contacting a sample with a fluorophore-labeled aptamer bound to a
solid support; (b) directly illuminating the aptamer with polarized
light whereby the direct illumination of the fluorophore directly
excites the fluorophore; (c) measuring the fluorescence anisotropy
of the fluorophore; and (d) identifying a presence or amount of the
analyte when said fluorescence anisotropy measurement is greater
than an anisotropy measurement obtained in the absence of the
analyte.
2. The method of claim 1 wherein the aptamer comprises between
about 10 and about 100 nucleotides.
3. The method of claim 1 wherein the aptamer is labeled with a
fluorophore selected from the group consisting of fluorescein
derivatives, eosin derivatives, coumarin derivatives, and rhodamine
derivatives.
4. The method of claim 3 wherein the aptamer is labeled with
carboxyfluorescein (FAM).
5. The method of claim 1 wherein the aptamer is part of an array of
aptamers.
6. The method of claim 5 wherein the array comprises two or more
addressable locations.
7. The method of claim 6 wherein each addressable location
comprises a single type of aptamer.
8. The method of claim 6 wherein each addressable location
comprises multiple types of aptamers.
9. The method of claim 8 wherein each type of aptamer is labeled
with a fluorophore with unique spectral characteristics.
10. The method of claim 1 wherein the polarized light is laser
light.
11. The method of claim 1 wherein the analyte is associated with a
disease or disorder.
12. The method of claim 1 wherein the sample is obtained from a
patient suspected of suffering from a disease or disorder.
13. The method of claim 1 wherein the analyte is a protein.
14. The method of claim 1 wherein the analyte is a metabolite.
15. The method of claim 1 wherein the sample is from a human
patient and the analyte is associated with a disease or disorder.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 10/628,879, filed Jul. 28, 2003, which claims
priority under 35 U.S.C. .sctn.119(e) to U.S. Provisional
Application No. 60/401,021, filed Aug. 2, 2002, the entireties of
both of which are incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for detecting the
binding of analytes of interest to aptamers by measuring changes in
fluorescence anisotropy.
DESCRIPTION OF THE RELATED ART
[0003] The introduction of immunoassays in 1959 and the
enzyme-linked immunosorbent assay (ELISA) in 1971 revolutionized
clinical diagnostic medicine. Until recently, molecules capable of
molecular recognition, and therefore useful in diagnostic assays,
have been limited to antibodies. The recent development of in vitro
methods to select high affinity ligands by combinatorial chemistry
methodologies promises unique novel therapeutics and diagnostic
reagents. These methods allow for the identification of a large
number of different oligonucleotide sequences with high affinity
and specificity. For example, oligonucleotide sequences can be
identified by the systematic evolution of ligands by exponential
enrichment (SELEX.TM.), a general method for identification of
oligonucleotide ligands, also known as aptamers, as potential
drugs. In this method, a pool of RNAs, completely randomized at
particular positions, is subjected to selection for binding to a
particular nucleic-acid binding protein which has been immobilized
onto a nitrocellulose filter. The bound RNAs or DNAs are then
recovered and further amplified as DNA. The DNA may be subjected to
further characterization. These oligonucleotides can be used as
probes for the identification of a variety of analytes,
particularly proteins including various enzymes of HIV, growth
factors and inflammation-inducing enzymes (Sun, S., Curr. Opin.
Mol. Ther., 2(1):100-5 (2000)).
[0004] The platforms that have typically been used to identify and
quantify aptamers bound to analytes are very limited in that they
require a method for separating the bound product from one or both
unbound reactants. Examples for separating the bound product from
unbound reactants include binding to a solid phase, liquid
chromatography and electrophoresis.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention provides a method for
detecting an analyte in a sample. A fluorophore-labeled aptamer
bound to a solid support is contacted with the sample and
illuminated with polarized light. The fluorescence anisotropy of
the fluorophore is measured. and the presence of the analyte is
identified when the fluorescence anisotropy value is greater than
an anisotropy value obtained in the absence of the sample.
[0006] In one embodiment the solid support to which the
fluorophore-labeled aptamer is bound is a bead, such as a silica
bead. The bead may have a diameter between about 1 .mu.m and about
10 .mu.m. In a particular embodiment the bead has a diameter of
about 5 .mu.m. The bead may be suspended in solution or arranged in
a two-dimensional array.
[0007] In another embodiment the aptamer is labeled with a
fluorophore selected from the group consisting of fluorescein
derivatives, eosin derivatives, coumarin derivatives and rhodamine
derivatives. In a particular embodiment the fluorophore is
carboxyfluorescein.
[0008] The aptamer may be part of an array of aptamers. In one
embodiment the array of aptamers comprises two or more addressable
locations. Each addressable location may comprise a single type of
aptamer. In another embodiment each addressable location comprises
multiple types of aptamers.
[0009] In one embodiment the polarized light used to illuminate the
aptamer is laser light.
[0010] In another embodiment the analyte of interest is associated
with a disease or disorder. The sample may be obtained from a
patient suspected of suffering from a disease or disorder.
[0011] In one embodiment the analyte is a protein. In another
embodiment the analyte is a metabolite.
[0012] In another aspect, a sample is obtained from a patient
suspected of suffering from a disease or disorder. An analyte is
identified that is associated with the disease or disorder and the
presence or absence of the analyte is determined in the sample. The
patient is diagnosed as suffering from the disease or disorder if
the analyte is determined to be present in the sample taken from
the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows kinetic data of binding of thrombin and
lysozyme to FAM-labeled anti-thrombin aptamer coupled beads.
[0014] FIG. 2 shows kinetic data of binding of GP 120 to FAM-518
aptamer coupled beads.
[0015] FIG. 3 shows kinetic data of binding of lysozyme to FAM-518
aptamer coupled beads.
[0016] FIG. 4 shows kinetic data of reversible binding of lysozyme
to FAM-518 aptamer coupled beads.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0017] In one aspect, the present invention provides methods of
identifying one or more analytes in a sample by measuring changes
in fluorescence anisotropy resulting from the binding of the
analytes to a labeled aptamer.
Definitions
[0018] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. One
skilled in the art will recognize many methods and materials
similar or equivalent to those described herein, which could be
used in the practice of the present invention. Indeed, the present
invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0019] As used herein, an "aptamer" is an oligonucleotide that is
able to specifically bind an analyte of interest other than by base
pair hybridization. Aptamers typically comprise DNA or RNA or a
mixture of DNA and RNA. Aptamers may be naturally occurring or made
by synthetic or recombinant means. The aptamers are typically
single stranded, but may also be double stranded or triple
stranded. They may comprise naturally occurring nucleotides,
nucleotides that have been modified in some way, such as by
chemical modification, and unnatural bases, for example
2-aminopurine. See, for example, U.S. Pat. No. 5,840,867. The
aptamers may be chemically modified, for example, by the addition
of a label, such as a fluorophore, or a by the addition of a
molecule that allows the aptamer to be crosslinked to a molecule to
which it is bound. Aptamers are of the same "type" if they have the
same sequence or are capable of specific binding to the same
molecule. The length of the aptamer will vary, but is typically
less than about 100 nucleotides.
[0020] A large number of aptamers are known in the art, and may be
selected for use in particular applications based on their known
properties. Alternatively, new aptamers may be prepared and
identified by selection from random pools of oligonucleotides based
on their ability to bind the specific molecule of interest. For
example, new aptamers may be prepared and identified by the
Systematic Evolution of Ligands by Exponential Enrichment
(SELEX.TM.) process, described, for example, in U.S. Pat. Nos.
5,475,096 and 5,270,613.
[0021] The aptamers are labeled with one or more fluorophores. A
fluorophore may also be referred to as a fluorescent probe or dye.
A "fluorophore" is any molecule that when excited with light of a
particular wavelength, emits light of a different wavelength.
Fluorophores include, but are not limited to, fluoresceins (U.S.
Pat. Nos. 5,188,934; 6,008,379; 6,020,481), eosin, coumarin,
rhodamines (U.S. Pat. Nos. 5,366,860; 5,847,162; 5,936,087;
6,051,719; 6,191,278), such as tetramethylrhodamine,
benzophenoxazines (U.S. Pat. No. 6,140,500), Texas Red and dansyl
derivatives. Fluorescent reporter dyes useful for labeling
biomolecules include energy-transfer dye pairs of donors and
acceptors (U.S. Pat. Nos. 5,863,727; 5,800,996; 5,945,526), and
cyanines (Kubista, WO 97/45539), as well as any other fluorescent
label capable of generating a detectable signal. Examples of
fluorescein dyes include 5-carboxyfluorescein ("FAM"),
6-carboxyfluorescein ("6-FAM"); 2',4',4,7,-tetrachlorofluorescein;
and 2',4',5',7',4,7-hexachlorofluorescein. See U.S. Pat. No.
5,118,934.
[0022] The fluorophore may be incorporated into the aptamer at any
position. For example, the aptamer may be labeled along its
backbone, at a position in the bases, or at either end. The aptamer
may be labeled by any method known in the art, such as by using
standard DNA synthesis techniques using fluorescently labeled
linker compounds. Alternatively, unlabeled linker compounds can be
used during synthesis and subsequently labeled with a fluorophore.
(See, for example, Ruth, J. (1991) Oligo and Analogues, pp.
255-282, Eckstein, F, Ed., IRL Press, Oxford, UK; Vinayak R. (1999)
Tetrahedron Lett. 40:7611-7813.) When aptamers are prepared by the
SELEX.TM. process, the fluorophore may be incorporated into the
selection process to insure that the fluorophore does not perturb
binding of the analyte to the aptamer. In addition, identification
of fluorophore positions that are part of the aptamer binding
domain may insure that binding of the analyte results in a
significant change in fluorescence anisotropy.
[0023] A "label" is any moiety which can be attached to a
polynucleotide and provide a detectable signal, such as a
fluorophore.
[0024] "Fluorescence" is the emission of light from a sample or
dye, which is longer in wavelength than light which falls on the
sample or dye. The light falling on the sample or dye, usually
referred to as the "excitation" light, is absorbed by the sample or
dye and then emitted as "emission" light.
[0025] "Anisotropy" is the difference in the property of a system
with changes in direction. In the context of "fluorescence
anisotropy," this means a difference in the polarization of the
emission light, as hereinabove defined.
[0026] As used herein, the terms "polynucleotide" and
"oligonucleotide" are used interchangeably and mean
single-stranded, double-stranded and triple-stranded polymers of
nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and
ribonucleotides (RNA). A polynucleotide may be composed entirely of
deoxyribonucleotides, entirely of ribonucleotides, or chimeric
mixtures thereof. Polynucleotides may be comprised of
internucleotide, nucleobase and sugar analogs, including unnatural
bases, sugars, L-DNA and modified internucleotide linkages.
[0027] "Linker" refers to a chemical moiety in a molecule
comprising a covalent bond or a chain of atoms that covalently
attaches one moiety or molecule to another, e.g. a fluorophore to
an aptamer or an aptamer to a solid support.
[0028] A linker may comprise a removable protective group. A
"protective group" is a material which is bound to a molecule and
may be removed upon selective exposure to an activator, such as
light.
[0029] The term "solid support" refers to any solid phase material
upon which an aptamer may be attached or immobilized. For example,
a solid support may comprise glass, metal, silicon, germanium,
GaAs, or plastic. Solid support encompasses terms such as "resin",
"solid phase", and "support". A solid support may be composed of
organic polymers such as polystyrene, polyethylene, polypropylene,
polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as
co-polymers and grafts thereof. A solid support may also be
inorganic, such as glass, silica, controlled-pore-glass (CPG), or
reverse-phase silica. The configuration of a solid support may be
in the form of beads, spheres, particles, granules, a gel, a fiber
or a surface. Surfaces may be planar, substantially planar, or
non-planar. Solid supports may be porous or non-porous, and may
have swelling or non-swelling characteristics. A solid support may
be configured in the form of a well, depression or other container,
vessel, feature or location. A plurality of solid supports may be
configured in an array.
[0030] "Array" or "microarray" means a predetermined spatial
arrangement of aptamers present on a substrate. The aptamers may be
directly attached to the substrate, or may be attached to a solid
support that is associated with the substrate. The aptamers may all
be identical, as in the case of an array that is designed to detect
a single analyte, or the aptamers may be different, such as in an
array that is designed to detect and/or identify a variety of
different molecules in a sample. The array may comprise one or more
"addressable locations," that is, physical locations that comprise
a known type of aptamer. In one embodiment an addressable location
comprises more than one type of aptamer. However, the types of
aptamers present at each location are known or may be
determined.
[0031] An array can comprise any number of addressable locations,
e.g. 1 to about 100 (low number), 100 to about 1000 (medium number)
or a thousand or more (high number). In addition, the density of
the addressable locations on the array may be varied. For example,
the density of the addressable locations on a substrate may be
increased to reduce the necessary substrate size. Typically, the
array format is a geometrically regular shape, which may
facilitate, for example, fabrication, handling, stacking, reagent
and sample introduction, detection, and storage. The array may be
configured in a row and column format, with regular spacing between
each location. Alternatively, the locations may be arranged in
groups, randomly, or in any other pattern. In one embodiment an
array comprises a plurality of addressable locations configured so
that each location is spatially addressable for high-throughput
handling.
[0032] In a two-dimensional array the addressable location is
determined by location on the surface. However, in one embodiment
the array comprises a number of particles, such as beads, in
solution. Each particle comprises a specific type or types of
aptamer. In this case the identity of the aptamer may be determined
by the characteristics of the particle. For example, the particle
may have an identifying characteristic, such as shape, pattern,
chromophore, or fluorophore.
[0033] "Target" and "analyte" both refer to a specific molecule or
compound, the presence, absence or amount of which is to be
detected, and that is capable of interacting with an aptamer. The
analyte may be any molecule, including, without limitation, a
polypeptide, protein, protein complex, oligonucleotide, DNA, RNA,
carbohydrate, polysaccharide, metabolite, nutrient, drug or small
molecule. The analyte may be naturally occurring or synthetic. In
one embodiment an analyte is a polypeptide derived from a living,
or once living, organism, including but not limited to prokaryote,
eukaryote, plant, animal, and virus.
[0034] "Substrate" when used herein refers to the underlying core
material of the arrays of the invention. Typically the substrate is
a solid support and has a rigid or semi-rigid surface. In one
embodiment the surface of the substrate is flat. In other
embodiments the surface of the substrate may comprise physical
features, such as wells, trenches and raised or sunken regions. The
aptamers that form the array may be attached directly to the
substrate, or may be attached to a solid support that is itself
associated with, such as attached to or contained by, the
substrate.
Exemplary Modes for Carrying Out the Invention
[0035] The present invention provides methods for determining the
presence, absence or quantity of one or more analytes in a sample
based on changes in fluorescence anisotropy upon binding of an
analyte to a fluorescently labeled aptamer. Fluorescence anisotropy
arises from fluorescence polarization, which reveals the average
angular displacement of the fluorophore, which occurs between
absorption and subsequent emission of a photon.
[0036] As discussed above, the analytes are not limited in any way,
and thus the methods disclosed herein are broadly applicable to
many different fields. The identity of the analytes will vary
depending on the nature of the analysis and one of skill in the art
in a particular field will be able to adapt the methods to their
specific circumstances.
[0037] The presence or absence of one or more analytes is
determined in a sample. The sample may be any composition that is
desired to be analyzed for the presence of an analyte and is not
limited in any way. For example, without limitation, in a medical
setting it may be desirable to analyze a sample of bodily fluid,
such as blood, from a patient for the presence of a polypeptide
associated with a disease or disorder, such as an infectious agent,
while in an industrial setting it may be desirable to analyze a
waste stream for the presence of a particular organic compound.
[0038] Aptamers that are specific for the analytes of interest are
identified. The aptamer may be one that has been previously
identified as capable of binding the analyte of interest. Many such
aptamers are known in the art. For example, a database of aptamers
with known selectivity is maintained at the University of Texas at
Austin (http://aptamer.icmp.utexas.edu). A number of different
aptamers can also be obtained commercially, such as from Somologic
(Colorado, USA) or Gilead Sciences, Inc. (California, USA). If an
aptamer that is specific for the analyte of interest has not been
described previously, it can be identified by any method known in
the art. Methods for identifying aptamers that interact with
specific compounds are well known. For example, an aptamer that is
specific for a particular ligand can be identified by the
Systematic Evolution of Ligands by Exponential Enrichment
(SELEX.TM.) process, described, for example, in U.S. Pat. Nos.
5,475,096 and 5,270,613. In the SELEX.TM. process a pool of
randomized RNA or single stranded DNA sequences are selected
against a desired target. The sequences that show tighter binding
with the target are isolated and amplified. The selection is
repeated several times using the enriched pool derived from the
previous round of selection to identify useful aptamers.
[0039] Once an aptamer has been identified, one or more copies of
the aptamer are produced for use in the assay to detect the
presence of the analyte. The aptamers may be produced by any method
known in the art. Thus, they may be chemically synthesized by
standard procedures. The aptamers may be labeled with a
fluorophore, either during or after synthesis. They may be labeled
by any method known in the art. For example, the aptamer may be
labeled by incorporation of a modified nucleotide during synthesis.
Alternatively, the aptamer may be chemically modified after
synthesis. Following labeling, the aptamers are contacted with the
sample to be analyzed. In another embodiment, the label can be
incorporated during the SELEX.TM. process. This may help to place
the label within the binding site for the analyte, which in turn
will ensure the greatest change in fluorescence anisotropy upon
analyte binding.
[0040] The aptamers are typically present on a solid support when
they are contacted with the sample to be analyzed. The aptamers can
be synthesized directly on the solid support, or, alternatively,
the aptamers may be bound to the solid support following synthesis.
See, for example, Southern et al., Nuc. Acids Res., 20(7):1679-1684
(1992); Southern et al., Genomics, 13:1008-10017 (1992);
WO02/30561; WO02/0750; WO96/40790; Maclean et al. Proc. Natl. Acad.
Sci. USA 94:2805-2810 (1997); and U.S. Pat. Nos. 5,744,305,
5,405,783, 5,445,934 and 6,261,776. The aptamers may be bound to
the solid support by any method known in the art. For example,
3'-amine-modified aptamers may be applied to activated surfaces of
the solid support (Potyrailo et al., Analytical Chemistry,
70(16):3419-3425 (1998)). In one embodiment a glass substrate is
activated with (glycidoxypropyl) trimethoxy silane (GOPS).
Fluorescently labeled aptamers, modified by attachment of an
alkylamino group to the 3' end, are subsequently covalently bound
to the glass substrate. Unreacted aptamers may be removed by
washing, and unreacted surface groups may be blocked. In one
embodiment unreacted surface groups are blocked by an incubation
with 0.1 M ethanolamine solution. In another particular embodiment,
aptamers with C6-aminolinker at the 5' end are bound to treated
silica particles.
[0041] In another embodiment the aptamers are attached to a solid
support via linker molecules. Linker molecules are provided on the
surface of the substrate. The linker molecules are then contacted
with the aptamers under conditions such that the aptamers are bound
to the substrate via the linker molecules.
[0042] In a particular embodiment, arrays of aptamers are formed on
a substrate comprising linker molecules wherein the distal ends of
the linker molecules comprise a functional group with a protective
group. The formation of such arrays is described, for example, in
U.S. Pat. Nos. 5,445,934, 5,744,305 and 5,405,783, the disclosures
of which are incorporated herein by reference. The protective group
may be removed to expose the functional group by exposing the
linker molecule to the proper conditions, such as light, radiation,
electric fields, electric currents or other activators. By
directing the activator to particular linker molecules, a defined
portion of the substrate can be activated. For example, if the
protective group is removable by light, a defined region of the
substrate may be illuminated, thus activating the linker molecules
in that area. A particular type of aptamer may then be contacted
with the activated linker molecules. Excess aptamer is then removed
and any unreacted linker molecules are blocked. In this way, an
addressable location comprising a particular type of aptamer is
produced. A different discrete area of linker molecules on the
substrate may then be activated. A second type of aptamer may then
be bound to the second discrete area. By repeating this process an
array comprising a number of addressable locations comprising known
aptamer types may be formed on the substrate. In an alternative
embodiment, rather than attaching aptamers that have been
previously synthesized, aptamers can be synthesized directly on the
activated areas of the substrate.
[0043] The aptamers may be bound to a solid support that is
suspended in solution. For example, the aptamers may be bound to
beads that are subsequently suspended in buffer. In one embodiment
the beads are suspended in wells, such as in the wells of a
microtiter plate. Each well may contain beads that each comprise
the same type of aptamer. In this way, multiple samples may be
analyzed simultaneously for the presence of a single analyte. In
another embodiment each well contains beads that comprise a
different type of aptamer, to allow for detection of a variety of
different analytes. In yet another embodiment, each well contains
beads comprising a variety of different aptamers, allowing for the
determination of the presence of one or more of a variety of
analytes.
[0044] The presence of an analyte of interest in a sample is
determined by identifying a change in the fluorescence anisotropy
of the fluorophore attached to the aptamer upon binding of the
analyte to the aptamer. Baseline fluorescence anisotropy in the
absence of the analyte is measured, as described below.
Subsequently, the aptamer is contacted with the sample to be
analyzed for the presence of the analyte and fluorescence
anisotropy is again measured. The presence of the analyte is
indicated by observation of a change in fluorescence anisotropy.
Alternatively, the baseline fluorescence anisotropy can be
determined from an aptamer-coated solid-phase that is run in
parallel and is not subsequently contacted with sample.
[0045] Fluorescent intensities are determined for selected
positions of the excitation and emission polarizers. The
fluorophore bound to the aptamer is excited by illumination with
polarized light. Any excitation source known in the art may be
used. For example, light from a xenon arc lamp may be polarized and
focused on the desired area. In another embodiment, a laser
excitation source is used, thus eliminating the need for an
excitation polarizer.
[0046] Fluorescent measurements may be made by any method known in
the art, such as with single channel detection and in either right
angle (90.degree.), in T or L format, or front face emission
collection geometry using a fluorescence spectrometer, for example,
a Flurolog-3.TM. instrument, which is commercially available from
SPEX (NJ, USA). See, for example, Lakowicz, "Principles of
Fluorescence Spectroscopy," p. 111-153, Plenum Press, New York, New
York (1986) and Potyrailo et al. Anal. Chem. 70:3419-3425
(1998).
[0047] A typical apparatus for measuring the change in fluorescence
anisotropy will thus comprise an excitation source, an excitation
filter that passes the desired wavelength of light, a polarizing
filter that polarizes the light from the excitation source prior to
the excitation light contacting the aptamer(s), and a lens for
focusing the excitation light on the aptamer(s) of interest, such
as at an addressable location on a substrate. In one embodiment, a
laser is used. In this embodiment the polarizer and lens may not be
required due to the nature of laser light.
[0048] The apparatus will also typically comprise a sample chamber,
one or more emission polarizers to polarize the emitted light, an
emission filter to pass the desired wavelength of emitted light and
a detector for collecting and quantitating the amount of emitted
light.
[0049] Fluorescence anisotropy arises from fluorescence
polarization, which reveals the average angular displacement of the
fluorophore, which occurs between absorption and subsequent
emission of photon.
[0050] Fluorescence anisotropy (FA) is calculated from measurements
of emission intensity, I, as
FA=(I.sub.vv-GI.sub.vh)/(I.sub.vv+2Gl.sub.vh) (I)
[0051] Where G is the instrumental correction factor,
G=I.sub.hv/I.sub.hh and the subscripts v and h refer to the
vertical and horizontal orientation of the polarizer,
respectively.
[0052] In one embodiment, measurements are taken with a detection
time constant of about 1 s at a temperature of about 25.degree.
C.
[0053] The change in polarization anisotropy of the fluorophore
labeled aptamer with time on binding to the analyte of interest may
be determined in order to determine the kinetics of analyte binding
to the aptamer.
[0054] The sensitivity of this method allows for the identification
of small amounts of an analyte of interest in a sample. For
example, for aptamers that bind to an analyte with high affinity,
detection down to 1 to 10 pM of analyte is achievable.
[0055] Bead Arrays
[0056] Arrays of aptamers may be used to identify the presence of
one or more analytes in a sample. In one aspect of the invention,
the arrays comprise aptamers that are immobilized onto the surface
of a substrate. In one embodiment, labeled aptamers that are
specific for the analytes of interest are bound to microspheres or
beads. The beads may then, be used to form an array useful for the
detection of one or more analytes. See Walton et al. Analytic.
Chem. 74:2240-2247 (2002); Zhou et al. Trends in Biotech.
19:S34-S39 (2001); WO02/24959; WO00/50903; WO00/61803 and U.S. Pat.
Nos. 5,340,422 and 6,074,609.
[0057] The aptamer-attached beads may be free in solution or
physically attached to or associated with a substrate. When free in
solution, the fluorescence anisotropy may be measured in a flow
cytometer. If the beads are attached to the surface of a solid
support or substrate, they may be attached at particular sites to
form an array with one or more addressable locations.
[0058] "Microspheres" or "beads" are small discrete particles.
While the beads generally have a spherical geometry, their shape
may be irregular. In one embodiment the irregular size or shape
identifies a bead as comprising a particular type of aptamer.
Typically beads have a diameter from about 1 .mu.m to about 10
.mu.m. Alternatively they may have a diameter from about 10 nm to
about 1 mm, or even from about 10 nm to about 100 .mu.m. The beads
may be comprised of any material to which an aptamer may be bound,
including, without limitation, plastic, glass, ceramic, metal,
cellulose, nylon or latex.
[0059] The number of aptamers on a single bead is not limited in
any way. Typically, however, each bead will comprise at least about
10.sup.5 aptamers. Each bead may comprise a single type of aptamer.
Alternatively, however, each bead may comprise more than one type
of aptamer. This may be useful, for example, if the presence of at
least one of a number of compounds is to be determined in a sample,
and it is not necessary to know which particular compound is
present. In another embodiment, each bead may contain several
different types of aptamers, each comprising a different
fluorophore. In such a manner, each bead may be capable of
detecting multiple analytes.
[0060] The aptamers may be synthesized directly on the beads, or
may be made and subsequently attached to the beads using linker
molecules. For example, the surface of the bead may be modified to
allow the attachment of the aptamer, such as with a chemically
reactive group like a thiol or an amine. Such modifications are
well known in the art. The aptamers bound to the beads may be
fluorescently labeled before or after attachment to the beads.
[0061] The aptamer-coupled beads may be used to detect the presence
of one or more analytes in a sample. Aptarner-coupled beads may
themselves be attached to a solid support. Typically however, the
beads are placed in solution in a vessel, for example, a cuvette or
a well of a microtiter plate.
[0062] An array of beads may be used to detect a single analyte in
a sample. In this case, all of the beads in the array will be of
the same type and thus will typically be derivatized with the same
type of aptamer.
[0063] In one embodiment, beads labeled with fluorescently labeled
aptamers that are specific for the analyte of interest are placed
in solution in a vessel, for example a cuvette or a well of a
microtiter plate. A sufficient number of aptamer-coupled beads must
be present to generate a detectable signal. At least about 10.sup.6
beads are generally present in solution in the vessel. The sample
to be analyzed is added to the vessel. Fluorescence anisotropy is
measured as described above, both before and after addition of the
sample. A change in fluorescence anisotropy following addition of
the sample is indicative of the presence of the analyte in the
sample.
[0064] For the simultaneous analysis of multiple samples for the
presence of a single analyte, a number of vessels may be used. A
number of individual vessels, such as a number of cuvettes, can be
used. Alternatively, the vessels can be associated with each other
to facilitate rapid handling, such as in a multi-well microtiter
plate. In one embodiment, each well of a multi-well microtiter
plate comprises the same type of aptamer-coupled beads in solution.
The samples are then added to the wells, one sample per well.
Fluorescence anisotropy is measured in each well before and after
addition of the sample, A change in fluorescence anisotropy upon
addition of the sample is indicative of the presence of the analyte
in the sample. Samples that were added to wells in which a change
in fluorescence anisotropy was measured are identified as
comprising the analyte of interest.
[0065] An array comprising aptamer-coupled beads may also be used
to determine if one or more of a variety of different analytes are
present in a sample. In this embodiment a number of different types
of aptamers, equal to or greater than the number of analytes to be
identified, will be represented in the population of beads.
[0066] Typically, the identity of any analytes present is of
interest. Thus, it is necessary to identify the type of aptamer on
each bead at each location in the array so that the binding of
different analytes can be distinguished. This may be achieved by
individually placing beads with known aptamers in the array.
Alternatively, the beads may be randomly distributed in the array
and the specific location of individual beads in the array
determined after the array is formed. This may be accomplished by
any method known in the art. For example, the beads may be coded
such as with a fluorophore, bar-code, IR tag. In one embodiment a
fluorescently labeled oligonucleotide that is complementary to a
particular aptamer sequence may be used to determine the exact
location of beads that comprise that aptamer.
[0067] In one embodiment, an array is used to detect the presence
of two or more analytes in a sample. Aptamer-coupled beads are
prepared that are specific for each analyte to be detected. The
different types of aptamer-coupled beads are then placed into
solution in separate vessels, so that each vessel contains only
beads comprising aptamers that are specific for a particular
analyte. In one embodiment, the aptamer coupled beads are placed in
solution in the wells of a microtiter plate. The location of the
wells comprising specific types of aptamer-coupled beads is noted.
A particular sample of interest is then divided between each of the
wells comprising a specific type of aptamer coupled beads. The
fluorescence anisotropy is measured in each well before and after
addition of the sample. The identity of analytes that are present
in the sample can then be determined by comparing positive results,
i.e. a change in fluorescence anisotropy, to the noted location of
the particular types of aptamer coupled beads.
[0068] In another embodiment, aptamer coupled beads are physically
attached to a substrate to form a two-dimensional array. The beads
may be attached in such a way that beads comprising the same type
of aptamer are localized together in addressable locations. The
different types of beads may be separated by a physical barrier,
such as by localization in different wells of a microtiter plate.
Alternatively, the different types of beads may be spatially
separated. The analyte specificity of the beads in each location is
noted or determined. The sample is contacted with the beads and any
change in fluorescence anisotropy is determined for each
addressable location in the array. The addressable locations that
are associated with a change fluorescent anisotropy are identified,
and the presence of a particular analyte in the sample is
determined based on the analyte specificity of the aptamers at
those location in the array.
[0069] In an alternative embodiment the presence of one or more
analytes from a group of analytes is to be determined in a sample
without determining the specific identity of the analytes that are
present. In this case, it is not necessary to separate the types of
beads in the array. However, localization of the same types of
beads together in the array may produce an increased signal and
thus facilitate detection of smaller quantities of the analytes of
interest.
[0070] Non-Bead Arrays
[0071] In non-bead arrays, aptamers are arranged directly on a
substrate for use in the detection of one or more analytes.
Non-bead arrays are described, for example, in U.S. Pat. Nos.
5,445,934, 5,405,783, 5,744,305, 6,365,418, The aptamers may be
attached to the substrate via linkers, using methods well known in
the art. Alternatively, the aptamers may be synthesized directly on
the substrate.
[0072] In a typical array a substrate comprises one or more
addressable locations of aptamers. The addressable locations may be
directly adjacent to each other or may be physically separated by a
gap or a barrier. In one embodiment, the addressable locations are
sufficiently separated that illumination of one addressable
location for the determination of fluorescence anisotropy will not
illuminate any part of an adjacent location. Thus, if the area of
illumination is smaller than the addressable location of aptamers,
the addressable locations may be directly adjacent, without any
space in between. On the other hand, if the size of the addressable
locations is smaller than the minimum area of illumination, the
distance between the locations will be large enough to prevent
overlapping illumination.
[0073] Typically, each addressable location comprises one type of
aptamer. However, in one embodiment, at least one addressable
location comprises more than one type of aptamer. This arrangement
is useful, for example, if the presence of one of a number of
analytes is to be determined, and the identity of the particular
analyte is not of concern. Alternatively, multi-plex analyte
detection can be accomplished using a set of aptamers, each with a
different fluorophore, attached at the same location. Each of the
fluorophores has different spectral characteristics, allowing the
binding of multiple analytes to be distinguished.
[0074] In other embodiments, each addressable location comprises a
single type of aptamer. In this case, the number of locations on
the array of aptamers is at least as great as the number of
different types of aptamers to be used, and thus at least as great
as the number of analytes to be detected. For example, if the
presence of ten analytes is to be detected in a sample, at least
ten addressable locations of aptamers will be present on the
substrate. The number of addressable locations is not limited in
any way and will be determined, for example, by the number of
analytes to be detected and the physical size of the substrate on
which the array is formed. In one embodiment, each addressable
location comprises at least about 10.sup.5 aptamers.
[0075] Typically, the aptamer composition and physical location of
each addressable location is known. In one embodiment, each of the
addressable locations in the array comprises a different type of
aptamer. For example, if ten analytes are to be assayed for, the
array will comprise ten addressable locations, each comprising a
different type of aptamer. In an alternative embodiment, more than
one addressable location comprising a particular type of aptamer is
present.
[0076] The addressable locations of aptamers in the array may be
any geometric shape. For example, the addressable locations may be
circular, rectangular, square or irregularly shaped. Typically the
shape of the addressable locations will be chosen to facilitate
attachment of the aptamers to the substrate and determinations of
fluorescence anisotropy.
[0077] The overall size of the array is not limited and will be
determined based on a variety of factors, including the number of
aptamers in each addressable locations, the number of addressable
locations, and physical constraints on the size of the substrate
arising from the system used to detect changes in fluorescence
anisotropy. In one embodiment, the addressable locations of the
array are all present on a substrate with an area of about 100
cm.sup.2 or less. In another embodiment the discrete areas of the
array are all present on a substrate with an area of about 10
cm.sup.2 or less.
[0078] For detecting the presence of one or more analytes in a
sample, the sample is contacted with the array of aptamers. Each of
the addressable locations of aptamers is individually illuminated
with polarized light before and after contacting the sample and any
change in fluorescence anisotropy is determined. If a change in
fluorescence anisotropy is measured for any particular addressable
location of aptamers, the compound with which that particular type
of aptamer interacts is identified as being present in the
sample.
Exemplary Applications
[0079] The analysis of samples for the presence of one or more
particular analytes finds uses in a wide range of fields, from
medical, basic biological research, pharmaceutical, agricultural,
environmental and industrial diagnostics to proteomics.
[0080] In another embodiment, the arrays of the invention may be
useful for diagnostic applications and for use in diagnostic
devices. In one embodiment the arrays are used to establish a
correlation between the expression level of a particular protein
and a disease or a particular stage of a disease. In a further
embodiment, once such a correlation between the expression level of
a protein and a particular disease or a particular stage of a
disease has been made, or is known, the arrays of the invention may
be used to diagnose a particular disease or a stage of a disease in
a tissue of an organism. Accordingly, in one embodiment, the
invention provides a method of diagnosing a disease or disorder in
a patient. One or more analytes that are known to be associated
with the disease or disorder from which a patient is believed to be
suffering are selected. For example, if a patient is suspected of
suffering from a tumor, the methods of the present invention may be
used to identify the presence of one or more proteins that are
known to be expressed in tumor cells, but not in normal cells.
Similarly, if a patient is suspected of having been exposed to an
infectious agent, proteins known to be associated with the
infectious agent are selected for identification. For example, a
sample from a patient suspected of being infected with HIV may be
analyzed for the presence of protein known to be associated with
HIV, such as GP120MN.
[0081] Aptamers that specifically recognize the selected analytes
are identified and synthesized. The aptamers are fluorescently
labeled and attached to a solid support in an array format that
allows for the identification of more than one analyte.
Fluorescence anisotropy is measured as described above for each
type of aptamer and the aptamers are then contacted with an
appropriate sample from the patient. A change in the fluorescence
anisotropy for a particular type of aptamer following contact with
the sample is taken as indicative of the presence of the particular
analyte for which that type of aptamer is specific.
[0082] Similarly, the assays of the invention may be used to
evaluate the efficacy of a treatment regimen. For example, the
presence of one or more analytes known to be associated with a
disease or disorder may be assayed for in a biological sample from
a patient prior to and after treatment. This may help determine the
efficacy of particular treatment options.
[0083] In another embodiment, the invention provides a method of
comparing the expression of particular proteins in two or more
cells or populations of cells. Methods include assaying in parallel
for a plurality of different proteins in a sample, such as
expression products, or fragments thereof, of a cell or a
population of cells from an organism. The methods involve
incubating the sample to be analyzed for the presence of specific
proteins to an aptamer array of the invention comprising aptamers
that specifically bind the proteins of interest. The presence
and/or amount of the proteins of interest in the sample is
detected. Such methods optionally comprise the additional step of
further characterizing the protein bound to at least one type of
aptamer in the array. In addition, the presence of analytes other
than proteins can be detected, for example metabolites.
[0084] The methods may involve the comparison of the protein
expression pattern of a cell or population of cells, optionally
subjected to different conditions, to the protein expression
pattern of a control cell or population. For example, the protein
expression pattern of a neoplastic cell may be compared to the
protein expression pattern of a control cell or population. In a
further example, the different conditions may include infecting one
cell or population with a pathogen, exposing one cell or population
to a stressor or exposing one cell or population to a drug, such as
a potential therapeutic. For such comparison, a sample containing
expression products, or fragments thereof, of the first cell or
population of cells is incubated with an array of aptamers under
conditions suitable for protein binding. In a similar manner, a
sample containing expression products, or fragments thereof, of a
second cell or population of cells is incubated with a second
aptamer array that is identical to the first aptamer array. The
types of proteins identified by changes in fluorescence anisotropy
upon binding to the fluorescently labeled aptamers of the first
aptamer array may be compared to the types of protein identified by
the corresponding second aptamer array.
[0085] The methods of comparing the protein expression between two
cells or two population of cells may be useful in the
identification and validation of new potential drug targets, as
well as for drug screening. In particular, the method may be used
to identify a protein which is overexpressed in disease, such as
tumor cells, but not in normal cells. Such a protein may be a
target for drug intervention, such as with inhibitors targeted to
such a differentially expressed protein.
[0086] The aptamer assays of the invention may further be used to
evaluate the efficacy and specificity of a particular drug. For
example, the expression pattern of particular proteins in a cell or
population of cells that have been exposed to a particular drug may
be compared to the expression pattern of a control cell or a
population of cells that have not been exposed to the drug.
[0087] In another embodiment, the arrays may be used to establish a
correlation between the expression of a particular protein and a
disease or a particular stage of a disease.
[0088] One of skill in the art will be able to readily adapt the
disclosed methods to particular uses.
EXAMPLES
[0089] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0090] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
Example 1
[0091] A. Methods for assessing binding of proteins to
aptamer-coupled beads
[0092] The polarization anisotropy of dye-labeled aptmers changes
with time upon binding to a specific protein. Changes in
fluorescence anisotropy of 3 aptamers bound to a solid support upon
binding of specific proteins was measured.
[0093] The three fluorophore labeled aptamers used in these
experiments were anti-thrombin aptamer (FAM-labeled, a 15 mer),
which is specific for thrombin from human plasma, 518-aptamer
(FAM-labeled, a 60 mer), which is specific for GP120MN, an HIV-1
protein and 650-aptamer (FAM-labeled, a 60 mer), which is specific
for recombinant human FGF basic protein.
[0094] 1. Anti-Thrombin Aptamer for Thrombin
[0095] Binding of thrombin to FAM-labeled anti-thrombin aptamers
was measured. 5 micron silica particles were purchased from Bangs
Laboratories (Ohio, USA) and pretreated with 1N NaCI solution at
100.degree. C. for 1 hour. The silica particles were then treated
for 30 minutes with 3-aminopropyltriethoxysilane (APTES; Sigma,
USA), diluted to 10 mM in 200 proof ethanol. After reaction,
particles were washed with ethanol three times and dried at
60.degree. C. in a Vacufuge (Eppendorf, Germany) overnight. Dried
silanized particles were treated with 10 mM solution of
Bis(thiopropyl N-hydroxy succinidimyl) (Sigma, USA) for 30 minutes
in DMF at 40.degree. C., followed by washing with DMF and ethanol
quickly. A 15 mer anti-thrombin aptamer with C6-aminolinker at 5'
and FAM at 3' was synthesized using an ABI304 synthesizer and
subjected to HPLC purification. 10 mg of reactive silica beads with
silane and NHS were incubated with 100 .mu.m aptamer at 4.degree.
C. for 12 hours. Samples were washed and dried in a vacuum.
[0096] For polarization experiments, 100 mg/ml (about
1.times.10.sup.5 particles) of FAM-labeled anti-thrombin aptamer
coupled bead solution in phosphate buffered saline (PBS) was placed
in a 160 .mu.L quartz cuvette at ambient condition. The cuvette was
placed in a SPEX Flurolog Fluorometer (NJ, USA) set to polarization
mode. The samples were mixed. well with a small magnetic stir bar
during the experiment. Different concentrations of thrombin analyte
were added to the aptamer-coupled bead solution The resultant
polarization or anisotropy was plotted against time during the
experiment.
[0097] 2. 518-Aptamer for GP120MN Protein
[0098] Binding between GP120MN protein and 518 aptamer coupled to
beads was examined. 518-aptamer with a C6-aminolinker at the 5' end
and FAM at the 3' end was synthesized using an ABI394 synthesizer.
Subsequently the labeled aptamer was HPLC purified. Reactive silica
beads prepared as described above were treated with 100 .mu.M
labeled 518-aptamer at 40.degree. C. for 12 hours. Samples were
washed and dried in vacuum.
[0099] Polarization experiments using the FAM-labeled 518 aptamer
coupled beads and GP120MN protein were performed as described
above.
[0100] 3. 650-Aptamer for Recombinant Human FGF basic Protein
[0101] Binding between recombinant human FGF basic protein and 650
aptamer coupled to beads was examined. 650 aptamer with
C6-aminolinker at 5' and FAM at 3' was synthesized using an ABI394
synthesizer and subsequently HPLC purified. Reactive silica beads
prepared as described above were treated with 100 .mu.M labeled
650-aptamer at 40.degree. C. for 12 hours. Samples were washed and
dried in vacuum.
[0102] Polarization experiments using the FAM-labeled 650 aptamer
coupled beads and recombinant human FGF basic protein were
performed as described above.
[0103] B. Results of binding of proteins to aptamer-coupled
beads
[0104] Binding of proteins to aptamers attached to 5 .mu.m-sized
particles as described above was assessed by fluorescence
anisotropy. Fluorescence anisotropy was analyzed using 5'-aminated
anti-thrombin aptamer that was 3'-FAM labeled and attached to
silica beads. The thrombin aptamer-coupled particles were exposed
to thrombin protein, and the change in fluorescence anisotropy was
measured. An increase in anisotropy (FIG. 1) was observed upon
addition of 100 nM thrombin. The change was similar to that
observed upon binding of thrombin to thrombin aptamer in solution.
Non-specific binding to lysozyme, a cationic protein that binds
non-specifically to all aptamers, was also observed in the assay
(FIG. 1).
[0105] Aptamer 518 (60 mer) is specific for G120MN (HIV-1) protein.
Upon addition of 100 nM GP120, a large increase in fluorescence
anisotropy was observed (FIG. 2). the increase was significantly
greater than, and easily distinguished from the small increase in
fluorescence anisotropy that resulted from non-specific binding
upon addition of 1 mg/ml BSA or 100 nM thrombin. Non-specific
binding of lysozyme to FAM-518 aptamer coupled beads was also
observed (FIG. 3). The binding of lysozyme to FAM-518 aptamer was
reversible upon addition of 0.5% SDS (FIG. 4).
* * * * *
References