WO2012006556A2

WO2012006556A2 - Detection of analytes

Info

Publication number: WO2012006556A2
Application number: PCT/US2011/043406
Authority: WO
Inventors: Barry Schweitzer; Bruce Branchaud
Original assignee: Life Technologies Corporation
Priority date: 2010-07-08
Filing date: 2011-07-08
Publication date: 2012-01-12
Also published as: WO2012006556A3

Abstract

Disclosed are methods of detecting target analytes using covalent analyte binding moieties in proximity detection assays. Specifically, two proximity detection probes, each comprises an analyte binding moiety and an oligonucleotide moiety, are used for the detection with one analyte binding moiety that covalently binds to the targeted analytes. Further disclosed are methods of coupling an analyte binding moiety with an oligonucleotide moiety to form a proximity detection probe during the detection assays. Reagents and kits for carrying out the methods are also provided.

Description

DETECTION OF ANALYTES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 61/362,616, filed July 8, 2010, which is hereby incorporated herein by reference.

TECHNICAL FIELD

Methods for detecting target analytes are provided herein. A method for detecting a target analyte using a covalent analyte binding moiety, a noncovalent analyte binding moiety and a proximity detection assay is provided herein. Regeants and kits for use in the methods are also provided.

BACKGROUND

Covalent analyte binding moiety (CABM) have been designed to target specific features of proteins (such as enzymes) such that a covalent bond is formed between the CABM and the protein. Detection of the covalently-bound CABM is generally done directly, for example, by linking a detectable tag to the CABM. When the protein to be detected is present in very small amounts, it may require large amounts of sample in order to have enough linked detectable tag to be observable. Further, if the detectable tag has little or no dynamic range, it can be difficult to determine the amount of protein present in any quantitative way.

SUMMARY

The inventors have developed a method of detecting analytes that takes advantage of the specificity of covalent analyte binding moieties, but provides the sensitivity and quantitative aspects of proximity detection assays, such as proximity ligation assays.

In some embodiments, methods of detecting at least one target analyte in a sample are provided. In some embodiments, a method comprises (a) forming a complex comprising at least one target analyte, a first proximity detection probe, and a second proximity detection probe, wherein the first proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety, and the second proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety; and (b) detecting an interaction between the first oligonucleotide moiety and the second oligonucleotide moiety.

In some embodiments, (a) comprises (i) contacting the sample with a covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a binding pair, to form a first complex comprising at least one target analyte and the covalent analyte binding moiety; (ii) contacting the complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the binding pair, to form a second complex comprising at least one target analyte and the first proximity detection probe; and (iii) contacting the second complex with the second proximity detection probe to form a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe. In some embodiments, (i) further comprises separating unbound covalent analyte binding moiety from the first complex. In some embodiments, (ii) further comprises separating unbound first oligonucleotide moiety from the second complex. In some embodiments, (iii) further comprises separating unbound second proximity detection probe from the third complex.

In some embodiments, (a) comprises (i) contacting the sample with a covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a first binding pair, to form a first complex comprising at least one target analyte and the covalent analyte binding moiety; (ii) contacting the first complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the first binding pair, to form a second complex comprising at least one target analyte and the first proximity detection probe; (iii) contacting the second complex with a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a second binding pair, to form a third complex comprising at least one target analyte, the first proximity detection probe, and the non-covalent analyte binding moiety; and (iv) contacting the third complex with a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the second binding pair, to form a fourth complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe. In some embodiments, (i) further comprises separating unbound covalent analyte binding moiety from the first complex. In some embodiments, (ii) further comprises separating unbound first oligonucleotide moiety from the second complex. In some embodiments, (iii) further comprises separating unbound non-covalent analyte binding moiety from the third complex. In some embodiments, (iv) further comprises separating the unbound second oligonucleotide moiety from the fourth complex.

In some embodiments, (a) comprises (i) contacting the sample with the first proximity detection probe, to form a first complex comprising at least one target analyte and the first proximity detection probe; and (ii) contacting the first complex with the second proximity detection probe, to form a second complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe. In some embodiments, (i) further comprises separating the unbound first proximity detection probe from the first complex. In some embodiments, (ii) further comprises separating the unbound second proximity detection probe from the second complex.

In some embodiments, (a) comprises (i) contacting the sample with the first proximity detection probe, to form a first complex comprising at least one target analyte and the first proximity detection probe; (ii) contacting the first complex with a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a binding pair, to form a second complex comprising at least one target analyte, the first proximity detection probe, and the non-covalent analyte binding moiety; and (iii) contacting the second complex with a second oligonucleotide moiety, wherein the second

oligonucleotide moiety comprises a second member of the binding pair, to form a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe. In some embodiments, (i) further comprises separating unbound first proximity detection probe from the first complex. In some embodiments, (ii) further comprises separating unbound non-covalent analyte binding moiety from the second complex. In some embodiments, (iii) further comprises separating unbound second oligonucleotide moiety from the third complex.

In some embodiments, (a) comprises (i) contacting the sample with a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a binding pair, to form a first complex comprising at least one target analyte and the non-covalent analyte binding moiety; (ii) contacting the first complex with a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the binding pair, to form a second complex comprising at least one target analyte and the second proximity detection probe; and (iii) contacting the second complex with the first proximity detection probe to form a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe. In some embodiments, (i) further comprises separating unbound non-covalent analyte binding moiety from the first complex. In some embodiments, (ii) further comprises separating unbound second oligonucleotide moiety from the second complex. In some embodiments, (iii) further comprises separating unbound first proximity detection probe from the third complex.

In some embodiments, (a) comprises (i) contacting the sample with a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a first binding pair, to form a first complex comprising at least one target analyte and the non-covalent analyte binding moiety; (ii) contacting the first complex with a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the first binding pair, to form a second complex comprising at least one target analyte and the second proximity detection probe; (iii) contacting the second complex with a covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a second binding pair, to form a third complex comprising at least one target analyte, the second proximity detection probe, and the covalent analyte binding moiety; and (iv) contacting the third complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the second binding pair, to form a fourth complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe. In some embodiments, (i) further comprises separating unbound non-covalent analyte binding moiety from the first complex. In some embodiments, (ii) further comprises separating unbound second oligonucleotide moiety from the second complex. In some embodiments, (iii) further comprises separating unbound covalent analyte binding moiety from the third complex. In some embodiments, (iii) further comprises separating unbound first oligonucleotide moiety from the fourth complex.

In some embodiments, (a) comprises (i) contacting the sample with the second proximity detection probe, to form a first complex comprising at least one target analyte and the second proximity detection probe; and (ii) contacting the first complex with the first proximity detection probe, to firm a second complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe. In some embodiments, (i) further comprises separating unbound second proximity detection probe from the first complex. In some embodiments, (ii) further comprises separating unbound first proximity detection probe from the second complex.

In some embodiments, (a) comprises (i) contacting the sample with the second proximity detection probe, to form a first complex comprising at least one target analyte and the second proximity detection probe; (ii) contacting the first complex with a covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a binding pair, to form a second complex comprising at least one target analyte, the second proximity detection probe, and the covalent analyte binding moiety; and (iii) contacting the second complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the binding pair, to form a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe. In some embodiments, (i) further comprises separating unbound second proximity detection probe from the first complex. In some embodiments, (ii) further comprises separating unbound covalent analyte binding moiety from the second complex. In some embodiments, (iii) further comprises separating unbound first oligonucleotide moiety from the third complex.

In some embodiments, methods of detecting at least one target analyte in a cell are provided. In some embodiments, a method comprises (a) contacting the cell with a covalent analyte binding moiety under conditions allowing formation of a first complex comprising at least one target analyte and the covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a first binding pair; (b) contacting the first complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the first binding pair, to form a second complex comprising at least one target analyte and a first proximity detection probe, wherein the first proximity detection probe comprises the covalent analyte binding moiety and the first oligonucleotide moiety; (c) contacting the second complex with a second proximity detection probe under conditions allowing formation of a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe; and (d) detecting an interaction between the first oligonucleotide moiety and the second oligonucleotide moiety.

In some embodiments, (a) further comprises separating unbound covalent analyte binding moiety from the first complex. In some embodiments, (b) further comprises separating unbound first oligonucleotide moiety from the second complex. In some embodiments, (c) further comprises separating unbound second proximity detection probe from the third complex. In some embodiments, the method further comprises lysing the cells between (a) and (b), between (b) and (c), or between (c) and (d).

In some embodiments, methods of detecting at least one target analyte in a subject are provided. In some embodiments, a method comprises (a) administering a covalent analyte binding moiety to the subject, wherein the covalent analyte binding moiety comprises a first member of a first binding pair; (b) isolating cells of the subject that are suspected of containing a first complex comprising the target analyte and the covalent analyte binding moiety; (c) contacting the first complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the first binding pair, to form a second complex comprising at least one target analyte and a first proximity detection probe, wherein the first proximity detection probe comprises the covalent analyte binding moiety and the first oligonucleotide moiety; (d) contacting the second complex with a second proximity detection probe under conditions allowing formation of a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe; and (e) detecting an interaction between the first oligonucleotide moiety and the second oligonucleotide moiety.

In some embodiments, (c) further comprises separating unbound first oligonucleotide moiety from the second complex. In some embodiments, (d) further comprises separating unbound second proximity detection probe from the third complex. In some embodiments, a method further comprises lysing the cells between (b) and (c), between (c) and (d), or between (d) and (e).

In some embodiments, a method of detecting at least one target analyte further comprises associating at least one target analyte with a solid phase. In some embodiments, the associating comprises contacting at least one target analyte with an antibody that binds the at least one target analyte, wherein the antibody is associated with the solid phase. In some embodiments, the solid phase is selected from microparticles and planar surfaces. In some embodiments, the planar surfaces are selected from microplates and microarray chips. In some embodiments, the solid phase is a microplate.

In some embodiments, the first member of at least one binding pair comprises biotin or a derivative thereof and the second member of at least one binding pair comprises streptavidin or a derivative thereof. In some embodiments, the first member of at least one binding pair comprises streptavidin or a derivative thereof and the second member of at least one binding pair comprises biotin or a derivative thereof. In some embodiments, the first and second members of at least one binding pair are capable of undergoing a click reaction. In some embodiments, the first member of at least one binding pair comprises an azido moiety and the second member of at least one binding pair comprises a moiety selected from an ethynyl moiety, a phosphine moiety, and a dibenzocyclooctyne (DIBO) moiety. In some embodiments, the first member of at least one binding pair comprises a moiety selected from an ethynyl moiety, a phosphine moiety, and a dibenzocyclooctyne (DIBO) moiety and the second member of at least one binding pair comprises an azido moiety.

In some embodiments, the interaction between the first oligonucleotide moiety and the second oligonucleotide moiety comprises at least one interaction selected from hybridization between the first and second oligonucleotide moieties and ligation of the first and second oligonucleotide moieties. In some embodiments, the interaction comprises ligation of the first and second oligonucleotide moieties. In some embodiments, detecting the interaction between the first and second oligonucleotide moieties comprises incubating with at least one splint oligonucleotide.

In some embodiments, the covalent analyte binding moiety and the non-covalent analyte binding moiety are capable of interacting with the same target analyte. In some embodiments, the target analyte is selected from a peptide, a protein, a hormone, a carbohydrate, a polysaccharide, a small molecule, a moiety on the surface of a cell, and a moiety on the surface of a microorganism. In some embodiments, the target analyte is a protein. In some embodiments, the target analyte is an enzyme. In some embodiments, the enzyme is selected from a metalloprotease, a cysteine protease, a ubiquitin-specific protease, a cysteine cathepsin, an esterase, a kinase, a histone deacetylase, a serine reductase, an oxidoreductase, an ATPase, and a GTPase.

In some embodiments, the covalent analyte binding moiety is capable of interacting with a first target analyte and the non-covalent analyte binding moiety is capable of interacting with a second target analyte. In some embodiments, the first target analyte is capable of interacting with the second target analyte. In some embodiments, the first target analyte and the second target analyte are independently selected from a peptide, a protein, a hormone, a carbohydrate, a polysaccharide, a small molecule, a moiety on the surface of a cell, and a moiety on the surface of a microorganism. In some embodiments, the first target analyte is a protein. In some embodiments, the first target analyte is an enzyme. In some embodiments, the enzyme is selected from a metalloprotease, a cysteine protease, a ubiquitin- specific protease, a cysteine cathepsin, an esterase, a kinase, a histone deacetylase, a serine reductase, an oxidoreductase, an ATPase, and a GTPase.

In some embodiments, detecting comprises a real-time PCR reaction. In some embodiments, detecting comprises determining the level of a target analyte. In some embodiments, determining the level of a target analyte comprises comparing the level to a standard.

In some embodiments, complexes comprising at least one target analyte and a first proximity detection probe are provided. In some embodiments, a first proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety. In some embodiments, a complex further comprises a second proximity detection probe, wherein the second proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety. In some embodiments, a complex further comprises a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a binding pair. In some such embodiments, a complex further comprises a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the binding pair.

In some embodiments, complexes comprising at least one target analyte, a covalent analyte binding moiety, and a proximity detection probe are provided. In some embodiments, the covalent analyte binding moiety comprises a first member of a binding pair, and the proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety. In some embodiments, the complex further comprises a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the binding pair.

In some embodiments, complexes comprising at least one target analyte, a covalent analyte binding moiety, and a non-covalent analyte binding moiety are provided. In some embodiments, the covalent analyte binding moiety comprises a first member of a first binding pair, and the a non-covalent analyte binding moiety comprises a first member of a second binding pair. In some embodiments, a complex further comprises a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the first binding pair. In some embodiments, a complex further comprises a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the second binding pair.

In some embodiments, a complex is associated with a solid phase.

In some embodiments, kits are provided. In some embodiments, a kit comprises a first proximity detection probe and a second proximity detection probe, wherein the first proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety, and the second proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety. In some embodiments, a kit comprises a proximity detection probe and a covalent analyte binding moiety, wherein the proximity detection probe comprises a non-covalent analyte binding moiety and a first oligonucleotide moiety and the covalent analyte binding moiety comprises a first member of a binding pair. In some embodiments, a kit further comprises a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the binding pair.

In some embodiments, a kit comprises a proximity detection probe and a non-covalent analyte binding moiety, wherein the proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety and the non-covalent analyte binding moiety comprises a first member of a binding pair. In some embodiments, a kit further comprises a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the binding pair.

In some embodiments, a kit comprises a covalent analyte binding moiety and a non- covalent analyte binding moiety, wherein the a non-covalent analyte binding moiety comprises a first member of a first binding pair and the non-covalent analyte binding moiety comprises a first member of a second binding pair. In some embodiments, a kit further comprises a first oligonucleotide moiety comprising a second member of the first binding pair, and a second oligonucleotide moiety comprising a second member of the second binding pair.

In some embodiments, a kit comprises a splint oligonucleotide. In some

embodiments, a kit comprises a ligase.

DETAILED DESCRIPTION

Methods of detecting analytes are provided. The methods comprise forming a complex comprising at least one analyte, a first proximity detection probe, and second proximity detection probe, wherein the first proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide and the second proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide. Interaction of the first oligonucleotide and the second oligonucleotide is then detected. In some embodiments, the methods allow sensitive and/or quantitative detection of analytes. In some embodiments, the methods allow sensitive and/or quantitative detection of active analytes. In some embodiments, the methods allow sensitive and/or quantitative detection of enzyme activity.

The sensitive and/or quantitative methods provided are suitable for a variety of analyte assays. For example, as described herein, the methods are useful in small molecule - analyte interaction and profiling. The methods are particularly useful in detection and/or quantitation of induction or inhibition of an active enzyme. Accordingly, provided are assays which use the methods for enzyme inhibitor design, identification, and/or screening.

Provided also are assays which use the methods for enzyme activator design, identification, and/or screening. The methods provided are suitable for high throughput assay and screening procedures. As described herein, the methods may be used in multiplex or singleplex assays.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Definitions

Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

Exemplary techniques used in connection with recombinant DNA, oligonucleotide synthesis, tissue culture, enzymatic reactions, and purification are known in the art. Many such techniques and procedures are described, e.g., in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), among other places. In addition, exemplary techniques for chemical syntheses are also known in the art.

In this application, the use of "or" means "and/or" unless stated otherwise. In the context of a multiple dependent claim, the use of "or" refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

The terms "nucleic acid" and "polynucleotide" may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, PNA, LNA and any other nucleotide polymer that can be ligated and is PCR competent. "Nucleic acid sequence" or "polynucleotide sequence" may be used interchangeably, and refer to the linear sequence of nucleotides in the nucleic acid or polynucleotide.

The terms "annealing" and "hybridizing" are used interchangeably and refer to the base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex, triplex, or other higher-ordered structure. In some embodiments, the primary interaction is base specific, e.g., A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding. Base-stacking and hydrophobic interactions may also contribute to duplex stability.

In this application, a statement that one sequence is the same as or is complementary to another sequence encompasses situations where both of the sequences are completely the same or complementary to one another, and situations where only a portion of one of the sequences is the same as, or is complementary to, a portion or the entire other sequence. Further, a statement that one sequence is complementary to another sequence encompasses situations in which the two sequences have mismatches. Despite the mismatches, the two sequences should selectively hybridize to one another under appropriate conditions.

The term "primer" or "oligonucleotide primer" as used herein, refers to an oligonucleotide from which a primer extension product can be synthesized under suitable conditions. In some embodiments, such suitable conditions comprise the primer being hybridized to a complementary nucleic acid and incubated in the presence of, for example, nucleotides, a polymerization-inducing agent, such as a DNA or RNA polymerase, at suitable temperature, pH, metal concentration, salt concentration, etc. In some embodiments, primers are 5 to 100 nucleotides long. In some embodiments, primers are 8 to 75, 10 to 60, 10 to 50, 10 to 40, or 10 to 35 nucleotides long.

The term "ligation" as used herein refers to the covalent joining of two polynucleotide ends. In some embodiments, ligation involves the covalent joining of a 3' end of a first polynucleotide to a 5' end of a second polynucleotide. In some embodiments, ligation results in a phosphodiester bond being formed between the polynucleotide ends. In some embodiments, ligation may be mediated by any enzyme, chemical, or process that results in a covalent joining of the polynucleotide ends. In some embodiments, ligation is mediated by a ligase enzyme.

The term "analyte" or "target analyte" as used herein refers to a substance to be detected using one or more proximity detection probes. Such substances include, but are not limited to, peptides, proteins, carbohydrates, polysaccharides, hormones, small molecules, moieties on the surface of cells, moieties on the surface of microorganisms, and any other substance for which a covalent analyte binding moiety and/or a non-covalent analyte binding moiety can be developed. In some embodiments, an analyte is a protein. In some embodiments, the protein may be a G-protein coupled receptor. In some embodiments, the protein is selected from an enzyme and a receptor. In some embodiments, the enzyme may be a cytochrome P450 or a kinase. An analyte is not a nucleic acid.

The term "sample" as used herein refers to any sample that is suspected of containing at least one target analyte. Exemplary samples include, but are not limited to, prokaryotic cells, eukaryotic cells, tissue samples, viral particles, bacteriophage, infectious particles, pathogens, fungi, food samples, bodily fluids (including, but not limited to, mucus, blood, plasma, serum, urine, saliva, and semen), water samples, and filtrates from, e.g., water and air. Exemplary samples also include, but are not limited to, lysates of prokaryotic cells, eukaryotic cells, tissue samples, viral particles, bacteriophage, infectious particles, pathogens, fungi, food samples, and bodily fluids.

A "proximity detection probe" as used herein, is a probe that comprises at least one non-covalent analyte binding moiety or at least one covalent analyte binding moiety, connected, either covalently or noncovalently, to at least one oligonucleotide moiety. In some embodiments, a non-covalent analyte binding moiety or covalent analyte binding moiety comprises a first member of a binding pair and the oligonucleotide moiety comprises a second member of a binding pair, wherein the first member of the binding pair and the second member of the binding pair are capable of stably associating under the conditions used for proximity detection probe binding and hybridization and/or ligation. In some embodiments, one skilled in the art can select an appropriate binding pair. In some embodiments, a proximity detection probe comprises one or more linkers connecting at least one non-covalent analyte binding moiety to at least one oligonucleotide moiety. In some embodiments, one skilled in the art can select an appropriate linker.

A "covalent analyte binding moiety" as used herein, refers to a moiety that binds specifically and non-covalently to an analyte and subsequently reacts to form a covalent bond to the analyte at or near the site of the non-covalent binding. The non-covalent binding may occur during enzyme catalysis, simple binding to an enzyme active site, or simple binding to any binding site on the analyte. In some embodiments, a covalent analyte binding moiety preferentially attaches to an active analyte, such as an active enzyme or a receptor that is able to bind ligand. In some embodiments, at least the portion of the covalent analyte binding moiety that covalently attaches to an analyte is a small molecule. In some embodiments, a covalent analyte binding moiety comprises a member of a binding pair.

A "non-covalent analyte binding moiety" as used herein, refers to a moiety that specifically and non-covalently binds to a target analyte, but does not covalently attach to the analyte. Such a moiety may bind to the analyte, with a dissociation constant of about 10^"3 M to about 10^"15 M. Exemplary moieties that may be used as non-covalent analyte binding moieties include, but are not limited to, monoclonal antibodies and fragments thereof that are capable of binding an analyte, polyclonal antibodies and fragments thereof that are capable of binding an analyte, proteins, peptides, lectins, nucleic acids, aptamers, carbohydrates, soluble cell surface receptors, small molecules, and any other binding moieties that are specific for a target analyte. In some embodiments, a non-covalent analyte binding moiety comprises a member of a binding pair. The term "proximity detection assay" or "PDA" as used herein refers to an assay that involves contacting an analyte with at least two proximity detection probes, wherein at least one probe comprises a non-covalent analyte binding moiety and an oligonucleotide moiety and at least one probe comprises a covalent analyte binding moiety and an oligonucleotide moiety. The oligonucleotide moiety of each probe may be the same or different. In some embodiments, the oligonucleotide moiety of each probe in a set of proximity detection probes comprises a different sequence. In some embodiments, the analyte is contacted with a set of proximity detection probes. In some embodiments, a set of proximity detection probes comprises 2, 3, 4, 5, or more than 5 proximity detection probes. In some embodiments, a set of proximity detection probes is a pair of proximity detection probes, or a "proximity detection probe pair." In some embodiments, the non-covalent analyte binding moiety and the covalent analyte binding moiety in a set of proximity detection probes are capable of interacting with the same analyte. In some embodiments, the non-covalent analyte binding moiety and the covalent analyte binding moiety in a set of proximity detection probes are capable of interacting with the different analytes.

In some embodiments, after contacting one or more analytes with at least two proximity detection probes, the oligonucleotide moieties of at least two of the proximity detection probes are capable of interacting with one another. In some embodiments, at least a portion of the oligonucleotide moieties of at least two of the proximity detection probes hybridize to one another. In some embodiments, such interaction may be mediated by one or more additional oligonucleotides. In some embodiments, at least a portion of each of the oligonucleotide moieties of the proximity detection probes hybridizes to another

oligonucleotide. For example, in some embodiments, at least one additional oligonucleotide is added (referred to herein as a "splint oligonucleotide"), which mediates the interaction between at least two proximity detection probes by hybridizing to at least a portion of the oligonucleotide moiety of each of the proximity detection probes. A proximity detection assay (PDA) in which the oligonucleotide moieties hybridize to one another, or hybridize to another oligonucleotide that forms a bridge between at least two oligonucleotide moieties, wherein the oligonucleotide moieties are not ligated to one another, may also be referred to as a "proximity interaction assay" or "PIA."

In some embodiments, the oligonucleotide moieties of at least two of the proximity detection probes are capable of being ligated together by a polynucleotide ligase enzyme. In some embodiments, the ligatable ends of each of the oligonucleotide moieties are brought together by a splint oligonucleotide that is capable of hybridizing to at least a portion of the oligonucleotide moiety of each proximity detection probe. A proximity detection assay (PDA) in which oligonucleotide moieties of the proximity detection probes are ligated together may also be referred to as a "proximity ligation assay" or "PLA."

Following hybridization and/or ligation of the oligonucleotide moieties of at least two proximity detection probes, the hybridized and/or ligated oligonucleotide moieties may be detected by any method known in the art. In some such embodiments, the hybridized and/or ligated oligonucleotide moieties are referred to as a "target nucleic acid." Exemplary methods of detecting the hybridized and/or ligated oligonucleotide moieties (or "target nucleic acid") include, but are not limited to, direct detection, real-time PCR (including, but not limited to, 5'-nuclease real-time PCR), rolling circle amplification, combinations of ligation and PCR, and amplification followed by a detection step (such as a second amplification, direct detection, ligation, etc.). Nonlimiting exemplary methods of detecting nucleic acids are described herein.

Exemplary proximity detection assays are described, e.g., in U.S. Pat. No. 6,511,809 B2; U.S. Pat. Pub. No. US 2002/0064779; PCT Pub. No. WO 2005/123963; and Gustafsdottir et al., Clin. Chem. 52: 1152-1160 (2006).

The term "quantitative nucleic acid detection assay" as used herein refers to an assay that is capable of quantitating the amount of a particular nucleic acid sequence in a sample. Nonlimiting exemplary quantitative nucleic acid detection assays are described herein.

As used herein, the term "detector probe" refers to a molecule used in an

amplification reaction that facilitates detection of the amplification product. Exemplary amplification reactions include, but are not limited to, quantitative PCR, real-time PCR, and end-point analysis amplification reactions. In some embodiments, such detector probes can be used to monitor the amplification of a target nucleic acid and/or control nucleic acid. In some embodiments, detector probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a function of time.

In some embodiments, a detector probe is "sequence-based," meaning that it detects an amplification product in a sequence-specific manner. As a non-limiting example, a sequence -based detector probe may comprise an oligonucleotide that is capable of hybridizing to a specific amplification product. In some embodiments, a detector probe is "sequence-independent," meaning that it detects an amplification product regardless of the sequence of the amplification product.

Detector probes may be "detectably different," which means that they are

distinguishable from one another by at least one detection method. Detectably different detector probes include, but are not limited to, detector probes that emit light of different wavelengths, detector probes that absorb light of different wavelengths, detector probes that scatter light of different wavelengths, detector probes that have different fluorescent decay lifetimes, detector probes that have different spectral signatures, detector probes that have different radioactive decay properties, detector probes of different charge, and detector probes of different size. In some embodiments, a detector probe emits a fluorescent signal.

"Endpoint polymerase chain reaction" or "endpoint PCR" is a polymerase chain reaction method in which the presence or quantity of nucleic acid target sequence is detected after the PCR reaction is complete, and not while the reaction is ongoing.

"Real-time polymerase chain reaction" or "real-time PCR" is a polymerase chain reaction method in which the presence or quantity of nucleic acid target sequence is detected while the reaction is ongoing. In some embodiments, the signal emitted by one or more detector probes present in a reaction composition is monitored at multiple time points during the PCR as an indicator of synthesis of a primer extension product. In some embodiments, fluorescence emitted at multiple time points during the PCR is monitored as an indicator of synthesis of a primer extension product. In some embodiments, the signal is detected during each cycle of PCR.

A "multiplex amplification reaction" is an amplification reaction in which two or more target nucleic acid sequences and/or control nucleic acid sequences are amplified in the same reaction. A "multiplex polymerase chain reaction" or "multiplex PCR" is a polymerase chain reaction method in which two or more target nucleic acid sequences and/or control nucleic acid sequences are amplified in the same reaction.

A "singleplex amplification reaction" is an amplification reaction in which only one target nucleic acid sequence or control nucleic acid sequence is amplified in the reaction. A "singleplex polymerase chain reaction" or "singleplex PCR" is a polymerase chain reaction method in which only one target nucleic acid sequence or control nucleic acid sequence is amplified in the reaction.

"Threshold cycle" or "C_T" is defined as the cycle number at which the observed signal from a quantitative nucleic acid detection assay exceeds a fixed threshold. In some embodiments, the fixed threshold is set as the amount of signal observed in a reaction lacking a target nucleic acid sequence or control nucleic acid sequence. In some embodiments, the fixed threshold is set at a level above the background noise signal. For example, in some embodiments, the fixed threshold is set at a value corresponding to 3 or more times the combination of the root mean squared of the background noise signal and the background noise signal. In some embodiments, the observed signal is from a detector probe. In some embodiments, the observed signal is from a fluorescent label.

The term "normalizer control" means a molecule present in a sample that can be used to normalize the amount of a target analyte detected in a proximity detection assay. In some embodiments, a normalizer control is an analyte. In some embodiments, a normalizer control is a nucleic acid.

The term "solid support" as used herein refers to any solid substance that can be mixed or contacted with a liquid and then separated from the liquid. Separation from the liquid may comprise, in some embodiments, centrifugation, use of a magnet, filtration, settling, pipetting, etc. Nonlimiting exemplary solid supports include microparticles (such as polymer beads, metal particles, magnetic beads, etc., microtiter plates (such as 96-well plates, 384-well plates, 1536-well plates, etc.), and microarray chips. In some embodiments, a solid support comprises a coating that facilitates binding of, for example, a covalent analyte binding moiety and/or a non-covalent analyte binding moiety and/or an oligonucleotide moiety. In some embodiments, the coating comprises a first member of a binding pair. In some such embodiments, a covalent analyte binding moiety and/or a non-covalent analyte binding moiety moiety and/or an oligonucleotide moiety comprises a second member of the binding pair.

Exemplary Reagents

Exemplary Proximity Detection Probes

A proximity detection probe comprises at least one non-covalent analyte binding moiety or at least one covalent analyte binding moiety, and at least one oligonucleotide moiety. A non-covalent analyte binding moiety is capable of binding to a selected analyte. A covalent analyte binding moiety is capable of covalently attaching to a selected analyte. In some embodiments, a proximity detection probe comprises one non-covalent analyte binding moiety and one oligonucleotide moiety. In some embodiments, a proximity detection probe comprises more than one non-covalent analyte binding moiety. In some embodiments, a proximity detection probe comprises one covalent analyte binding moiety and one oligonucleotide moiety. In some embodiments, a proximity detection probe comprises more than one covalent analyte binding moiety. In some embodiments, a proximity detection probe comprises more than one oligonucleotide moiety. Nonlimiting exemplary multivalent proximity probes are described, e.g., in U.S. Pat. Pub. No. US 2005/0003361 to Fredriksson.

In some embodiments, the oligonucleotide moiety of a proximity detection probe comprises one or more of ribonucleotides, deoxyribonucleotides, analogs of ribonucleotides, and/or analogs of deoxyribonucleotides. Exemplary analogs of ribonucleotides and analogs of deoxyribonucleotides include, but are not limited to, analogs that comprise one or more modifications to the nucleotide sugar, phosphate, and/or base moiety. Exemplary

oligonucleotide analogs include, but are not limited to, LNA (see, e.g., U.S. Pat. No.

6,316,198), PNA (see, e.g., U.S. Pat. No. 6,451,968), and any other nucleotide analogs known in the art. See, e.g., Loakes, Nucleic Acids Res. 2001 Jun. 15; 29(12):2437-47; and Karkare et al., Appl. Microbiol. Biotechnol. 2006 August; 71(5):575-86. Epub 2006 May 9.

In some embodiments, the oligonucleotide moiety of the proximity detection probe comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 75, or at least 100 nucleotides. In some embodiments, the oligonucleotide moiety of the proximity detection probe comprises 10 to 1000 nucleotides, 10 to 500 nucleotides, 10 to 200, or 10 to 100 nucleotides.

The oligonucleotide moiety and the non-covalent analyte binding moiety or the covalent analyte binding moiety of the proximity detection probe may be covalently or non- covalently associated with one another. Many ways of covalently and non-covalently associating a non-covalent analyte binding moiety or a covalent analyte binding moiety and an oligonucleotide moiety are known in the art.

In some embodiments, the oligonucleotide moiety comprises a first member of a binding pair and the non-covalent analyte binding moiety or the covalent analyte binding moiety comprises a second member of a binding pair, wherein the first member of the binding pair and the second member of the binding pair are capable of stably associating under the conditions used for proximity detection probe binding and/or oligonucleotide hybridization and/or oligonucleotide ligation. In some embodiments, the binding pair stably associates through a non-covalent interaction. In some embodiments, the binding pair stably associates through a covalent interaction. In some embodiments, the binding pair need not stably associate during detection of the hybridized and/or ligated oligonucleotide moieties. In some embodiments, the binding pair need not stably associate during the initial binding of the covalent analyte binding moiety and/or the non-covalent analyte binding moiety to the analyte.

Exemplary binding pairs include, but are not limited to, antibody/antigen, biotin and bio tin derivatives/avidin and avidin derivatives, biotin and biotin derivatives/strep tavidin and streptavidin derivatives, hybridizing nucleic acids, receptor/ligand, folic acid/folate binding protein, vitamin B12/intrinsic factor, protein A/Fc, and protein G/Fc, metal/chelator, and moieties capable of undergoing a click reaction, etc. In some embodiments, the non-covalent analyte binding moiety or the covalent analyte binding moiety is associated with an oligonucleotide moiety through a biotin or biotin derivative and a streptavidin or streptavidin derivative. In some embodiments, the non- covalent analyte binding moiety or the covalent analyte binding moiety comprises biotin or a biotin derivative. In some such embodiments, the oligonucleotide moiety comprises streptavidin or a streptavidin derivative. In some embodiments, the non-covalent analyte binding moiety or the covalent analyte binding moiety comprises streptavidin or a streptavidin derivative. In some such embodiments, the oligonucleotide moiety comprises biotin or a biotin derivative. Nonlimiting exemplary biotin derivatives and streptavidin derivatives are described, e.g., in U.S. Pub. No. US 2008/0255004. In some embodiments, streptavidin or a streptavidin derivative may be attached to an oligonucleotide moiety by the use of a sulfo-SMCC reagent (see, e.g., Pierce Catalog #22322). In some embodiments, biotin or a biotin derivative may be attached to an oligonucleotide moiety, for example, by a method described in Misiura et al, Nucl. Acids Res., 18: 4345-4354 (1990); Alves et al, Tetrahedron Letters, 30: 3089-3092 (1989); Pon, R.T., Tetrahedron Letters, 32: 1715-1718 (1991); or U.S. Pat. No. 5,567,811. In some embodiments, the non-covalent analyte binding moiety may be attached to oligonucleotide moiety using hydrazone chemistry, as exemplified by use of S-HyNic and sulfo-S-4FB (Solulink™ Antibody- Oligonucleotide All-in-One Conjugation Kit, Catalog #A-9202-001).

In some embodiments, a non-covalent analyte binding moiety or a covalent analyte binding moiety comprises a moiety capable of undergoing a click reaction. In some such embodiments, an oligonucleotide comprises a complementary moiety capable of undergoing a click reaction. A complementary moiety capable of undergoing a click reaction refers to a second moiety that is capable of undergoing a click reaction with a first moiety. Nonlimiting complementary moieties capable of undergoing a click reaction include azido

moieties/ethynyl moieties, azido moieties/phosphine moieties, azido

moieties/dibenzocyclooctyne (DIBO) and DIBO-like moieties, and other Click-based chemistries. Nonlimiting exemplary moieties capable of undergoing a click reaction are described, e.g., in U.S. Pat. No. 7,375,234; PCT Pub. No. WO 01/68565; and PCT Pub. No. WO 2009/067663.

In some embodiments, the non-covalent analyte binding moiety or the covalent analyte binding moiety and the oligonucleotide moiety of the proximity detection probe are covalently associated. The non-covalent analyte binding moiety or the covalent analyte binding moiety and the oligonucleotide moiety of the proximity detection probe may be covalently associated following a click reaction as discussed above, or may be covalently associated through other methods. Certain methods of forming covalent bonds between various molecules are known in the art. For example, nonlimiting exemplary methods of making proximity detection probes are described, e.g., in Gullberg et. al., Proc. Natl. Acad. Sci. 101(22): 8420-8424 (2004).

In some embodiments, the 3' end or the 5' end of the oligonucleotide moiety is associated with the non-covalent analyte binding moiety. In some embodiments, the oligonucleotide moiety is associated with the non-covalent analyte binding moiety at a location other than the 3' end or the 5' end of the oligonucleotide moiety, for example, through one or more nucleotides or modified nucleotides in the oligonucleotide sequence.

In some embodiments, two or more proximity detection probes are combined to form a proximity detection probe set. Each proximity detection probe set comprises at least a first proximity detection probe that comprises covalent analyte binding moiety and a first oligonucleotide moiety, and a second proximity detection probe that comprises an non- covalent analyte binding moiety and a second oligonucleotide moiety. A proximity detection probe set that comprises a first proximity detection probe and a second proximity detection probe may be referred to as a proximity detection probe pair. The covalent analyte binding moiety (which is part of the first proximity detection probe) and the non-covalent analyte binding moiety (which is part of the second proximity detection probe) in a proximity detection probe set may interact with the same analyte, or may interact with different analytes. In some embodiments, when the covalent analyte binding moiety (which is part of the first proximity detection probe) interacts with a first analyte, and the non-covalent analyte binding moiety (which is part of the second proximity detection probe) interacts with a second analyte, the first and second analyte are capable of associating with one another. In some embodiments, such a proximity detection probe set may, for example, be used to detect the association of the first and second analytes.

In some embodiments, a proximity detection probe is capable of binding to more than one analyte. In some embodiments, when a proximity detection probe comprises a covalent analyte binding moiety, the covalent analyte binding moiety may be capable of covalently attaching to more than one analyte. For example, in some embodiments, a covalent analyte binding moiety is capable of covalently attaching to a particular class or subclass of analytes. Nonlimiting exemplary classes or subclasses of analytes include metalloproteases, cysteine proteases, ubiquitin-specific proteases, cysteine cathepsins, esterases, kinases, histone deacetylases, serine reductases, oxidoreductases, ATPases, and GTPases. Additional exemplary classes or subclasses of analytes include serine hydrolases and serine proteases (such as urokinase plasminogen activator, tissue-plasminogne activator, granzymes, fatty acid amide hydrolase, dipeptidylpeptidases (including, but not limited to, dipeptidylpeptidase IV and dipeptidylpeptidase VII)), glycosidases, phosphatases, and cytochrome P450 enzymes.

Nonlimiting exemplary covalent analyte binding moieties are described, e.g., in

Bachovchin et al., Nat. Biotech. 27: 387-394 (2009); Cravatt et al., Ann. Rev. Biochem. 77: 383-414 (2008); Fonovic et al., Curr. Pharmac. Des. 13: 253-261 (2007); Kato et al., Nat. Chem. Biol. 1: 33-38 (2005); Patricelli et al., Biochem. 46: 350-358 (2007); Paulick et al., Curr. Opin. Genet. Dev. 18: 97-106 (2008); Saghatelian et al., PNAS 101: 10000-10005 (2004); Salisbury et al., /. Am. Chem. Soc. 130: 2184-2194 (2008); Salisbury et al. PNAS 104: 1171-1176 (2007); Wright et al., Chem. & Biol. 14: 1043-1051 (2007); Wright et al., JACS 131: 10692-10700 (2009); U.S. Pat. No. 6,872,574 B2; and U.S. Pub. Nos. US 2009/0252677 Al and US 2008/0176841 Al, incorporated herein by reference. Such exemplary covalent analyte binding moieties include, but are not limited to, those comprising fluorphosphonates, aryl phosphonates, sulfonyl fluorides, carbamates for use, for example, in detecting serine hydrolases and serine proteases. For use in detecting analytes such as cysteine proteases, cysteine cathepsins, and/or ubiquitin-specific proteases, such exemplary covalent analyte binding moieties include, but are not limited to, those comprising epoxides, vinyl sulfones, diazomethyl ketones, alpha-halo ketones, and acyloxymethyl ketones. For use in detecting analytes such as metalloproteases and/or histone deacetylases, such exemplary covalent analyte binding moieties include, but are not limited to, HxBP-Rh, HxBP with alkyne group, suberoylanilide hydroxamic acid modified with benzophenone and alkyne groups (SAHA-BPyne), and others comprising a photoreactive moiety whereby covalent labeling is accomplished by exposure to light, such as UV light. For use in detecting analytes such as kinases, such exemplary covalent analyte binding moieties include, but are not limited to, those comprising acyl phosphate-containing nucleotides, Wortmannin, and resorcylic acid lactones. For use in detecting analytes such as cytochrome P540, such exemplary covalent analyte binding moieties include, but are not limited to, those comprising aryl acetylenes, such as 2-ethynylnaphthalene (2EN).

In some embodiments, a non-covalent analyte binding moiety of a proximity detection probe is capable of binding to more than one analyte. In some such embodiments, a non- covalent analyte binding moiety is capable of binding to a particular motif or epitope that is found in multiple analytes, such as when the non-covalent analyte binding moiety is an antibody or antibody fragment. In some embodiments, a proximity detection probe comprises multiple covalent analyte binding moieties and/or non-covalent analyte binding moieties such that the proximity detection probe interacts with multiple analytes.

Exemplary oligonucleotide moieties and splint oligonucleotides

In some embodiments, at least a portion of an oligonucleotide moiety of a first proximity detection probe is capable of hybridizing to at least a portion of an oligonucleotide moiety of a second proximity detection probe. In some embodiments, the hybridized region comprises at least 5 base pairs, at least 10 base pairs, at least 15 base pairs, at least 20 base pairs, at least 25 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, or at least 100 base pairs.

In some embodiments, an oligonucleotide moiety of a first proximity detection probe is not capable of hybridizing to an oligonucleotide moiety of a second proximity detection probe. In some such embodiments, the first and second proximity detection probes may be contacted with a splint oligonucleotide that is capable of hybridizing to at least a portion of the oligonucleotide moiety of the first proximity detection probe, and is also capable of hybridizing to at least a portion of the oligonucleotide moiety of the second proximity detection probe. In some embodiments, the hybridized region between the splint oligonucleotide(s) and an oligonucleotide moiety of a proximity detection probe comprises at least 5 base pairs, at least 10 base pairs, at least 15 base pairs, at least 20 base pairs, at least 25 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, or at least 100 base pairs. In some embodiments, a splint oligonucleotide is symmetric, e.g., it hybridizes to an equal number of bases of each oligonucleotide moiety. In some embodiments, a splint oligonucleotide is asymmetric, e.g., it hybridizes to a greater number of bases of one oligonucleotide moiety than of the other oligonucleotide moiety. Nonlimiting exemplary splint oligonucleotides are described, e.g., in PCT Pub. No. WO 2005/123963.

In some embodiments, a splint oligonucleotide hybridizes to the first and second oligonucleotide moieties in such a way that the 3' end of one of the oligonucleotide moieties is adjacent to the 5' end of the other oligonucleotide moieties. In some embodiments, the 3' and 5' ends of the oligonucleotide moieties of the proximity detection probe pair are capable of being ligated together. In some embodiments, the 3' end of one of the oligonucleotide moieties is separated from the 5' end of the other oligonucleotide moieties by a gap of 1 or more nucleotides. In some embodiments, the gap is filled in using a polymerase such that the filled-in ends are capable of being ligated together. In some embodiments, a splint oligonucleotide may comprise one or more of ribonucleotides, deoxyribonucleotides, analogs of ribonucleotides, and/or analogs deoxyribonucleo tides. Exemplary analogs of ribonucleotides and analogs of

deoxyribonucleotides include, but are not limited to, analogs that comprise one or more modifications to the nucleotide sugar, phosphate, and/or base moiety. Exemplary oligonucleotide analogs include, but are not limited to, LNA (see, e.g., U.S. Pat. No.

6,316,198), PNA (see, e.g., U.S. Pat. No. 6,451,968), and any other nucleotide analogs discussed herein or known in the art (see, e.g., Loakes, Nucleic Acids Res. 2001 Jun. 15; 29(12):2437-47, and Karkare et al., Appl Microbiol Biotechnol. 2006 August; 71(5):575-86. Epub 2006 May 9). In some embodiments, a splint oligonucleotide comprises at least one deoxy-uracil (dU) nucleotide in place of at least one deoxy-thymine (dT) nucleotide.

One skilled in the art can select appropriate sequences and lengths for the

oligonucleotide moieties of proximity detection probes and/or splint oligonucleotides, according to the intended use. A discussion of nonlimiting exemplary methods of selecting oligonucleotide moieties for proximity detection probes and/or split oligonucleotides can be found, e.g., in U.S. Pat. No. 6,511,809 B2 and PCT Pub. No. WO 2005/123963.

Exemplary Detector Probes

In some embodiments, detection of the interaction between an oligonucleotide moiety of a first proximity detection probe and an oligonucleotide moiety of a second proximity detection probe comprises amplification. In some embodiments, a detector probe is used in an amplification reaction to facilitate detection of the amplification product. Nonlimiting exemplary detector probes include, but are not limited to, probes used in a 5'-nuclease assay (for example, TaqMan® probes, described, e.g., in U.S. Pat. No. 5,538,848); stem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature Biotechnology 14:303-308); stemless or linear beacons (see, e.g., WO

99/21881), PNA Molecular Beacons™ (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091); linear PNA beacons (see, e.g., Kubista et al., 2001, SPIE 4264:53-58); non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097); Sunrise®/Amplifluor® probes (U.S. Pat. No. 6,548,250); stem-loop and duplex Scorpion® probes (Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743); bulge loop probes (U.S. Pat. No. 6,590,091); pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752); MGB Eclipse™ probe (Epoch Biosciences); hairpin probes (U.S. Pat. No. 6,596,490); peptide nucleic acid (PNA) light-up probes; self-assembled nanoparticle probes; and ferrocene-modified probes. Nonlimiting exemplary detector probes are described, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al., 1999, Nature Biotechnology 17:804-807; Isacsson et al., 2000, Molecular Cell Probes 14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35; Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002, Nuc. Acids Res. 30:4208-4215; Riccelli et al., 2002, Nuc. Acids Res. 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, /. Am. Chem. Soc. 124:9606-9612; Broude et al., 2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res. Toxicol. 15: 118-126; and Yu et al., 2001, /. Am. Chem. Soc. 14: 11155-11161.

In some embodiments, detector probes comprise quenchers. Exemplary quenchers include, but are not limited to, black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers

(Epoch). In some embodiments, detector probes comprise two probes, wherein, for example, one probe comprises a fluorescent moiety and another probe comprises a quencher, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization of the two probes on a target alters the signal via a change in fluorescence. Nonlimiting exemplary detector probes comprising two probes are described, e.g., in U.S. Pat. Pub. No. US 2006/0014191 to Lao et al. Exemplary detector probes also include, but are not limited to, sulfonate derivatives of fluorescein dyes with SO₃ instead of the carboxylate group, phosphoramidite forms of fluorescein, and phosphoramidite forms of CY 5

(commercially available, e.g., from Amersham).

In some embodiments, detector probes comprise intercalating labels. Exemplary intercalating labels include, but are not limited to, ethidium bromide, SYBR® Green I (Molecular Probes), and PicoGreen® (Molecular Probes), which allow visualization in realtime, or at an end point, of an amplification product in the absence of a nucleic acid probe. In some embodiments, a detector probe comprising an intercalating label is a sequence- independent detector probe. In some embodiments, real-time visualization can comprise a sequence-independent intercalating detector probe and a sequence -based detector probe.

In some embodiments, a detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction, and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction. In some embodiments, detector probes further comprise various modifications, such as, for example, a minor groove binder (see, e.g., U.S. Pat. No. 6,486,308) to further provide desirable thermodynamic characteristics. In some embodiments, detector probes can correspond to identifying portions or identifying portion complements, also referred to as zip- codes. Identifying portions are described, e.g., in U.S. Pat. Nos. 6,309,829 (referred to as a "tag segment" therein); 6,451,525 (referred to as a "tag segment" therein); 6,309,829 (referred to as a "tag segment" therein); 5,981,176 (referred to as "grid oligonucleotides" therein); 5,935,793 (referred to as "identifier tags" therein); and PCT Pub. No. WO 01/92579 (referred to as "addressable support-specific sequences" therein).

Exemplary Methods

Methods provided herein may be carried out in any order of the recited events that is logically possible, as well as the recited order of events.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications and/or as commonly accomplished in the art and/or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods known in the art and as described in various general and more specific references, including but not limited to, those that are cited and discussed throughout the present specification. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

N.Y. (1989)); Lehninger, Biochemistry (Worth Publishers, Inc.); Methods in Enzymology (S. Colowick and N. Kaplan Eds., Academic Press, Inc.); Oligonucleotide Synthesis (N. Gait, ed., 1984); A Practical Guide to Molecular Cloning (2.sup.nd Ed., Wily Press, 1988). Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, biology, biochemistry, analytical chemistry, and synthetic organic chemistry described herein are those known and used in the art.

Methods of detecting an analyte in a sample are provided. In some embodiments, the methods facilitate detection of active analyte in a sample. The methods comprise forming a complex comprising an analyte, a first proximity detection probe, and a second proximity detection probe, wherein the first proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety and the second proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety. In some embodiments, a method comprises detecting an interaction between the first oligonucleotide moiety and the second oligonucleotide moiety. In some embodiments, the interaction between the first oligonucleotide moiety and the second oligonucleotide moiety comprises hybridization between the first oligonucleotide moiety and the second

oligonucleotide moiety. In some embodiments, the first oligonucleotide moiety and the second oligonucleotide moiety are contacted with a splint oligonucleotide that hybridizes with at least a portion of the first oligonucleotide and at least a portion of the second oligonucleotide. In some embodiments, the first oligonucleotide and the second

oligonucleotide are ligated together in the presence of the splint oligonucleotide. In some embodiments, detecting the interaction of the first oligonucleotide moiety and the second oligonucleotide moiety comprises amplification. In some embodiments, detecting the interaction comprises quantitative PCR.

Formation of a complex comprising an analyte, a first proximity detection probe, and a second proximity detection probe can be accomplished using many different reagents and through a series of steps carried out in many different orders. That is, in some embodiments, the complex is formed by (a) contacting a target analyte (TA) with a covalent analyte binding moiety (CABM) that comprises a first member of a binding pair; (b) contacting the TA-

CABM complex with a first oligonucleotide moiety (01) that comprises a second member of the binding pair; (c) contacting the TA-CABM-Ol complex with a proximity detection probe comprising a non-covalent analyte binding moiety (NABM) and a second oligonucleotide moiety (NABM-02), thus forming complex 02-NABM-TA-CABM-Ol, which comprises an analyte, a first proximity detection probe, and a second proximity detection probe, wherein the first proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety and the second proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety.

In some embodiments, steps (b) and (c) are carried out in reverse order (in which case, an 02-NABM-TA-CABM complex is formed after the second step) or are carried out simultaneously. In some embodiments, step (c) is carried out before steps (a) and (b), in which case, an 02-NABM-TA complex is formed before step (a) is carried out (which forms an 02-NABM-TA-CABM complex). In some embodiments, all of the steps are carried out simultaneously.

In some embodiments, the complex is formed by (a) contacting a target analyte (TA) with a covalent analyte binding moiety (CABM) that comprises a first member of a first binding pair; (b) contacting the TA-CABM complex with a non-covalent analyte binding moiety (NABM) that comprises a first member of a second binding pair; (c) contacting the NABM-TA-CABM complex with a first oligonucleotide moiety (01) that comprises a second member of the first binding pair; (d) contacting the NABM- TA-CABM-Ol complex with a second oligonucleotide moiety (02) that comprises a second member of the second binding pair, thus forming complex 02-NABM-TA-CABM-Ol, which comprises an analyte, a first proximity detection probe, and a second proximity detection probe. In some embodiments, steps (a) and (b) are carried out in reverse order (in which case, an NABM-TA complex is formed after the first step) or simultaneously. In some embodiments, steps (c) and (d) are carried out in reverse order (in which case, an 02-NABM-TA-CABM is formed after the third step) or are carried out simultaneously. In some embodiments, the order of steps is (a) and (c), in that order or simultaneously (forming a TA-CABM-01 complex), then (b) and (d), in that order or simultaneously. In some embodiments, the order of steps is (b) and (d), in that order or simultaneously (forming an 02-NABM-TA complex), then (a) and (c), in that order or simultaneously. In some embodiments, all of the steps are carried out simultaneously.

In some embodiments, the complex is formed by (a) contacting a target analyte (TA) with a proximity detection probe that comprises a covalent analyte binding moiety and a first oligonucleotide moiety; (b) contacting the TA-CABM-01 complex with a non-covalent analyte binding moiety (NABM) that comprises a first member of a binding pair; (c) contacting the NABM-TA-CABM-Ol complex with a second oligonucleotide moiety (02) that comprises a second member of the binding pair, thus forming complex 02-NABM-TA - CABM-Ol, which comprises an analyte, a first proximity detection probe, and a second proximity detection probe. In some embodiments, steps (a) and (b) are carried out in reverse order (in which case, an NABM-TA is formed after the first step) or are carried out simultaneously. In some embodiments, steps (b) and (c) are carried out before step (a), in which case, an 02-NABM-TA complex is formed before step (a) is carried out. In some embodiments, all of the steps are carried out simultaneously.

In some embodiments, a complex is formed by (a) contacting a target analyte (TA) with a proximity detection probe that comprises a covalent analyte binding moiety and a first oligonucleotide moiety; and (b) contacting the TA-CABM-Ol complex with a proximity detection probe comprising a non-covalent analyte binding moiety and a second

oligonucleotide moiety (NABM-02), thus forming complex 02-NABM-TA-CABM-Ol, which comprises an analyte, a first proximity detection probe, and a second proximity detection probe. In some embodiments, steps (a) and (b) are carried out in reverse order (thus forming an 02-NABM-TA complex after the first step), or are carried out simultaneously.

In some embodiments, one or more of the steps described above for forming a complex comprising an analyte, a first proximity detection probe, and a second proximity detection probe is carried out in a lysate of a biological sample. In some embodiments, the lysate is a prokaryotic cell lysate, a eukaryotic cell lysate, a viral lysate, a bacteriophage lysate, or a tissue lysate. In some embodiments, one or more of the steps described above for forming the complex are carried out on whole cells. In some such embodiments, the target analyte is located on the surface of a cell, and one or more of the steps described above for forming the complex are carried out without lysing the cells. In some embodiments, all of the steps described above for forming the complex are carried out without lysing the cells. In some embodiments, detecting the interaction between the first oligonucleotide moiety and the second oligonucleotide moiety is carried out without lysing the cells.

In some embodiments, the target analyte is located within cells, and at least one of the steps described above for forming a complex comprising an analyte, a first proximity detection probe, and a second proximity detection probe is carried out without lysing the cells. In some embodiments, a TA-CABM complex is formed without lysing cells. In some embodiments, following formation of the TA-CABM complex, the cells are lysed before the remaining components are bound to the complex. In some such embodiments, the first member of a binding pair comprised in the covalent analyte binding moiety is a moiety capable of undergoing a click reaction. In some such embodiments, an oligonucleotide moiety comprises a second member of the binding pair, such as a complementary click moiety. In some embodiments, the first member of a binding pair comprised in the covalent analyte binding moiety is a biotin or biotin derivative. In some such embodiments, an oligonucleotide moiety comprises a second member of the binding pair, such as a streptavidin or streptavidin derivative.

In some embodiments, the target analyte is located within a multicellular organism and at least one of the steps described above for forming a complex comprising an analyte, a first proximity detection probe, and a second proximity detection probe is carried out in the living organism. In some embodiments, the organism is administered, or contacted with, a covalent analyte binding moiety comprising a first member of a binding pair. In some embodiments, following formation of a TA-CABM complex, a sample is removed from the organism and the remaining components are bound to the complex. In some embodiments, the remaining components are bound to the complex following lysis of the sample removed from the organism. In some embodiments, the remaining components are bound to the complex without lysing the sample removed from the organism.

In some embodiments, more than one proximity detection probe sets are bound to their respective target analytes in the same mixture. That is, in some embodiments, the steps described above for forming a complex are carried out to form more than one different complex simultaneously. Thus, for example, in some embodiments, a first covalent analyte binding moiety comprising a first member of a first binding set and a second covalent analyte binding moiety comprising a first member of a second binding set are incubated with the same sample to form a TA1-CABM1 complex and a TA2-CABM2 complex. The TA1- CABM1 complex and TA2-CABM2 complex are then contacted with a first oligonucleotide moiety comprising a second member of the first binding set and a second oligonucleotide moiety comprising a second member of the second binding set, to form a TAl-CABMl-Ol complex and a TA2-CABM2-02 complex. The TAl-CABMl-Ol complex and TA2-

CABM2-02 complex are then contacted with a first proximity detection probe comprising a first non-covalent analyte binding moiety and a third oligonucleotide moiety, and a second proximity detection probe comprising a second non-covalent analyte binding moiety and a fourth oligonucleotide moiety, to form an 03-NABMl-TAl-CABMl-Ol complex and an 04-NABM2-TA2-CABM2-02 complex. As discussed above, many different ways of forming the final complexes are contemplated, and every complex in a mixture need not have been formed in the same way.

In some embodiments, one or more of the steps discussed above for forming a complex comprising an analyte, a first proximity detection probe, and a second proximity detection probe, are carried out on a solid support. In some embodiments, a covalent analyte binding moiety and/or a non-covalent analyte binding moiety and/or an oligonucleotide moiety is bound to a solid support, a covalent analyte binding moiety and/or a non-covalent analyte binding moiety and/or an oligonucleotide moiety may be bound to a solid support noncovalently or covalently. In some embodiments, a covalent analyte binding moiety and/or a non-covalent analyte binding moiety and/or an oligonucleotide moiety is reversibly bound to a solid support. In some embodiments, a covalent analyte binding moiety and/or a non- covalent analyte binding moiety and/or an oligonucleotide moiety is bound to a solid support using a binding pair. In some embodiments, when a covalent analyte binding moiety and/or a non-covalent analyte binding moiety and/or an oligonucleotide moiety is bound to a solid support, one or more steps in forming a complex comprising an analyte, a first proximity detection probe, and a second proximity detection probe is followed and/or preceded by at least one wash step. In some embodiments, each step is followed and/or preceded by a wash step. In some embodiments, not all of the steps are followed and/or preceded by a wash step.

In some embodiments, at least a portion of detecting the interaction between a first oligonucleotide moiety and a second oligonucleotide moiety occurs on a solid phase. In some embodiments, detecting the interaction between a first oligonucleotide moiety and a second oligonucleotide moiety occurs in solution.

In some embodiments, at least one splint oligonucleotide is added to the sample before, at the same time as, or after addition of at least one proximity detection probe. In some embodiments, a ligation mix is added to the sample after addition of at least one proximity detection probe set. In some embodiments, the ligation mix comprises a ligase enzyme suitable for ligating the ends of the oligonucleotide moieties of a proximity detection probe set together, and a suitable buffer. In some embodiments, the ligation mix is added after addition of at least one splint oligonucleotide. In some embodiments, the ligation mix is added at the same time as the at least one splint oligonucleotide.

After the ligation mix is added to the sample, in some embodiments, the ligation reaction is incubated for at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, or at least 1 hour. In some embodiments, the ligation reaction is incubated for 5 to 10 minutes. In some embodiments, after addition of the ligation mix, the ligation reaction is incubated at a temperature between 0°C to 25°C. In some embodiments, the ligation reaction is incubated at a temperature greater than 25°C. In some embodiments, the ligation reaction is incubated at a temperature between 0°C and 10°C, between 4 °C and 15 °C, between 4°C and 20°C, between 10°C and 20°C, or between 15°C and 25°C. In some embodiments, a ligation reaction is terminated. In some embodiments, a ligation reaction is terminated by adding at least one protease. When a splint oligonucleotide comprises at least one dU nucleotide, in some embodiments, a ligation reaction is terminated by adding uracil- DNA glycosylase.

In some embodiments, at least one splint oligonucleotide is added to the sample before, at the same time as, or after at least one proximity detection probe is added to the sample. In some embodiments, the ligation step discussed above is omitted. For example, in some embodiments, where at least one splint oligonucleotide is added after the proximity detection probe set addition and incubation, the sample is further incubated at a temperature and for a time sufficient to allow hybridization of the at least one splint oligonucleotide to at least one proximity detection probe set. In some embodiments, one skilled in the art can select an appropriate time and temperature for such hybridization. In some embodiments, hybridization conditions include temperatures between 0°C to 75°C In some embodiments, the incubation is carried out at between 0°C and 65°C, between 4°C and 50°C, between 10°C and 45°C, or between 15°C and 40°C. In some embodiments, the incubation is carried out for at least 4 hours. In some embodiments, the incubation is carried out for at least 5 minutes, at least 10 minutes, at least 30 minutes, at least an hour, or at least 2 hours.

In some embodiments, after the complex comprising an analyte, a first proximity detection probe, and a second proximity detection probe is formed, the sample is treated with at least one protease. In some embodiments, after addition of the at least one protease, the sample is incubated for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, or at least 4 hours. In some embodiments, the sample is incubated at at least one temperature between 0°C to 65°C, between 0°C and 55°C, between 4°C and 50°C, between 10°C and 45°C, or between 15°C and 40°C. In some embodiments, at least one protease is inactivated after incubation. In some embodiments, at least one protease is heat inactivated, e.g., by incubating the sample for at least 5 minutes at at least 50°C. In some embodiments, the sample is incubated at at least 55°C, at least 60°C, at least 65°C, at least 70°C, or at least 75°C to heat inactivate the protease. In some

embodiments, at least one protease is inactivated, e.g., by addition of at least one chemical. In some embodiments, at least one protease is inactivated by addition of PMSF.

In some embodiments, after inactivation of the at least one protease, the hybridized and/or ligated proximity detection probe sets are detected. In some embodiments, one or more proximity detection probe sets are detected using the same detection method. In some embodiments, one or more proximity detection probe sets are detected simultaneously. In some embodiments, detection of the at least one hybridized and/or ligated proximity detection probe sets comprises multiplex quantitative PCR. In some embodiments, detection of the at least one hybridized and/or ligated proximity detection probe sets comprises singleplex quantitative PCR. In some embodiments, the method does not comprise a nucleic acid purification step prior to detection of the one or more proximity detection probe sets. For example, in some embodiments, a different label is used to detect each different proximity detection probe set.

Exemplary Proximity Detection Assays

Exemplary proximity detection assays are described, e.g., in U.S. Pat. No. 6,511,809 B2; U.S. Pat. Pub. No. US 2002/0064779; PCT Pub. No. WO 2005/123963; U.S. Pat. Pub. No. US 2005/0003361 Al; U.S. Pat. Pub. No. US 2007/0026430; Fredricksson et al., Nature Biotech. 20: 473-477 (2002); and Gustafsdottir et al., Clin. Chem. 52: 1152-1160 (2006).

In some embodiments, a proximity detection assay comprises forming at least one complex comprising a target analyte and at least one proximity detection probe set. When the proximity detection assay is a proximity ligation assay, in some embodiments, a complex is contacted with at least one splint oligonucleotide and the mixture is incubated under conditions allowing hybridization between the at least one splint oligonucleotide and the oligonucleotide moieties of the proximity detection probe set. In some embodiments, the splint oligonucleotide hybridizes to two oligonucleotide moieties such that the 3 ' end of a first oligonucleotide moiety is adjacent to the 5' end of a second oligonucleotide moiety. In some embodiments, the 3' end of the first oligonucleotide moiety and the 5' end of the second oligonucleotide moiety are ligated together. In some embodiments, ligation is mediated by a ligase enzyme.

In some embodiments, the ligated product is detected by at least one method discussed herein. In some embodiments, the ligated product and the hybridized splint oligonucleotide are subjected to a primer extension reaction as part of, or prior to, the detection method. In some embodiments, the primer extension reaction produces a double- stranded oligonucleotide. In some embodiments, the primer extension reaction comprises at least one oligonucleotide primer complimentary to the ligated product. In some

embodiments, the splint oligonucleotide serves as a primer in the primer extension reaction, along with a second oligonucleotide primer. In some embodiments, two oligonucleotide primers other than the splint oligonucleotide are included in the primer extension reaction. In some embodiments, following a primer extension reaction that produces a double-stranded oligonucleotide, a first strand of the double stranded oligonucleotide comprises the ligated oligonucleotide moieties, and the second strand comprises the sequence of the splint oligonucleotide connected to (i) a first sequence that is complementary to at least a portion of the first oligonucleotide moiety, and also connected to (ii) a second sequence that is complementary to at least a portion of the second oligonucleotide moiety.

In some embodiments, when the detection method involves hybridization of one or more oligonucleotides (such as, for example, one or more oligonucleotide primers and/or detector probes comprising oligonucleotides), one skilled in the art can select an appropriate nucleotide sequence such that the oligonucleotide can be used to specifically detect the ligated product. For example, in some embodiments, where the ligated oligonucleotide moieties are subjected to a primer extension reaction, one or more oligonucleotides that hybridize to the primer extension product and not to the oligonucleotide moieties or the splint oligonucleotide can be selected. Such oligonucleotides may be used, in some embodiments, in a direct detection method and/or in a detection method involving an amplification step. In some embodiments, one or more oligonucleotides can be selected to amplify the ligated oligonucleotide moieties such that amplification will only occur if the moieties are ligated together.

In some embodiments, when the proximity detection assay is a proximity interaction assay, an oligonucleotide moiety of a first proximity detection probe is capable of hybridizing to an oligonucleotide moiety of a second proximity detection probe. Alternatively, in some embodiments, at least one splint oligonucleotide is added to the mixture for each proximity detection probe set. In some embodiments, the mixture is then incubated under conditions allowing hybridization between the hybridizable oligonucleotide moieties, and/or between the oligonucleotide moieties and the at least one splint oligonucleotide.

In some embodiments, the hybridized oligonucleotides are subjected to a primer extension reaction as part of, or prior to, the detection method. In some embodiments, when the oligonucleotide moieties hybridize to one another, the primer extension reaction extends from the end of each oligonucleotide moiety to produce a double- stranded oligonucleotide that comprises a first strand that comprises the first oligonucleotide moiety connected to a sequence that is complementary to at least a portion of the second oligonucleotide moiety, and a second strand that comprises the second oligonucleotide moiety connected to a sequence that is complementary to at least a portion of the first oligonucleotide moiety. In some embodiments, the double-stranded oligonucleotide is subjected to a further primer extension reaction using at least one oligonucleotide primer. In some embodiments, the double- stranded oligonucleotide is subjected to a further primer extension reaction using at least two oligonucleotide primers.

In some embodiments, when at least one splint oligonucleotide hybridizes to the oligonucleotide moieties, the splint oligonucleotide serves as a primer in the primer extension reaction, along with a second oligonucleotide primer, to produce a double-stranded oligonucleotide. In some embodiments, the double-stranded oligonucleotide comprises a first strand comprising at least a portion of the sequence of each of the oligonucleotide moieties, and a second strand comprising the sequence of the splint oligonucleotide connected to a sequence that is complementary to at least a portion of one of the oligonucleotide moieties. Exemplary Normalizer Controls for Proximity Detection Assays

In some embodiments, the amount of a target analyte may be normalized to at least one normalizer control. Nonlimiting exemplary normalizer controls are described, e.g., herein and in PCT Pub. No. WO 2005/123963. In some embodiments, one skilled in the art can select one or more normalizer controls for a particular application.

A normalizer control may be "exogenous" or "endogenous." In some embodiments, an exogenous normalizer control is added to a sample after the sample is collected. In some embodiments, the sample naturally comprises an amount of the same analyte that is used as an exogenous normalizer control, but the normalizer control is considered to be exogenous because an additional amount of analyte has been added.

In some embodiments, an endogenous normalizer control is already present in a sample at the time the sample is collected for analysis. A normalizer control is referred to as "housekeeping," in some embodiments, when it is present at a high level in a biological sample without having been added. In some embodiments, a housekeeping normalizer control is present at a high level in more than one different type of biological sample.

In some embodiments, a normalizer control is an endogenous analyte. In some embodiments, a normalizer control is an endogenous protein. In some embodiments, a normalizer control is an endogenous enzyme. In some embodiments, a normalizer controls is an endogenous housekeeping protein. Exemplary endogenous housekeeping protein normalizer controls include, but are not limited to, GAPDH, acidic ribosomal protein, beta- actin, HPRT, beta-glucuronidase, cystatin B, ICAM1, and p53.

In some embodiments, a normalizer control is an exogenous analyte. In some embodiments, a normalizer control is an exogenous protein. In some embodiments, a normalizer control is an exogenous enzyme. Exemplary exogenous protein normalizer controls include, but are not limited to, bacterial proteins, protein tags, viral proteins, intact virions, insect proteins, mammalian proteins not normally expressed in the selected biological sample, and mammalian proteins normally expressed at a low level in the selected biological sample. In some embodiments, a normalizer control is an enzyme. In some embodiments, a normalizer control is the same class or subclass of enzyme as the target analyte. In some embodiments, a normalizer control is a receptor. In some embodiments, a normalizer control is the same class or subclass of receptor as the target analyte.

In some embodiments, a sample comprises at least one normalizer control, at least two normalizer controls, at least three normalizer controls, at least four normalizer controls, or at least five normalizer controls. In some embodiments, a sample comprises at least one endogenous normalizer control and at least one exogenous normalizer control. In some embodiments, all of the normalizer controls in a sample are endogenous. In some embodiments, all of the normalizer controls in a sample are exogenous.

In some embodiments, a normalizer control is detected in the same sample in which a target analyte is detected. In some embodiments, a normalizer control is detected in the same vessel in which a target analyte is detected, using the same or different methods. In some embodiments, the sample is split or divided and a normalizer control and a target analyte are detected in separate vessels, using the same or different methods. In some embodiments, a normalizer control is detected at the same time that a target analyte is detected.

In some embodiments, the amount of a target analyte may be normalized to a normalizer control using the "comparative CT method" or "ACT method," which involves calculating the ACT- In some embodiments, the ACT is calculated by subtracting the CT of a quantitative nucleic acid detection assay used to detect a normalizer control from the CT of a quantitative nucleic acid detection assay used to detect a target analyte. In some

embodiments, the fold difference in the amounts of the normalizer control and target analyte is calculated from the ACT- In some embodiments, the fold difference in the amounts of the normalizer control and target analyte is calculated from the ACT according to the formula 2^"

ACT

In some embodiments, the AGr is calculated by subtracting the ACT of a "calibrator sample" from the ACT of a "test sample." Exemplary calibrator samples include, but are not limited to, a sample prepared from untreated cells and a sample prepared from a particular tissue. Exemplary test samples include, but are not limited to, a sample prepared from treated cells and a sample prepared from a tissue other than the tissue from which a calibrator sample was prepared. In some embodiments, the AACT is calculated by subtracting the ACT of a calibrator sample from the ACT of a test sample.

In some embodiments, the fold difference in the amount of target nucleic acid in the calibrator and test samples is calculated from the AACT according to the formula 2^~AACT. In some embodiments, the fold difference in the amount of target analyte in the calibrator and test samples is calculated from the AACT according to the formula 2^~AACT. Use of the AACT method is described, e.g., in Applied Biosystems, "Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR" (2008); and Applied Biosystems, User Bulletin #2: ΑΒΙ Prism 7700 Sequence Detection System (Dec. 11, 1997 (updated October 2001)).

In some embodiments, the use of a normalizer control may eliminate the need to prepare an external standard curve using an analyte, which may produce a CT value that differs from the CT value observed when there is an identical level of the analyte in a sample. In some embodiments, the use of a normalizer control may control for a variable in a proximity detection assay. Exemplary variables in proximity detection assays include, but are not limited to, nucleic acid degradation, analyte degradation, the extent to which analyte activity and/or structure has been maintained, the efficiency with which a proximity detection probe interacts with an analyte, the efficiency of a ligation reaction, and the efficiency of a real-time PCR reaction.

In some embodiments, an analyte normalizer control is detected using a proximity detection assay. Nonlimiting exemplary proximity detection assays are described herein. In some embodiments, an analyte normalizer control is detected using the same method (using appropriate proximity detection probes) and in the same vessel as a target analyte. In some embodiments, an analyte normalizer control is detected using the same method (using appropriate proximity detection probes) but in a different vessel as a target analyte.

Exemplary Ligation Reaction Termination

In some embodiments, a ligation reaction in a proximity ligation assay is terminated prior to detection of the ligated product. In some embodiments, a ligation reaction is terminated prior to storing the proximity ligation assay. The proximity ligation assay may be stored before or after detection of the ligated product. In some embodiments, termination of the ligation reaction reduces the amount of additional ligated products that may accumulate over time, for example, during storage of a proximity ligation assay.

In some embodiments, the ligation reaction is terminated by treatment with a protease.

In some embodiments, a protease is selected based on one or more of the following characteristics: the ease with which the protease can be inactivated, whether the protease requires metal ions for activity, whether the protease requires detergents for activity, whether protease digestion results in a degradation of nucleic acids, and whether the protease releases the target nucleic acid.

In some embodiments, a protease is selected which can be heat-inactivated. In some embodiments, a protease is selected which can be chemically-inactivated. Nonlimiting exemplary chemicals that can be used to inactivate a protease include, but are not limited to, AEBSF, aprotinin, bestatin, chymostatin, E-64, EDTA, EGTA, leupeptin, pepstatin A, 1,10- phenanthroline, phosphoramidon, and PMSF. In some embodiments, one or more serine proteases are used. In some embodiments, one or more proteases are selected from subtilisin carlsberg protease, streptomyces griseus protease, and proteinase K. When streptomyces griseus protease is selected, in some embodiments, the protease is heat-inactivated. When proteinase K is selected, in some embodiments, the protease is chemically inactivated.

In some embodiments, more than one protease is used. When more than one protease is used, the proteases may be added at the same or different times. In some embodiments, when more than one protease is used, the method may comprise one inactivation step or more than one inactivation step. Furthermore, in various embodiments, the inactivation steps may be the same or different, e.g., one or more inactivation steps may be heat treatment, while one or more inactivation steps may be chemical treatment.

In some embodiments, e.g., when the target analyte is a protein or a peptide, a protease is added after hybridization and/or ligation of the proximity detection probe sets.

In some embodiments, a ligation reaction is terminated by altering the splint oligonucleotide. In some embodiments, a splint oligonucleotide used in a proximity detection assay comprises deoxy-uracil (dU) in place of deoxy-thymine (dT). In some embodiments, the dU-containing splint oligonucleotide is altered by adding uracil-DNA glycosylase (UNG) to the proximity detection assay after the ligation step. In some embodiments, altering the splint oligonucleotide reduces unwanted primer extension products that may form during detection of ligated products.

Exemplary Detection of Proximity Detection Probe Sets

In some embodiments, the hybridized and/or ligated oligonucleotide moieties of the proximity detection probes are subjected to a pretreatment prior to detection. Exemplary pretreatments include, but are not limited to, ligation and primer extension reactions. In some embodiments, when detection of the hybridized and/or ligated oligonucleotide moieties involves amplification, the pretreatment primer extension reaction may not be necessary, because the amplification conditions will allow the primer extension reaction to occur prior to, or simultaneously with, amplification.

In some embodiments, multiple hybridized and/or ligated oligonucleotide moieties of proximity detection probes are detected simultaneously in the same vessel. In some embodiments, multiple hybridized and/or ligated oligonucleotide moieties of proximity detection probes are detected simultaneously in a multiplex amplification reaction. In some embodiments, different labels are used to identify the different proximity detection probe sets. For example, in some embodiments, if five target analytes are being detected in a sample, and a single detection reaction is used to detect the hybridized and/or ligated oligonucleotide moieties of the five different proximity detection probe sets, five different labels may be used to separately identify the different detection reaction products. In some embodiments, such labels may be in the form of detector probes, discussed herein, or any other label known in the art that is suitable for use in the detection methods. One skilled in the art can select an appropriate label or labels, according to some embodiments.

In some embodiments, the hybridized and/or ligated oligonucleotide moieties of the proximity detection probes are detected using real-time PCR. Exemplary methods of performing real-time PCR include, but are not limited to, 5' nuclease real-time PCR, and multiplexed versions thereof. Nonlimiting exemplary methods of 5' nuclease real-time PCR are known in the art and are described, e.g., in Livak, Methods Mol. Biol. 212: 129-47 (2003); Lee et al., Biotechniques 27(2):342-9 (1999); Livak, Genet. Anal. 14(5-6): 143-9 (1999); Heid et al., Genome Res. 6(10):986-94 (1996); and Lee et al., Nucleic Acids Res. l l ;21(16):3761-6 (1993). Nonlimiting exemplary quantitative PCR is described, e.g., in A-Z Quantitative PCR, Bustin, S., Ed., IUL Biotechnology Series (2004). Nonlimiting exemplary methods of real- time PCR are also described, e.g., in Watson et al., Int J Toxicol. 2005 May- June; 24(3): 139- 45; and U.S. Pat. Nos. 6,890,718; 6,773,817; and 6,258,569. In some embodiments, a target nucleic acid is detected using TaqMan One-step qRT-PCR (Applied Biosystems).

In some embodiments, passive reference dyes may be used in quantitative PCR methods. Nonlimiting exemplary passive reference dyes are described, e.g., in U.S. Pat. No. 5,736,333. In some embodiments, external controls may be used in quantitative PCR methods. Nonlimiting exemplary quantitative controls are described, e.g., in U.S. Pat. No. 6,890,718.

In some embodiments, the hybridized and/or ligated oligonucleotide moieties of the proximity detection probes are detected using a combination of PCR and ligation. As a non- limiting example, hybridized and/or ligated oligonucleotide moieties of the proximity detection probes may be detected by first amplifying by PCR, and then applying a ligation inquiry. Certain exemplary such methods are known in the art, and are described, e.g., in Chen et al., Genome Res. 8(5):549-56 (1998). As a further non-limiting example, hybridized and/or ligated oligonucleotide moieties of the proximity detection probes may be detected by first performing a ligation reaction, followed by PCR amplification. Certain exemplary such methods are known in the art and are described, e.g., in U.S. Pat. No. 4,797,470. In some embodiments, the ligation assay may comprise a flap endonuclease, e.g., as described in U.S. Pat. No. 6,511,810.

In some embodiments, the hybridized and/or ligated oligonucleotide moieties of the proximity detection probes are amplified in a first "pre-amplification reaction" (described, e.g., in PCT Pub. No. WO2004/051218), and then decoded in a second amplification reaction. Certain exemplary such methods are known in the art and are described, e.g., in U.S. Pat. No. 6,605,451 ; U.S. Pat. No. 7,604,937; and U.S. Pat. No. 7,601,821.

Nonlimiting exemplary methods of detecting the hybridized and/or ligated oligonucleotide moieties of the proximity detection probes are also described, e.g., in U.S. Pat. No. 6,511,809 B2; U.S. Pub. No. US 2002/0064779 Al; and PCT Pub. No. WO

2005/123963. Nonlimiting exemplary multiplex detection methods are described, e.g., in U.S. Pub. No. US 2006/0216737.

In some embodiments, a detector probe is used to facilitate detection of the hybridized and/or ligated oligonucleotide moieties of the proximity detection probes. Nonlimiting exemplary detector probes are discussed herein. In some embodiments, one skilled in the art can select one or more suitable detector probes according to the intended application.

Exemplary Kits In some embodiments, kits comprising at least one component for carrying out the methods exemplified herein are provided. In some embodiments, a kit comprises a first proximity detection probe that comprise a covalent analyte binding moiety and an oligonucleotide moiety, and a second proximity detection probe that comprises a non- covalent analyte binding moiety and an oligonucleotide moiety. In some embodiments, a kit comprises a covalent analyte binding moiety that comprises a first member of a binding pair, and a proximity detection probe that comprises a non-covalent analyte binding moiety and an oligonucleotide moiety. In some embodiments, a kit comprises a proximity detection probe that comprises a covalent analyte binding moiety and an oligonucleotide moiety, and a non- covalent analyte binding moiety that comprises a first member of a binding pair. In some embodiments, a kit comprises a covalent analyte binding moiety that comprises a first member of a first binding pair, and a non-covalent analyte binding moiety that comprises a first member of a second binding pair. In some embodiments, a kit comprises one or more oligonucleotide moieties that comprise second members of binding pairs. In some embodiments, a kit comprises at least one ligase. In some embodiments, a kit comprises a splint oligonucleotide. In some embodiments, a kit comprises at least one normalizer control.

In some embodiments, a kit comprises at least one component for detecting a proximity detection probe set. In some embodiments, a kit comprises at least one component for detecting a normalizer control. Exemplary components include, but are not limited to, detector probes, primers, polymerases, and reverse transcriptases.

EXAMPLES

The examples discussed below are intended to be purely exemplary of the invention and should not be considered to limit the invention in any way. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1: Homogeneous Activity-Based Proximity Ligation Assay

Cell lysates are prepared as described in Patricelli et al. Biochemistry 46: 350-8 (2007). Gel filtration of cleared lysates is carried out to remove endogenous nucleotides. A kinase-reactive covalent analyte binding moiety (kinase-CABM; such as biotin-hex-acyl-ATP (BHAcATP)), is then added to the cleared and filtered lysates at a concentration of 20 μΜ, and the samples are incubated for 5 minutes. For small molecule profiling experiments, various concentrations of known or putative inhibitors of a kinase (such as ERK2) are preincubated with the cleared and filtered lysates for 5 minutes before addition of kinase- CABM.

Unreacted kinase-CABM is removed by gel filtration, and then streptavidin-5 ' (or 3')- oligonucleotide 1 is added to a final concentration of 10 nM, along with 10 nM of an antibody that binds a selected kinase (such as ERK2). Prior to addition, the antibody has been complexed near the 5' or 3' end of an oligonucleotide 2, e.g., through a biotin- streptavidin linkage. After a 60 minute incubation at 37°C, a "splint" oligonucleotide is added, which hybridizes to the 5' end of one of the bound oligonucleotides and the 3' end of the other, bringing the ends in proximity to one another. Oligonucleotide 1 and

oligonucleotide 2 are then ligated together using T4 DNA ligase for 10 minutes at 37°C. After ligation, aliquots of the reaction mixture are subjected to real-time PCR (TaqMan) assays.

If the assay is used as a small molecule profiling assay, then a reduction in signal from real-time PCR (or an increase in the number of cycles required to reach a predetermined signal threshold) in the reaction mixture that includes a small molecule relative to a reaction mixture without a small molecule indicates that the small molecule may be a kinase inhibitor.

If the assay is used to quantify kinase activity, the results of the real-time PCR can be compared to one or more assays carried out on samples containing known amounts of kinase activity.

Example 2: Heterogeneous Activity-Based Proximity Ligation Assay

Cell lysates are prepared as described in Patricelli et al. Biochemistry 46: 350-8 (2007). Gel filtration of cleared lysates is carried out to remove endogenous nucleotides. A kinase-reactive covalent analyte binding moiety (kinase-CABM, such as biotin-hex-acyl-ATP (BHAcATP)), is then added to the cleared and filtered lysates at a concentration of 20 μΜ, and the samples are incubated for 5 minutes. For small molecule profiling experiments, various concentrations of known or putative inhibitors of a kinase (such as ERK2) are preincubated with the cleared and filtered lysates for 5 minutes before addition of kinase- CABM.

Following incubation with kinase-CABM, streptavidin-coated magnetic beads (such as MyOne SA Tl (Dynal)) that have been complexed with biotinylated anti-kinase (such as ERK2) antibody are added to the mixture and incubated at 37°C for 1 hour to capture kinase. The beads are then washed to remove unreacted kinase-CABM, and then incubated with streptavidin-5'(or 3 ^-oligonucleotide 1. After washing to remove unbound oligonucleotide 1 , the beads are incubated with a second biotinylated anti -kinase (such as ERK2) antibody that binds to a different epitope than the antibody on the beads. Beads are washed again to remove unbound antibody, and then incubated with streptavidin-3 '(or 5')-oligonucleotide 2. (Alternatively, the second anti-kinase antibody has been complexed with an equimolar amount of streptavidin-3' (or 5')-oligonucleotide 2 prior to addition to the beads.) The beads are then washed to remove unbound oligonucleotide 2. A "splint" oligonucleotide is then added, which hybridizes to the 5' end of one of the bound oligonucleotides and the 3' end of the other, bringing the ends in proximity to one another. Oligonucleotide 1 and

As above, if the assay is used as a small molecule profiling assay, then a reduction in signal from real-time PCR (or an increase in the number of cycles required to reach a predetermined signal threshold) in the reaction mixture that includes a small molecule relative to a reaction mixture without a small molecule indicates that the small molecule may be a kinase inhibitor. If the assay is used to quantify kinase activity, the results of the realtime PCR can be compared to one or more assays carried out on samples containing known amounts of kinase activity.

Example 3: Cellular Activity-Based Proximity Ligation Assay

To measure kinases (or other CABM targets) in living cells or tissues, a kinase- CABM comprising a "click" moiety (such as an alkyne group) is incubated with the cells or tissue. The kinase-CABM diffuses or is transported across the cell membrane, where it can react with one or more kinases. The cells or tissue are then lysed, and the lysate subjected to gel filtration to remove unreacted kinase-CABM. The cleared lysate is then incubated with a 5 '(or 3') oligonucleotide 1 comprising a complementary click moiety (such as an azide group), which allows covalent attachment of the oligonucleotide 1 to the kinase-CABM via a click reaction. Gel filtration is then used to remove unreacted oligonucleotide 1.

Alternatively, the cleared lysate can be incubated with a biotin comprising a complementary click moiety (such as carboxamide-6-azidohexanyl biotin) to attach a biotin to the kinase-CABM via a click reaction. A streptavidin-5'(or 3') oligonucleotide 1 is then added and allowed to bind to the kinase-CABM-biotin.

Binding of an anti-kinase antibody complexed with streptavidin-3' (or 5')- oligonucleotide 2, ligation, and real time PCR assays are carried out as described above in Example 1. Alternatively, some or all of the steps of this assay can be carried out in a heterogeneous format, e.g., as described in Example 2.

Example 4: Cytochrome P-450 Activity-Based Proximity Ligation Assay

Insect cells expressing one or more recombinant human cytochrome P450s, human hepatocytes, or human liver samples are treated with test agents to determine the effect of test agents on cytochrome P450 activity. Cells are lysed and the lysate cleared as described above. The cleared lysate is then incubated with a cytochrome P450-reactive CABM (P450- CABM) comprising a "click" moiety (such as alkyne-modified 2-ethynylnaphthalene (2EN- CABM)). Alternatively, the cells may be incubated with the P450-CABM prior to lysis. The mixture is subjected to gel filtration to remove unreacted P450-CABM, and then incubated with 5 '(or 3') oligonucleotide 1 comprising a complementary click moiety (such as an azide group), which allows covalent attachment of the oligonucleotide 1 to the P450-CABM via a click reaction. Gel filtration is then used to remove unreacted oligonucleotide 1.

Alternatively, the cleared lysate can be incubated with a biotin comprising a complementary click moiety (such as carboxamide-6-azidohexanyl biotin) to attach a biotin to the P450-CABM via a click reaction. A streptavidin-5'(or 3') oligonucleotide 1 is then added and allowed to bind to the P450-CABM-biotin.

Binding of an anti-cytochrome P450 antibody complexed with streptavidin-3' (or 5')- oligonucleotide 2, ligation, and real time PCR assays are carried out as described above in Example 1. Alternatively, some or all of the steps of this assay can be carried out in a heterogeneous format, e.g., as described in Example 2.

Although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications may be made without departing from the teachings herein. The foregoing examples are provided to better illustrate the present teachings and are not intended to limit the scope of the teachings herein. Certain aspects of the present teachings may be further understood in light of the following claims.

Claims

1. A method of detecting at least one target analyte in a sample, comprising:

(a) forming a complex comprising at least one target analyte, a first proximity detection probe, and a second proximity detection probe, wherein the first proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety, and the second proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety; and

(b) detecting an interaction between the first oligonucleotide moiety and the second oligonucleotide moiety.

2. The method of claim 1, wherein (a) comprises:

(i) contacting the sample with a covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a binding pair, to form a first complex comprising at least one target analyte and the covalent analyte binding moiety;

(ii) contacting the complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the binding pair, to form a second complex comprising at least one target analyte and the first proximity detection probe;

(iii) contacting the second complex with the second proximity detection probe to form a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe.

3. The method of claim 2, wherein (i) further comprises separating unbound covalent analyte binding moiety from the first complex.

4. The method of claim 2 or claim 3, wherein (ii) further comprises separating unbound first oligonucleotide moiety from the second complex.

5. The method of any one of claims 2, 3, and 4, wherein (iii) further comprises separating unbound second proximity detection probe from the third complex.

6. The method of claim 1, wherein (a) comprises:

(i) contacting the sample with a covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a first binding pair, to form a first complex comprising at least one target analyte and the covalent analyte binding moiety;

(ii) contacting the first complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the first binding pair, to form a second complex comprising at least one target analyte and the first proximity detection probe;

(iii) contacting the second complex with a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a second binding pair, to form a third complex comprising at least one target analyte, the first proximity detection probe, and the non-covalent analyte binding moiety; and

(iv) contacting the third complex with a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the second binding pair, to form a fourth complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe.

7. The method of claim 6, wherein (i) further comprises separating unbound covalent analyte binding moiety from the first complex.

8. The method of claim 6 or claim 7, wherein (ii) further comprises separating unbound first oligonucleotide moiety from the second complex.

9. The method of any one of claims 6, 7, and 8, wherein (iii) further comprises separating unbound non-covalent analyte binding moiety from the third complex.

10. The method of any one of claims 6, 7, 8, and 9, wherein (iv) further comprises separating the unbound second oligonucleotide moiety from the fourth complex.

11. The method of claim 1, wherein (a) comprises:

(i) contacting the sample with the first proximity detection probe, to form a first complex comprising at least one target analyte and the first proximity detection probe;

(ii) contacting the first complex with the second proximity detection probe, to firm a second complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe.

12. The method of claim 11, wherein (i) further comprises separating the unbound first proximity detection probe from the first complex.

13. The method of claim 11 or claim 12, wherein (ii) further comprises separating the unbound second proximity detection probe from the second complex.

14. The method of claim 1, wherein (a) comprises:

(ii) contacting the first complex with a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a binding pair, to form a second complex comprising at least one target analyte, the first proximity detection probe, and the non-covalent analyte binding moiety; and

(iii) contacting the second complex with a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the binding pair, to form a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe.

15. The method of claim 14, wherein (i) further comprises separating unbound first proximity detection probe from the first complex.

16. The method of claim 14 or claim 15, wherein (ii) further comprises separating unbound non-covalent analyte binding moiety from the second complex.

17. The method of any one of claims 14, 15, and 16, wherein (iii) further comprises separating unbound second oligonucleotide moiety from the third complex.

18. The method of claim 1, wherein (a) comprises:

(i) contacting the sample with a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a binding pair, to form a first complex comprising at least one target analyte and the non-covalent analyte binding moiety;

(ii) contacting the first complex with a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the binding pair, to form a second complex comprising at least one target analyte and the second proximity detection probe;

(iii) contacting the second complex with the first proximity detection probe to form a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe.

19. The method of claim 18, wherein (i) further comprises separating unbound non-covalent analyte binding moiety from the first complex.

20. The method of claim 18 or claim 19, wherein (ii) further comprises separating unbound second oligonucleotide moiety from the second complex.

21. The method of any one of claims 18, 19, and 20, wherein (iii) further comprises separating unbound first proximity detection probe from the third complex.

22. The method of claim 1, wherein (a) comprises:

(i) contacting the sample with a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a first binding pair, to form a first complex comprising at least one target analyte and the non-covalent analyte binding moiety;

(ii) contacting the first complex with a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the first binding pair, to form a second complex comprising at least one target analyte and the second proximity detection probe; (iii) contacting the second complex with a covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a second binding pair, to form a third complex comprising at least one target analyte, the second proximity detection probe, and the covalent analyte binding moiety; and

(iv) contacting the third complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the second binding pair, to form a fourth complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe.

23. The method of claim 22, wherein (i) further comprises separating unbound non-covalent analyte binding moiety from the first complex.

24. The method of claim 22 or claim 23, wherein (ii) further comprises separating unbound second oligonucleotide moiety from the second complex.

25. The method of any one of claims 22, 23, and 24, wherein (iii) further comprises separating unbound covalent analyte binding moiety from the third complex.

26. The method of any one of claims 22, 23, 24, and 25, wherein (iii) further comprises separating unbound first oligonucleotide moiety from the fourth complex.

27. The method of claim 1, wherein (a) comprises:

(i) contacting the sample with the second proximity detection probe, to form a first complex comprising at least one target analyte and the second proximity detection probe;

(ii) contacting the first complex with the first proximity detection probe, to form a second complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe.

28. The method of claim 27, wherein (i) further comprises separating unbound second proximity detection probe from the first complex.

29. The method of claim 27 or claim 28, wherein (ii) further comprises separating unbound first proximity detection probe from the second complex.

30. The method of claim 1, wherein (a) comprises:

(ii) contacting the first complex with a covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a binding pair, to form a second complex comprising at least one target analyte, the second proximity detection probe, and the covalent analyte binding moiety; and

(iii) contacting the second complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the binding pair, to form a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe.

31. The method of claim 30, wherein (i) further comprises separating unbound second proximity detection probe from the first complex.

32. The method of claim 30 or claim 31, wherein (ii) further comprises separating unbound covalent analyte binding moiety from the second complex.

33. The method of any one of claims 30, 31, and 32, wherein (iii) further comprises separating unbound first oligonucleotide moiety from the third complex.

34. A method of detecting at least one target analyte in a cell, comprising:

(a) contacting the cell with covalent analyte binding moiety under conditions allowing formation of a first complex comprising at least one target analyte and the covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a first binding pair;

(b) contacting the first complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the first binding pair, to form a second complex comprising at least one target analyte and a first proximity detection probe, wherein the first proximity detection probe comprises the covalent analyte binding moiety and the first oligonucleotide moiety;

(c) contacting the second complex with a second proximity detection probe under conditions allowing formation of a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe; and

(d) detecting an interaction between the first oligonucleotide moiety and the second oligonucleotide moiety.

35. The method of claim 34, wherein (a) further comprises separating unbound covalent analyte binding moiety from the first complex.

36. The method of claim 34 or claim 35, wherein (b) further comprises separating unbound first oligonucleotide moiety from the second complex.

37. The method of any one of claims 34, 35, and 36, wherein (c) further comprises separating unbound second proximity detection probe from the third complex.

38. The method of any one of claims 34 to 37, further comprising lysing the cells between (a) and (b), between (b) and (c), or between (c) and (d).

39. A method of detecting at least one target analyte in a subject, comprising: (a) administering a covalent analyte binding moiety to the subject, wherein the covalent analyte binding moiety comprises a first member of a first binding pair;

(b) isolating cells of the subject that are suspected of containing a first complex comprising the target analyte and the covalent analyte binding moiety;

(c) contacting the first complex with a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the first binding pair, to form a second complex comprising at least one target analyte and a first proximity detection probe, wherein the first proximity detection probe comprises the covalent analyte binding moiety and the first oligonucleotide moiety;

(d) contacting the second complex with a second proximity detection probe under conditions allowing formation of a third complex comprising at least one target analyte, the first proximity detection probe, and the second proximity detection probe; and

(e) detecting an interaction between the first oligonucleotide moiety and the second oligonucleotide moiety.

40. The method of claim 39, wherein (c) further comprises separating unbound first oligonucleotide moiety from the second complex.

41. The method of any claim 39 or claim 40, wherein (d) further comprises separating unbound second proximity detection probe from the third complex.

42. The method of any one of claims 39 to 41, further comprising lysing the cells between (b) and (c), between (c) and (d), or between (d) and (e).

43. The method of any one of the preceding claims, wherein the method further comprises associating at least one target analyte with a solid phase.

44. The method of claim 43, wherein the associating comprises contacting at least one target analyte with an antibody that binds the at least one target analyte, wherein the antibody is associated with the solid phase.

45. The method of claim 43 or claim 44, wherein the solid phase is selected from microparticles and planar surfaces.

46. The method of claim 45, wherein the planar surfaces are selected from microplates and microarray chips.

47. The method of claim 46, wherein the solid phase is a microplate.

48. The method of any one of claims 2 to 10, 14 to 26, and 30 to 42, wherein the first member of at least one binding pair comprises biotin or a derivative thereof and the second member of at least one binding pair comprises streptavidin or a derivative thereof.

49. The method of any one of claims 2 to 10, 14 to 26, and 30 to 42, wherein the first member of at least one binding pair comprises streptavidin or a derivative thereof and the second member of at least one binding pair comprises biotin or a derivative thereof.

50. The method of any one of claims 2 to 10, 14 to 26, and 30 to 42, wherein the first and second members of at least one binding pair are capable of undergoing a click reaction.

51. The method of claim 50, wherein the first member of at least one binding pair comprises an azido moiety and the second member of at least one binding pair comprises a moiety selected from an ethynyl moiety, a phosphine moiety, and a dibenzocyclooctyne (DIBO) moiety.

52. The method of claim 50, wherein the first member of at least one binding pair comprises a moiety selected from an ethynyl moiety, a phosphine moiety, and a

dibenzocyclooctyne (DIBO) moiety and the second member of at least one binding pair comprises an azido moiety.

53. The method of any one of the preceding claims, wherein the interaction between the first oligonucleotide moiety and the second oligonucleotide moiety comprises at least one interaction selected from hybridization between the first and second oligonucleotide moieties and ligation of the first and second oligonucleotide moieties.

54. The method of claim 53, wherein the interaction comprises ligation of the first and second oligonucleotide moieties.

55. The method of claim 54, wherein detecting the interaction between the first and second oligonucleotide moieties comprises incubating with at least one splint oligonucleotide.

56. The method of any one of the preceding claims, wherein the covalent analyte binding moiety and the non-covalent analyte binding moiety are capable of interacting with the same target analyte.

57. The method of claim 56, wherein the target analyte is selected from a peptide, a protein, a hormone, a carbohydrate, a polysaccharide, a small molecule, a moiety on the surface of a cell, and a moiety on the surface of a microorganism.

58. The method of claim 57, wherein the target analyte is a protein.

59. The method of claim 58, wherein the target analyte is an enzyme.

60. The method of claim 59, wherein the enzyme is selected from a

metalloprotease, a cysteine protease, a ubiquitin-specific protease, a cysteine cathepsin, an esterase, a kinase, a histone deacetylase, a serine reductase, an oxidoreductase, an ATPase, and a GTPase.

61. The method of any one of claims 1 to 55, wherein the covalent analyte binding moiety is capable of interacting with a first target analyte and the non-covalent analyte binding moiety is capable of interacting with a second target analyte.

62. The method of claim 61, wherein the first target analyte is capable of interacting with the second target analyte.

63. The method of claim 61, wherein the first target analyte and the second target analyte are independently selected from a peptide, a protein, a hormone, a carbohydrate, a polysaccharide, a small molecule, a moiety on the surface of a cell, and a moiety on the surface of a microorganism.

64. The method of claim 63, wherein the first target analyte is a protein.

65. The method of claim 64, wherein the first target analyte is an enzyme.

66. The method of claim 65, wherein the enzyme is selected from a

67. The method of any one of the preceding claims, wherein the detecting comprises a real-time PCR reaction.

68. The method of any one of the preceding claims, wherein the detecting comprises determining the level of a target analyte.

69. The method of claim 68, wherein determining the level of a target analyte comprises comparing the level to a standard.

70. A complex comprising at least one target analyte and a first proximity detection probe, wherein the first proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety.

71. The complex of claim 70, further comprising a second proximity detection probe, the second proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety.

72. The complex of claim 70, further comprising a non-covalent analyte binding moiety, wherein the non-covalent analyte binding moiety comprises a first member of a binding pair.

73. The complex of claim 72, further comprising a second oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the binding pair.

74. A complex comprising at least one target analyte, a covalent analyte binding moiety, and a proximity detection probe, wherein the covalent analyte binding moiety comprises a first member of a binding pair, and wherein the proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety.

75. The complex of claim 74, further comprising a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the binding pair.

76. A complex comprising at least one target analyte, a covalent analyte binding moiety, and a non-covalent analyte binding moiety, wherein the covalent analyte binding moiety comprises a first member of a first binding pair, and the a non-covalent analyte binding moiety comprises a first member of a second binding pair.

77. The complex of claim 76, further comprising a first oligonucleotide moiety, wherein the first oligonucleotide moiety comprises a second member of the first binding pair.

78. The complex of claim 76 or claim 77, further comprising a second

oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the second binding pair.

79. The complex of any one of claims 70 to 78, wherein the complex is associated with a solid phase.

80. The complex of any one of claims 73, 75, 77, and 78, wherein the first member of at least one binding pair comprises biotin or a derivative thereof and the second member of at least one binding pair comprises streptavidin or a derivative thereof.

81. The complex of any one of claims 73, 75, 77, and 78, wherein the first member of at least one binding pair comprises streptavidin or a derivative thereof and the second member of at least one binding pair comprises biotin or a derivative thereof.

82. The complex of any one of claims 73, 75, 77, and 78, wherein the first and second members of at least one binding pair are capable of undergoing a click reaction.

83. The complex of any one of claims 71 to 82, wherein the covalent analyte binding moiety and the non-covalent analyte binding moiety are capable of interacting with the same target analyte.

84. The complex of any one of claims 71 to 82, wherein the covalent analyte binding moiety is capable of interacting with a first target analyte and the non-covalent analyte binding moiety is capable of interacting with a second target analyte.

85. A kit comprising a first proximity detection probe and a second proximity detection probe, wherein the first proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety, and the second proximity detection probe comprises a non-covalent analyte binding moiety and a second oligonucleotide moiety.

86. A kit comprising a proximity detection probe and a covalent analyte binding moiety, wherein the proximity detection probe comprises a non-covalent analyte binding moiety and a first oligonucleotide moiety and the covalent analyte binding moiety comprises a first member of a binding pair.

87. The kit of claim 86, wherein the kit further comprises a second

oligonucleotide moiety, wherein the second oligonucleotide moiety comprises a second member of the binding pair.

88. The kit of claim 87, wherein the first member of the binding pair and the second member of the binding pair are capable of undergoing a click reaction.

89. A kit comprising a proximity detection probe and a non-covalent analyte binding moiety, wherein the proximity detection probe comprises a covalent analyte binding moiety and a first oligonucleotide moiety and the non-covalent analyte binding moiety comprises a first member of a binding pair.

90. The kit of claim 89, wherein the kit further comprises a second

91. A kit comprising a covalent analyte binding moiety and a non-covalent analyte binding moiety, wherein the a non-covalent analyte binding moiety comprises a first member of a first binding pair and the non-covalent analyte binding moiety comprises a first member of a second binding pair.

92. The kit of claim 91, wherein the kit further comprises a first oligonucleotide moiety comprising a second member of the first binding pair, and a second oligonucleotide moiety comprising a second member of the second binding pair.

93. The kit of any one of claims 85 to 92, wherein the kit comprises a splint oligonucleotide.

94. The kit of any one of claims 85 to 93, wherein the kit comprises a ligase.

95. The kit of any one of claims 85 to 94, wherein the covalent analyte binding moiety and the non-covalent analyte binding moiety are capable of interacting with the same target analyte.

96. The kit of claim 95, wherein the target analyte is selected from a peptide, a protein, a hormone, a carbohydrate, a polysaccharide, a small molecule, a moiety on the surface of a cell, and a moiety on the surface of a microorganism.

97. The method of claim 96, wherein the target analyte is a protein.

98. The kit of claim 97, wherein the target analyte is an enzyme.

99. The kit of claim 98, wherein the enzyme is selected from a metalloprotease, a cysteine protease, a ubiquitin-specific protease, a cysteine cathepsin, an esterase, a kinase, a histone deacetylase, a serine reductase, an oxidoreductase, an ATPase, and a GTPase.

100. The kit of any one of claims 85 to 94, wherein the covalent analyte binding moiety is capable of interacting with a first target analyte and the non-covalent analyte binding moiety is capable of interacting with a second target analyte.

101. The kit of claim 100, wherein the first target analyte is capable of interacting with the second target analyte.

102. The kit of claim 101, wherein the first target analyte and the second target analyte are independently selected from a peptide, a protein, a hormone, a carbohydrate, a polysaccharide, a small molecule, a moiety on the surface of a cell, and a moiety on the surface of a microorganism.

103. The kit of claim 102, wherein the first target analyte is a protein.

104. The kit of claim 103, wherein the first target analyte is an enzyme.

105. The kit of claim 104, wherein the enzyme is selected from a metalloprotease, a cysteine protease, a ubiquitin-specific protease, a cysteine cathepsin, an esterase, a kinase, a histone deacetylase, a serine reductase, an oxidoreductase, an ATPase, and a GTPase.