WO2021034971A1 - Methods and compositions for analyte detection and quantification - Google Patents

Abstract

The invention relates generally to methods for the detection and/or quantification of an analyte in sample using (i) a first probe comprising a binding agent that binds the analyte conjugated to an oligonucleotide, (ii) a second probe comprising a binding agent that binds the analyte conjugated to the oligonucleotide, and (iii) a lock probe that simultaneously hybridizes to the oligonucleotide of the first and second probes if the first and second probe are in sufficient proximity to each other (e.g., are both bound to the analyte). The bound probes can be used as a template for the generation of amplification products which, if present, are indicative of the presence and/or amount of the analyte in the sample.

Description

METHODS AND COMPOSITIONS FOR ANALYTE DETECTION AND

QUANTIFICATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/888,597, filed August 19, 2019, which is incorporated herein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The invention relates generally to methods for the detection and/or quantification of an analyte in sample.

BACKGROUND

[0003] Over the years, the detection and quantification of analytes has been critical in the diagnosis and treatment of numerous diseases or disorders, as well as the development of new therapies and treatment modalities. Significant progress has been made in the development of analyte detection and quantification systems, including solid or solution based assays, such as blotting-based technologies such as Western blots, enzyme linked immunoassays (ELISAs), digital ELISAs, micro-fluidic-based ELISA technologies, and automated bead-based assays.

[0004] In one example of a analyte detection system, a pair of oligonucleotide-labeled antibodies (“probes”) are allowed to pair-wise bind to a target analyte present in a sample. When the two probes are in close proximity, a new PCR target sequence is formed by a proximity-dependent DNA polymerization event. The resulting sequence is subsequently detected and quantified using real-time PCR. In another example of an analyte detection system, antibody-conjugated bead sets are used to detect analytes in a multiplexed sandwich immunoassay format. Each bead in the set is identified by a unique set of two addressing dyes, with a third dye used to read out binding of the analyte via a biotin-conjugated antibody and streptavidin-conjugated second step detector. Data is acquired on a dedicated flow cytometry-based platform. See, e.g., Mckinnon (2018) CURR. PROTOC. IMMUNOL. 120: 5.1.1-5.1.11.

[0005] However, the above technologies have limitations. In particular, these assays can suffer from high background, do not provide a high level of multiplexing coupled with a high signal to noise ratio, and/or require expensive instrumentation to implement. Accordingly there is an ongoing need for methods and systems that facilitates the detection and quantification of one or more analytes with the requisite sensitivity.

SUMMARY OF THE INVENTION

[0006] The present disclosure relates, in general, to methods and compositions for the detection and quantification of one or more analytes in a sample. The disclosed methods allow for efficient detection and/or quantification of multiple analytes while maintaining a high signal to noise ratio.

[0007] In one aspect, the invention provides a method for detecting the presence or amount of an analyte in a sample. The method comprises one or more of the following steps: (i) contacting the sample with a first probe comprising a first binding agent capable of binding the analyte conjugated to first oligonucleotide and a second probe comprising a second binding agent capable of binding the analyte conjugated to a second oligonucleotide, under conditions to permit the first probe and the second probe to bind the analyte if the analyte is present in the sample, wherein the first binding agent and the second binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated to each respective binding agent via the UAA, and the first and second oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region; (ii) contacting the sample with a lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe, under conditions to permit the lock probe to simultaneously hybridize to the first and second probe to form a duplex if the first and second probe are in sufficient proximity to each other; (iii) ligating the first probe and the second probe together with a DNA ligase to form a ligation product; (iv) optionally cleaving the ligation product with a cleavage agent capable of cleaving the cleavage site of the first and second probe thereby removing the first and second binding agents to form a cleaved ligation product; (v) using the ligation product or the cleaved ligation product as a template for an amplification reaction; and (vi) detecting a product form the amplification reaction, which if present is indicative of the presence and/or amount of the analyte in the sample. [0008] In certain embodiments, the first binding agent and the second binding agent bind the same epitope on the analyte, e.g. , the first binding agent and the second binding agent are substantially identical. In other embodiments, the first binding agent and the second binding agent bind different epitopes on the analyte.

[0009] In certain embodiments, the UAA comprises an azide or alkyne functional group or hydroxytryptophan. In certain embodiments, the oligonucleotide is conjugated to the UAA by a linker (e.g., a DBCO linker).

[0010] In certain embodiments, the first and second binding agents are antibodies, e.g, anti- HER2 antibodies. For example, the first and second binding agent may comprise a trastuzumab antibody comprising the UAA.

[0011] In certain embodiments, the spacer sequence of the first probe and the spacer sequence of the second probe are substantially the same length. In other embodiments, the spacer sequence of the first probe and the spacer sequence of the second probe have different lengths. For example, the spacer sequence of the first probe may be from about 50 to about 500, about 50 to about 400, about 50 to about 300, about 50 to about 300, about 50 to about 200, about 50 to about 100, about 100 to about 500, about 100 to about 400, about 100 to about 300, about 100 to about 300, about 100 to about 200, about 200 to about 500, about 200 to about 400, about 200 to about 300, about 200 to about 300, about 300 to about 500, about 300 to about 400, or about 400 to about 500 nucleotides in length and/or the spacer sequence of the second probe may be from about 50 to about 500, about 50 to about 400, about 50 to about 300, about 50 to about 300, about 50 to about 200, about 50 to about 100, about 100 to about 500, about 100 to about 400, about 100 to about 300, about 100 to about 300, about 100 to about 200, about 200 to about 500, about 200 to about 400, about 200 to about 300, about 200 to about 300, about 300 to about 500, about 300 to about 400, or about 400 to about 500 nucleotides in length. It is contemplated that the spacer sequence of the first probe and the spacer sequence of the second probe may be any combination of the foregoing nucleotide lengths.

[0012] In certain embodiments, the complementary region of the first probe and the complementary region of the second probe are substantially identical. In other embodiments, the complementary region of the first probe and the complementary region of the second probe are different. [0013] In certain embodiments, the lock probe comprises a 3’ dideoxy nucleotide.

[0014] In certain embodiments, the DNA ligase is T4 DNA ligase. In certain embodiments, the cleavage site is a deoxyuracil and/or the cleavage agent is a uracil-DNA glycosylase.

[0015] In certain embodiments, the amplification reaction is a polymerase chain reaction (PCR) or a rolling chain amplification (RCA).

[0016] In certain embodiments, where the amplification reaction is a PCR reaction, the first probe comprises a first PCR primer binding site and/or the second probe comprises a second PCR primer binding site. The first PCR primer binding site may, for example, be 5’ to the spacer sequence of the first probe, within the spacer sequence of the first probe, or partially within the spacer sequence of the first probe. The second PCR primer binding site may, for example, be 3’ to the spacer sequence of the second probe, within the spacer sequence of the second probe, or partially within the spacer sequence of the second probe. In certain embodiments, the PCR reaction comprises incubating the ligation product or the cleaved ligation product with a first primer capable of binding the first PCR primer binding and a second primer capable of binding the second PCR primer binding site.

[0017] In certain embodiments, the first probe comprises the cleavage site, the spacer sequence, the unique identifying sequence, and the complementary region in a 5’ to 3’ orientation. In certain embodiments, the second probe comprises the cleavage site, the spacer sequence, the unique identifying sequence, and the complementary region in a 3’ to 5’ orientation.

[0018] In another aspect, the invention provides a method for detecting the presence or amount of multiple ( e.g 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100) analytes in a sample. The method comprises, for the detection of each analyte, one or more of the following steps: (i) contacting the sample with a first probe comprising a first binding agent capable of binding the analyte conjugated to a first oligonucleotide and a second probe comprising a second binding agent capable of binding the analyte conjugated to a second oligonucleotide, under conditions to permit the first probe and the second probe to bind the analyte if the analyte is present in the sample, wherein the first binding agent and the second binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated (e.g., covalently linked) to each respective binding agent via the UAA, and the first and second oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region; (ii) contacting the sample with a lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe, under conditions to permit the lock probe to simultaneously hybridize to the first and second probe to form a duplex if the first and second probe are in sufficient proximity to each other; (iii) ligating the first probe and the second probe together (e.g., with a DNA ligase) to form a ligation product; (iv) optionally cleaving the ligation product with a cleavage agent capable of cleaving the cleavage site of the first and second probe thereby removing the first and second binding agents to form a cleaved ligation product; (v) using the ligation product or the cleaved ligation product as a template for an amplification reaction; and (vi) detecting a product form the amplification reaction, which if present is indicative of the presence and/or amount of the analyte in the sample.

[0019] In another aspect, the invention provides a method for detecting the presence or amount of a first analyte and a second analyte in a sample. The method comprises one or more of the following steps: (i) contacting the sample with a first probe comprising a first binding agent capable of binding the first analyte conjugated to a first oligonucleotide, a second probe comprising a second binding agent capable of binding the first analyte conjugated to a second oligonucleotide, a third probe comprising a third binding agent capable of binding the second analyte conjugated to a third oligonucleotide, and a fourth probe comprising a fourth binding agent capable of binding the second analyte conjugated to a fourth oligonucleotide, under conditions to permit the first probe and the second probe to bind the first analyte if the first analyte is present in the sample and to permit the third probe and the fourth probe to bind the second analyte if the second analyte is present in the sample, wherein the first, second, third, and fourth binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated (e.g., covalently coupled) to each respective binding agent via each respective UAA, and the first, second, third, and fourth oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region; (ii) contacting the sample with a first lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe, and a second lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the third probe and (b) a second region that is capable of hybridizing to the complementary region of the fourth probe, under conditions to permit the first lock probe to simultaneously hybridize to the first and second probe to form a duplex if the first and second probe are in sufficient proximity to each other, and to permit the second lock probe to simultaneously hybridize to the third and fourth probe to form a duplex if the third and fourth probe are in sufficient proximity to each other; (iii) ligating the first probe and the second probe together (e.g., with a DNA ligase) to form a first ligation product and the third and fourth probe together (e.g., with a DNA ligase) to form a second ligation product; (iv) optionally cleaving the first and second ligation product with at least one cleavage agent capable of cleaving the cleavage site of the first, second, third, and fourth probes thereby removing the first, second, third, and fourth binding agents to form a first and second cleaved ligation product; (v) using the first and second ligation products or cleaved ligation products as a template for an amplification reaction to produce corresponding amplification products; and (vi) detecting the presence and/or amount of amplification products, which if present are indicative of the presence and/or amount of the first and second analyte in the sample.

[0020] These and other aspects and features of the invention are described in the following detailed description and claims.

DESCRIPTION OF THE DRAWINGS

[0021] The invention can be more completely understood with reference to the following drawings.

[0022] FIGURE 1 depicts a schematic overview of an exemplary detection method. The method employs a first probe (also referred herein to as a left probe) and a second probe (also referred to herein as a right probe) including an oligonucleotide with a deoxyuracil cleavage site (U), a spacer sequence (also referred to herein as a primary sequence; first/left primary (LP) or second/right primary (RP)), a unique identifying sequence (I) and a complementary region (first/left complementary (LC) or second/right complementary (RC)). The first/left and second/right oligonucleotides are conjugated to a binding agent, e.g., an antibody (Ab), e.g, via an unnatural amino acid in the antibody that allows for site specific conjugation, as shown in FIGURE 1A. Exemplary methods of UAA incorporation and oligonucleotide conjugation are described in Italia et al. (2017) NAT. CHEM. BIOL. 13(4):446-450, Liu et al. (2010) ANNU. REV BIOCHEM. 79:413-44, Dumas et al. (2015) CHEM. SCI. 6(l):50-69, and Zheng et al. (2018) BIOCHEMISTRY 57(4):441-445. The method also employs a lock probe comprising a first region that is capable of hybridizing to the complementary region of the first/left probe (LC) and a second region that is capable of hybridizing to the complementary region of the second/right probe (RC). Following a general ELISA workflow, a target analyte ( e.g ., a protein) can be contacted with the first/right and second/left probes. The target analyte can be captured on a plate, surface, bead, etc. or be free in solution. When present, the target analyte is capable of being bound by the first/right and second/left probes, as shown in FIGURE IB. It is understood that the method will typically include washes (e.g., between each step depicted in FIGURE 1) to remove excess or unbound components. When the first/right and second/left probes are in close enough proximity (e.g, when both are bound to the same analyte or to two different analytes in sufficient proximity) the lock probe is capable of simultaneously hybridizing to the first and second probe to form a duplex, in effect circularizing the structure as shown in FIGURE 1C. Binding of the first/left and second/right probes to the lock probe allows the first/left and second/right probes to together form a single template for an amplification reaction (e.g, a PCR reaction). Optionally, the 3’ end of the first/left oligonucleotide and the 5’ end of the second/right oligonucleotide are ligated together (e.g, using a DNA ligase) when in sufficient proximity (e.g, when both bound to the lock probe). Optionally, all non-reacted, linear probes can be digested enzymatically or chemically or washed away as shown in FIGURE ID. The oligonucleotide may need to be liberated from the binding agent-oligonucleotide conjugate to allow for subsequent amplification. Accordingly, optionally, the binding agent is released by cleavage of the deoxyuracil cleavage site using uracil-DNA glycosylase, as shown in FIGURE IE.

The resulting oligonucleotide is used as a template for an amplification reaction (e.g, a PCR reaction, e.g, a qPCR reaction), as shown in FIGURE IF, the product of which is indicative of the presence and/or amount of the analyte. The amplification product may be sequenced. In a multiplexed assay, the unique identifying sequence may be used to distinguish between multiple analytes. In a multiplexed assay, the first/left and second/right probes for different target analytes may contain universal forward and reverse PCR primer binding sites, allowing for simultaneous amplification of multiple templates using the same set of PCR primers. [0023] FIGURE 2 depicts a schematic overview of an exemplary detection method. A target analyte (e.g., HER2 as shown in FIGURE 2A) can be free in solution or immobilized on a solid support such as a bioassay plate ( e.g ., a 96-well plate). This immobilization can be by non-specific binding, i.e., through adsorption to the surface. Alternatively, immobilization can be by specific binding, i.e., through binding by a capture antibody (e.g., via an antibody that binds the analyte that is different from the binding agent in the first/left and/or second/right probes). Once bound to the solid support, the support is blocked with a blocking agent (e.g., milk). It is understood that the method will typically include washes (e.g, between each step depicted in FIGURE 2) to remove excess or unbound components. Next, as shown in FIGURE 2B, the first/right and second/left probes, including a binding agent (e.g, an antibody) conjugated to an oligonucleotide (e.g, including a cleavage site, a spacer sequence, a unique identifying sequence, and a complementary region, as show in FIGURE 1) are added. Next, as shown in FIGURE 2C, a ligase (e.g, T4 DNA ligase; T4) and lock probe (e.g, including a first region that is capable of hybridizing to the complementary region of the first/left probe and a second region that is capable of hybridizing to the complementary region of the second/right probe, as shown in FIGURE 1) are added. Optionally, as shown in FIGURE 2D, a cleavage agent (e.g, uracil DNA glycosylase; UDG) is used to cleave the oligonucleotide from the binding agent and release the oligonucleotide, as shown in FIGURE 2E. It is also contemplated that heat or proteolytic digestion may be used to denature or degrade the binding agent and release the oligonucleotide. Supernatant containing free-ligated oligo construct is isolated and used as a template for an amplification reaction (e.g, qPCR using taq polymerase; Taq), following standard manufacturer’s protocols, as shown in FIGURE 2F. Detection of PCR products can be via a fluorescent plate reader or DNA agarose gel, as shown in FIGURE 2G. As would be understood by a person of skill in the art, the reaction can be multiplexed with orthogonal probes targeting different analytes.

[0024] FIGURE 3 depicts a schematic representation of an exemplary set of probes (a first/left probe, a second/right probe, and a lock probe). The first/left oligonucleotide is designed to have a 5’ conjugation handle compatible with conjugation to the binding agent (e.g, targeting mAh). The first/left oligonucleotide includes a linker region of variable length (e.g, about 50bp) followed by a deoxyuridine region containing a deoxyuracil for cleavage. 3’ to the deoxyuridine region is a spacer region of variable length (e.g, about 50bp). The spacer region can be used to adjust the size of a target amplification product to eliminate any background. 3’ to the spacer region is a unique identifier sequence (Pair Unique Seq) for qPCR analysis and a complementary region to the 3’ end of the lock probe. The second/right probe contains the same features as the first/left probe but in the opposite 5’ to 3’ orientation. Arrows indicate PCR primers. The lock probe can contain one 3’ dideoxy nucleotide in order to prevent the lock probe from functioning as an unintentional primer (amplifying the first/left oligo) or typical mismatch pairing of the last two 3’ nucleotides of the lock probe can eliminate any background amplification (FIGURE 3). [0025] FIGURE 4 depicts an SDS page gel showing PCR amplification products following incubation of the indicated components as described in Example 1.

[0026] FIGURE 5A depicts a schematic representative of an antibody oligonucleotide conjugation scheme. FIGURES 5B and 5C depict intermediates in the conjugation scheme and FIGURES 5D and 5E depict final conjugates as analyzed by HPLC-HIC. FIGURE 5F depicts intermediates or final conjugates as analyzed by SDS-PAGE, where lanes 1-3 correspond to the intermediates or conjugates shown in FIGURES 5C-E respectively.

[0027] FIGURE 6 depicts an SDS page gel showing PCR amplification products following incubation of the indicated components as described in Example 3.

[0028] FIGURE 7 depicts a schematic overview of genetic code expansion using unnatural amino acids (UAAs).

[0029] FIGURES 8A-8C depicts a subset of leucyl analog UAAs.

[0030] FIGURE 9 depicts a subset of tryptophanyl analog UAAs.

PET ATT /ED DESCRIPTION

[0031] The present disclosure relates, in general, to methods and compositions for the detection and quantification of one or more analytes in a sample, for example, a liquid sample. The disclosed methods allow for efficient detection and/or quantification of multiple analytes while maintaining a high signal to noise ratio.

[0032] In one aspect, the invention provides a method for detecting the presence or amount of an analyte in a sample. The method comprises one or more of the following steps: (i) contacting the sample with a first probe comprising a first binding agent capable of binding the analyte conjugated to first oligonucleotide and a second probe comprising a second binding agent capable of binding the analyte conjugated to a second oligonucleotide, under conditions to permit the first probe and the second probe to bind the analyte if the analyte is present in the sample, wherein the first binding agent and the second binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated (for example, covalently coupled) to each respective binding agent via the UAA, and the first and second oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region; (ii) contacting the sample with a lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe, under conditions to permit the lock probe to simultaneously hybridize to the first and second probe to form a duplex if the first and second probe are in sufficient proximity to each other; (iii) ligating the first probe and the second probe together (e.g., with a DNA ligase) to form a ligation product; (iv) optionally cleaving the ligation product with a cleavage agent capable of cleaving the cleavage site of the first and second probe thereby removing the first and second binding agents to form a cleaved ligation product; (v) using the ligation product or the cleaved ligation product as a template for an amplification reaction; and (vi) detecting a product form the amplification reaction, which if present is indicative of the presence and/or amount of the analyte in the sample.

[0033] It is contemplated that the methods described herein can provide for multiplexing, i.e., the simultaneous detection of multiple analytes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 analytes) in a sample.

[0034] For example, in another aspect, the invention provides a method for detecting the presence or amount of multiple analytes in a sample. The method comprises, for the detection of each analyte, one or more of the following steps: (i) contacting the sample with a first probe comprising a first binding agent capable of binding the analyte conjugated (e.g., covalently coupled) to a first oligonucleotide and a second probe comprising a second binding agent capable of binding the analyte conjugated (e.g., covalently coupled) to a second oligonucleotide, under conditions to permit the first probe and the second probe to bind the analyte if the analyte is present in the sample, wherein the first binding agent and the second binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated to each respective binding agent via the UAA, and the first and second oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region; (ii) contacting the sample with a lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe, under conditions to permit the lock probe to simultaneously hybridize to the first and second probe to form a duplex if the first and second probe are in sufficient proximity to each other; (iii) ligating the first probe and the second probe together (e.g., with a DNA ligase) to form a ligation product; (iv) optionally cleaving the ligation product with a cleavage agent capable of cleaving the cleavage site of the first and second probe thereby removing the first and second binding agents to form a cleaved ligation product; (v) using the ligation product or the cleaved ligation product as a template for an amplification reaction; and (vi) detecting a product form the amplification reaction, which, if present, is indicative of the presence and/or amount of the analyte in the sample.

[0035] In another aspect, the invention provides a method for detecting the presence or amount of a first analyte and a second analyte in a sample. The method comprises one or more of the following steps: (i) contacting the sample with a first probe comprising a first binding agent capable of binding the first analyte conjugated (e.g., covalently coupled) to a first oligonucleotide, a second probe comprising a second binding agent capable of binding the first analyte conjugated (e.g., covalently coupled) to a second oligonucleotide, a third probe comprising a third binding agent capable of binding the second analyte conjugated (e.g., covalently coupled) to a third oligonucleotide, and a fourth probe comprising a fourth binding agent capable of binding the second analyte conjugated (e.g., covalently coupled) to a fourth oligonucleotide, under conditions to permit the first probe and the second probe to bind the first analyte if the first analyte is present in the sample and to permit the third probe and the fourth probe to bind the second analyte if the second analyte is present in the sample, wherein the first, second, third, and fourth binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated (e.g., covalently coupled) to each respective binding agent via each respective UAA, and the first, second, third, and fourth oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region; (ii) contacting the sample with a first lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe, and a second lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the third probe and (b) a second region that is capable of hybridizing to the complementary region of the fourth probe, under conditions to permit the first lock probe to simultaneously hybridize to the first and second probe to form a duplex if the first and second probe are in sufficient proximity to each other, and to permit the second lock probe to simultaneously hybridize to the third and fourth probe to form a duplex if the third and fourth probe are in sufficient proximity to each other; (iii) ligating the first probe and the second probe together (e.g., with a DNA ligase) to form a first ligation product and the third and fourth probe together (e.g., with a DNA ligase) to form a second ligation product; (iv) optionally cleaving the first and second ligation product with at least one cleavage agent capable of cleaving the cleavage site of the first, second, third, and fourth probes thereby removing the first, second, third, and fourth binding agents to form a first and second cleaved ligation product; (v) using the first and second ligation products or cleaved ligation products as a template for amplification to produce corresponding amplification products; and (vi) detecting the presence and/or amount of amplification products, which if present is indicative of the presence and/or amount of the first and second analyte in the sample.

[0036] Various features and aspects of the invention are discussed in more detail below.

I. Analytes

[0037] The systems and methods described herein may be used to detect the presence, or to quantify the amount, of an analyte in a sample of interest, for example, a liquid or tissue sample.

[0038] Analytes may be detected and/or quantified in a variety of samples. In certain embodiments, the sample is derived from a subject. As used herein, the terms “subject” and “patient” refer to an organism that is the source of a sample that is interrogated by the methods described herein Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0039] The sample can be in any form that allows for measurement of the analyte. In other words, the sample must be sufficient for analyte extraction or processing to permit detection of the analyte, such as preparation of thin sections. Accordingly, the sample can be fresh, preserved through suitable cryogenic techniques, or preserved through non-cry ogenic techniques.

[0040] In certain embodiments, the sample is a body fluid sample, such as a blood, serum, plasma, urine, saliva, cerebrospinal fluid, or interstitial fluid sample.

[0041] In certain embodiments, the sample is a tissue sample, such as a biopsy sample. A biopsy sample can be obtained by using conventional biopsy instruments and procedures. Endoscopic biopsy, excisional biopsy, incisional biopsy, fine needle biopsy, punch biopsy, shave biopsy and skin biopsy are examples of recognized medical procedures that can be used by one of skill in the art to obtain tissue samples. A standard process for handling clinical biopsy tissue specimens is to fix the tissue sample in formalin and then embed the sample in paraffin. Samples in this form are commonly known as formalin-fixed, paraffin- embedded (FFPE) tissue. Suitable techniques of tissue preparation for subsequent analysis are well-known to those of skill in the art.

[0042] In certain embodiments, the sample is a cell sample, or a cell supernatant sample.

[0043] Exemplary analytes include cells, organic compounds, antibodies, antigens, virus particles, pathogenic bacteria, metals, metal complexes, ions, spores, yeasts, molds, cellular metabolites, enzyme inhibitors, receptor ligands, nerve agents, peptides, proteins, fatty acids, steroids, hormones, narcotic agents, synthetic molecules, medications, enzymes, nucleic acid single-stranded or double-stranded polymers. Analytes include biological molecules, for example, a protein, peptide, carbohydrate, glycoprotein, glycopeptide, lipid, lipoprotein, nucleic acid, or nucleoprotein.

[0044] In certain embodiments the analyte is a cytokine. Examples of cytokines include, but are not limited to, interferons (e.g., IFNa, PTNίb, and IFNy), interleukins (e.g., IL-1, IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I2, IL-I7 and IL-20), tumor necrosis factors (e.g., TNFa and TNFp), erythropoietin (EPO), FLT-3 ligand, glpIO, TCA-3, MCP-I, MIF, MIR-Ia, MIR-Ib, Rantes, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF), as well as functional fragments of any of the foregoing.

[0045] In certain embodiments the analyte is a hormone. Examples of hormones include, but are not limited to, epinephrine, melatonin, norepinephrine, triiodothyronine, thyroxine, dopamine, prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin (or islet amyloid polypeptide), anti-miillerian hormone (or miillerian inhibiting factor or hormone), adiponectin, adrenocorticotropic hormone (or corticotropin), angiotensinogen and angiotensin, antidiuretic hormone (or vasopressin, arginine vasopressin), atrial-natriuretic peptide (or atriopeptin), brain natriureticc peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, cortistatin, enkephalin, endothelin, erythropoietin, follicle-stimulating hormone, galanin, gastric inhibitory polypeptide, gastrin, ghrelin, glucagon, glucagon-like peptide- 1, gonadotropin-releasing hormone, growth hormone-releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor (or somatomedin), leptin, lipotropin, luteinizing hormone, melanocyte stimulating hormone, motilin, orexin, osteocalcin, oxytocin, pancreatic polypeptide, parathyroid hormone, pituitary adenylate cyclase-activating peptide, prolactin, prolactin releasing hormone, relaxin, renin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (or thyrotropin), thyrotropin releasing hormone, vasoactive intestinal peptide, guanylin, uroguanylin, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, aldosterone, estradiol, estrone, estriol, cortisol, progesterone, calcitriol (1,25-dihydroxyvitamin D3), and calcidiol (25-hydroxyvitamin D3).

[0046] In certain embodiments the analyte is a cancer antigen. Examples of cancer antigens include, but are not limited to, adenosine A2a receptor (A2aR), A kinase anchor protein 4 (AKAP4), B melanoma antigen (B AGE), brother of the regulator of imprinted sites (BORIS), breakpoint cluster region Abelson tyrosine kinase (BCR/ABL), CA125, CAIX,

CD 19, CD20, CD22, CD30, CD33, CD52, CD73, CD 137, carcinoembryonic antigen (CEA), CS1, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), estrogen receptor binding site associated antigen 9 (EBAG9), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), EGF -like module receptor 2 (EMR2), epithelial cell adhesion molecule (EpCAM) (17-1A), FR-alpha, G antigen (GAGE), disialoganglioside GD2 (GD2), glycoprotein 100 (gplOO), human epidermal growth factor receptor 2 (Her2), hepatocyte growth factor (HGF), human papillomavirus 16 (HPV-16), heat-shock protein 105 (HSP105), isocitrate dehydrogenase type 1 (IDH1), idiotype (NeuGcGM3), indoleamine-2,3- dioxygenase 1 (IDOl), IGF-1, IGF1R, IGG1K, killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG-3), lymphocyte antigen 6 complex K (LY6K), Matrix-metalloproteinase- 16 (MMP16), melanotransferrin (MFI2), melanoma antigen 3 (MAGE- A3), melanoma antigen C2 (MAGE-C2), melanoma antigen D4 (MAGE-D4), melanoma antigen recognized by T-cells 1 (Mel an- A/MART- 1), N-methyl-N’-nitroso- guanidine human osteosarcoma transforming gene (MET), mucin 1 (MUC1), mucin 4 (MUC4), mucin 16 (MUC16), New York esophageal squamous cell carcinoma 1 (NY-ESO- 1), prostatic acid phosphatase (PAP), programmed cell death receptor 1 (PD-1), programmed cell death receptor ligand 1 (PD-L1), phosphatidylserine, preferentially expressed antigen of melanoma (PRAME), prostate specific antigen (PSA), protein tyrosine kinase 7 (PTK7, also known as colon carcinoma kinase 4 (CCK4)), receptor tyrosine kinase orphan receptor 1 (ROR1), scatter factor receptor kinase, sialyl-Tn, sperm-associated antigen 9 (SPAG-9), synovial sarcoma X-chromosome breakpoint 1 (SSX1), survivin, telomerase, T-cell immunoglobulin domain and mucin domain-3 (TIM-3), vascular endothelial growth factor (VEGF) (e.g, VEGF-A), vascular endothelial growth factor Receptor 2 (VEGFR2), V- domain immunoglobulin-containing suppressor of T-cell activation (VISTA), Wilms’ Tumor- 1 (WT1), X chromosome antigen lb (XAGE-lb), 5T4, Mesothelin, Glypican 3 (GPC3), Prostate Specific Membrane Antigen (PSMA), cMET, CD38, B Cell Maturation Antigen (BCMA), CD123, CLDN6, CLDN9, LRRC15, PRLR (Prolactin Receptor), RING finger protein 43 (RNF43), Uroplakin-1 B (UPK1 B), tumor necrosis factor superfamily member 9 (TNFSF9), tumor necrosis factor receptor superfamily member 21 (TNFSRF21), bone morphogenetic protein receptor type- IB (BMPR1B), Kringle domain-containing transmembrane protein 2 (KREMEN2), Delta-like protein 3 (DLL3), Siglec7 and Siglec9. Additional exemplary cancer antigens include those found on cancer stem cells, e.g, SSEA3, SSEA4, TRA-1-60, TRA-1-81, SSEA1, CD133 (AC133), CD90 (Thy-1), CD326 (EpCAM), Cripto-1 (TDGF1), PODXL-1 (Podocalyxin-like protein 1), ABCG2, CD24, CD49f (Integrin a6), Notch2, CD146 (MCAM), CD10 (Neprilysin), CD117 (c-KIT), CD26 (DPP-4), CXCR4, CD34, CD271, CD 13 (Alanine aminopeptidase), CD56 (NCAM), CD 105 (Endoglin), LGR5, CD114 (CSF3R), CD54 (ICAM-1), CXCR1, 2, TIM-3 (HAVCR2), CD55 (DAF), DLL4 (Delta-like ligand 4), CD20 (MS4A1), and CD96.

II. Probes

A. Binding Agents

[0047] Probes useful in the practice of invention include a binding agent. The term “binding agent” as used herein refers to an agent that binds preferentially or specifically to an analyte of interest. The terms “bind preferentially,” or “binds specifically” as used in connection with a binding agent refers to an agent that binds and/or associates (i) more stably, (ii) more rapidly, (iii) with stronger affinity, (iv) with greater duration, or (v) or a combination of any two or more of (i)-(iv), with a particular target analyte it does with a molecule other than the target analyte. For example, a binding agent that specifically or preferentially binds a target analyte is a binding domain that binds a target analyte, e.g., with stronger affinity, avidity, more readily, and/or with greater duration than it binds a different analyte. The binding agent have affinity for the analyte of about 100 nM, 50 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, or 0.01 nM, or stronger, as determined by surface plasmon resonance. For example, the binding agent may have an affinity for the analyte within the range from about 0.01 nM to about 100 nM, from about 0.1 nM to about 100 nM, or from about 1 nM to about 100 nM. It is understood that a binding agent that binds preferentially to a first target analyte may or may not preferentially bind to a second target analyte. As such, “preferential binding” does not necessarily require (although it can include) exclusive binding.

[0048] Exemplary binding agents include enzymes (for example, that bind substrates and inhibitors), antibodies (for example, that bind antigens), antigens (for example, that bind target antibodies), receptors (for example, that bind ligands), ligands (for example, that bind receptors), nucleic acid single-strand polymers (for example, that bind nucleic acid molecules to form, for example, DNA-DNA, RNA-RNA, or DNA-RNA double strands), and synthetic molecules that bind with target analytes. Natural, synthetic, semi -synthetic, and genetically- altered macromolecules may be employed as binding agents. Binding agents include biological binding agents, for example, an antibody, an aptamer, a receptor, an enzyme, or a nucleic acid.

[0049] As used herein, unless otherwise indicated, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody) or antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody or antigen-binding fragment that has been modified, engineered, or chemically conjugated. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. [0050] In certain embodiments, an antibody binds to its target with a K_D of about 300 pM, 250 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM,

110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM, or lower.

[0051] An antibody may have a human IgGl, IgG2, IgG3, IgG4, or IgE isotype.

[0052] In certain embodiments, the antibody is selected from, or the antibody is derived from antibody selected from, adecatumumab, ascrinvacumab, cixutumumab, conatumumab, daratumumab, drozitumab, duligotumab, durvalumab, dusigitumab, enfortumab, enoticumab, epratuxumab, figitumumab, ganitumab, glembatumumab, intetumumab, ipilimumab, iratumumab, icrucumab, lexatumumab, lucatumumab, mapatumumab, narnatumab, necitumumab, nesvacumab, ofatumumab, olaratumab, panitumumab, patritumab, pritumumab, radretumab, ramucirumab, rilotumumab, robatumumab, seribantumab, tarextumab, teprotumumab, tovetumab, vantictumab, vesencumab, votumumab, zalutumumab, flanvotumab, altumomab, anatumomab, arcitumomab, bectumomab, blinatumomab, detumomab, ibritumomab, minretumomab, mitumomab, moxetumomab, naptumomab, nofetumomab, pemtumomab, pintumomab, racotumomab, satumomab, solitomab, taplitumomab, tenatumomab, tositumomab, tremelimumab, abagovomab, atezolizumab, durvalumab, avelumab, igovomab, oregovomab, capromab, edrecolomab, nacolomab, amatuximab, bavituximab, brentuximab, cetuximab, derlotuximab, dinutuximab, ensituximab, futuximab, girentuximab, indatuximab, isatuximab, margetuximab, rituximab, siltuximab, ublituximab, ecromeximab, abituzumab, alemtuzumab, bevacizumab, bivatuzumab, brontictuzumab, cantuzumab, cantuzumab, citatuzumab, clivatuzumab, dacetuzumab, demcizumab, dalotuzumab, denintuzumab, elotuzumab, emactuzumab, emibetuzumab, enoblituzumab, etaracizumab, farletuzumab, ficlatuzumab, gemtuzumab, imgatuzumab, inotuzumab, labetuzumab, lifastuzumab, lintuzumab, lirilumab, lorvotuzumab, lumretuzumab, matuzumab, milatuzumab, moxetumomab, nimotuzumab, obinutuzumab, ocaratuzumab, otlertuzumab, onartuzumab, oportuzumab, parsatuzumab, pertuzumab, pidilizumab, pinatuzumab, polatuzumab, sibrotuzumab, simtuzumab, tacatuzumab, tigatuzumab, trastuzumab, tucotuzumab, urelumab, vandortuzumab, vanucizumab, veltuzumab, vorsetuzumab, sofituzumab, catumaxomab, ertumaxomab, depatuxizumab, ontuxizumab, blontuvetmab, tamtuvetmab, nivolumab, pembrolizumab, epratuzumab, MEDI9447, urelumab, utomilumab, hu3F8, hul4.18-IL-2, 3F8/OKT3BsAb, lirilumab, BMS- 986016 pidilizumab, AMP-224, AMP-514, BMS-936559, atezolizumab, and avelumab.

[0053] In certain embodiments, the binding agent comprises, or is derived from, trastuzumab. For example, in certain embodiments, the binding agent comprises an antibody comprising (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 4, or an amino acid that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4, and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid that has at least 85%, 90%, 95% 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

5.

[0054] Where the binding agent comprises, or is derived from, a protein ( e.g ., an antibody), and the binding agent comprises a protein sequence comprising an unnatural amino acid (UAA), and an oligonucleotide is conjugated to the binding agent via the UAA, it is understood that the UAA may be incorporated (and the oligonucleotide may therefore be conjugated) to any appropriate location within the protein sequence. For example, where the binding agent comprises, or is derived from, trastuzumab, and trastuzumab comprises a protein sequence comprising an unnatural amino acid (UAA) and an oligonucleotide is conjugated to trastuzumab via the UAA, it is understood that the UAA may be incorporated (and the oligonucleotide may therefore be conjugated) to any appropriate location within the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0055] Sequence identity may be determined in various ways that are within the skill of a person skilled in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin etal., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36:290-300; Altschul etal, (1997) NUCLEIC ACIDS RES. 25:3389-3402, incorporated by reference herein) are tailored for sequence similarity searching. For a discussion of basic issues in searching sequence databases see Altschul el al, (1994) NATURE GENETICS 6: 119-129, which is fully incorporated by reference herein. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al ., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, fully incorporated by reference herein).

Four blastn parameters may be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=l (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent blastp parameter settings may be Q=9; R=2; wink=l; and gapw=32. Searches may also be conducted using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameter ( e.g . : -G, Cost to open gap [Integer]: default = 5 for nucleotides/ 11 for proteins; -E, Cost to extend gap [Integer]: default = 2 for nucleotides/ 1 for proteins; -q, Penalty for nucleotide mismatch [Integer]: default = - 3; -r, reward for nucleotide match [Integer]: default = 1; -e, expect value [Real]: default = 10; -W, wordsize [Integer]: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins; -y, Dropoff (X) for blast extensions in bits: default = 20 for blastn/ 7 for others; -X, X dropoff value for gapped alignment (in bits): default = 15 for all programs, not applicable to blastn; and -Z, final X dropoff value for gapped alignment (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, e.g., Blosum62 matrix and Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1). A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty). The equivalent settings in Bestfit protein comparisons are GAP=8 and LEN=2.

[0056] Typically, where the binding agent is an antibody, between each assay step, the bound analyte is washed, for example, with a mild detergent solution. Protocols may also include one or more blocking steps, which involve use of a non-specifically-binding protein such as bovine serum albumin to block unwanted non-specific binding of protein reagents.

[0057] Methods for producing antibodies are known in the art. For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be synthesized chemically or by recombinant DNA methodologies. For example, the sequences of the antibodies can be cloned from hybridomas by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using the appropriate synthetic nucleic acid primers. The resulting DNA molecules encoding the variable regions of interest can be ligated to other appropriate nucleotide sequences, including, for example, constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired antibodies. Production of defined gene constructs is within routine skill in the art.

[0058] Nucleic acids encoding desired antibodies can be incorporated (e.g., ligated) into suitable expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BEK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions.

[0059] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli , it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed secreted protein accumulates in refractile or inclusion bodies, and can be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the proteins refolded and cleaved by methods known in the art.

[0060] If the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct may contain enhancers and introns. This expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be introduced into eukaryotic host cells using conventional techniques. The host cells express VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a moiety having another function (e.g., cytotoxicity). In some embodiments, a host cell is transfected with a single vector expressing a polypeptide expressing an entire, or part of, a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region). In some embodiments, a host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In some embodiments, a host cell is co-transfected with more than one expression vector (e.g., one expression vector expressing a polypeptide comprising an entire, or part of, a heavy chain or heavy chain variable region, and another expression vector expressing a polypeptide comprising an entire, or part of, a light chain or light chain variable region).

[0061] A polypeptide comprising an immunoglobulin heavy chain variable region or light chain variable region can be produced by growing (culturing) a host cell transfected with an expression vector encoding such a variable region, under conditions that permit expression of the polypeptide. Following expression, the polypeptide can be harvested and purified or isolated using techniques known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) or histidine tags.

[0062] Exemplary nucleic acid based binding agents include aptamers and spiegelmers. Aptamers are nucleic acid-based sequences that have strong binding activity for a specific target molecule. Spiegelmers are similar to aptamers with regard to binding affinities and functionality but have a structure that prevents enzymatic degradation, which is achieved by using nuclease resistant L-oligonucleotides rather than naturally occurring, nuclease sensitive D-oligonucleotides.

[0063] Aptamers are specific nucleic acid sequences that bind to target molecules with high affinity and specificity and are identified by a method commonly known as Selective Evolution of Ligands by Evolution (SELEX), as described, for example, in U.S. Patent Nos. 5,475,096 and 5,270,163. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the observation that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.

[0064] The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule. Thus, this method allows for the screening of large random pools of nucleic acid molecules for a particular functionality, such as binding to a given target molecule.

[0065] The SELEX method also encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability and protease resistance. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Patent Nos. 5,660,985 and 5,580,737, which include highly specific nucleic acid ligands containing one or more nucleotides modified at the T position with, for example, a T -amino, 2^,-fluoro, and/or T -O-methyl moiety.

[0066] Instead of using aptamers, which may require additional modifications to become more resistant to nuclease activity, it is contemplated that spiegelmers, mirror image aptamers composed of L-ribose or L-2’deoxyribose units (see, U.S. Patent Nos. 8,841,431, 8,691,784, 8367,629, 8,193,159 and 8,314,223) can be used in the practice of the invention. The chiral inversion in spiegelmers results in an improved plasma stability compared with natural D- oligonucleotide aptamers. L-nucleic acids are enantiomers of naturally occurring D-nucleic acids that are not very stable in aqueous solutions and in biological systems or biological samples due to the widespread presence of nucleases. Naturally occurring nucleases, particularly nucleases from animal cells are not capable of degrading L-nucleic acids.

Because of this, the biological half-life of the L-nucleic acid is significantly increased in such a system, including the animal and human body. Due to the lacking degradability of L- nucleic acids, no nuclease degradation products are generated and thus no side effects arising therefrom observed.

[0067] Using in vitro selection, an oligonucleotide that binds to the synthetic enantiomer of a target molecule, e.g., a D-peptide, can be selected. The resulting aptamer is then resynthesized in the L-configuration to create a spiegelmer (from the German “spiegel” for mirror) that binds the physiological target with the same affinity and specificity as the original aptamer to the mirror-image target. This approach has been used to synthesize spiegelmers that bind, for example, hepcidin (see, U.S. Patent No. 8,841,431), MCP-1 (see, U.S. Patent Nos. 8,691,784, 8367,629 and 8,193,159) and SDF-1 (see, U.S. Patent No. 8,314,223).

B. Oligonucleotides

[0068] Probes useful in the practice of invention comprise an oligonucleotide, for example, an oligonucleotide conjugated to a binding agent.

[0069] The terms "nucleic acid," "polynucleotides," and "oligonucleotides" refer to biopolymers of nucleotides and, unless the context indicates otherwise, includes modified and unmodified nucleotides, and both DNA and RNA, and modified nucleic acid backbones. For example, in certain embodiments, the nucleic acid is a peptide nucleic acid (PNA) or a locked nucleic acid (LNA). Typically, the methods as described herein are performed using DNA as the nucleic acid template for amplification. However, nucleic acid whose nucleotide is replaced by an artificial derivative or modified nucleic acid from natural DNA or RNA is also included in the nucleic acid of the present invention insofar as it functions as a template for synthesis of complementary chain. Unless denoted otherwise, whenever a oligonucleotide sequence is represented, it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, "T" denotes deoxythymidine, and "U¹ denotes uridine. Oligonucleotides are said to have "5' ends" and "3' ends" because mononucleotides are typically reacted to form oligonucleotides via attachment of the 5' phosphate or equivalent group of one nucleotide to the 3' hydroxyl or equivalent group of its neighboring nucleotide, optionally via a phosphodiester or other suitable linkage.

[0070] The term, "complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) or hybridize with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. As used herein "hybridization," refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under low, medium, or highly stringent conditions, including when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. See e.g. Ausubel, etal., Current Protocols In Molecular Biology, John Wiley & Sons, New York, N.Y., 1993. If a nucleotide at a certain position of a polynucleotide is capable of forming a Watson-Crick pairing with a nucleotide at the same position in an anti parallel DNA or RNA strand, then the polynucleotide and the DNA or RNA molecule are complementary to each other at that position. The polynucleotide and the DNA or RNA molecule are "substantially complementary" to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hybridize or anneal with each other in order to affect the desired process.

[0071] Oligonucleotides contemplated herein may include one or more of the following features: a cleavage site, a spacer sequence, a unique identifying sequence, and a complementary region. In certain embodiments a probe ( e.g ., a first probe) comprises the cleavage site, the spacer sequence, the unique identifying sequence, and the complementary region in a 5’ to 3’ orientation. In certain embodiments, a probe (e.g., a second probe) comprises the cleavage site, the spacer sequence, the unique identifying sequence, and the complementary region in a 3’ to 5’ orientation.

[0072] The cleavage site allows for the potential removal of the binding agent prior to any downstream steps, for example, prior to an amplification reaction (e.g, PCR). The cleavage site may comprise any nucleotide or nucleotide sequence that is capable of being cleaved by a cleavage agent. In certain embodiments, the cleavage site is a deoxyuracil and/or the cleavage agent is a uracil-DNA glycosylase. In certain embodiments, the cleavage agent is a restriction enzyme. It is also contemplated that a protein-based binding agent (e.g, an antibody) may be removed from an oligonucleotide by digestion of the protein with a protease (e.g, trypsin) or by denaturation of the protein with heat.

[0073] The spacer sequence may, for example (i) allow for optimal access or activity of the cleavage agent, (ii) include, wholly or partially, a primer binding site for an amplification reaction (e.g. PCR), (iii) allow for control of the length of the product from an amplification reaction (e.g, PCR) and/or (iv) allow for control of oligonucleotide probe stability and/or hydrophobicity. The spacer sequence may be any appropriate length. For example, the spacer sequence of a probe (for example, a first and/or second probe) may be from about 50 to about 500, about 50 to about 400, about 50 to about 300, about 50 to about 300, about 50 to about 200, about 50 to about 100, about 100 to about 500, about 100 to about 400, about 100 to about 300, about 100 to about 300, about 100 to about 200, about 200 to about 500, about 200 to about 400, about 200 to about 300, about 200 to about 300, about 300 to about 500, about 300 to about 400, or about 400 to about 500 nucleotides in length, greater than about 50, about 100, about 200, about 300, about 400 or about 500 nucleotides in length, or about 50, about 100, about 200, about 300, about 400 or about 500 nucleotides in length. In certain embodiments, the spacer sequence of a first probe and the spacer sequence of a second probe are substantially the same length. In other embodiments, the spacer sequence of a first probe and the spacer sequence of a second probe have different lengths. For example, it is contemplated that the spacer sequence of the first probe and the spacer sequence of the second probe may be any combination of the foregoing nucleotide lengths.

[0074] It is contemplated that an oligonucleotide may contain or more unique identifying sequence. The unique identifying sequence (also referred to as a barcode) enables independent identification of an oligonucleotide probe following amplification and sequencing. Independent identification provided by the unique identifying sequence allows, for example, for distinguishing between multiple target analytes in a multiplexed assay. It is contemplated that an oligonucleotide may contain more than one unique identifying sequence. For example, the complementary region may include a second unique identifying sequence.

[0075] The complementary region allows for binding to a lock probe (as discussed below) under certain assay conditions. In certain embodiments, the complementary region of the first probe and the complementary region of the second probe are substantially identical. In other embodiments, the complementary region of the first probe and the complementary region of the second probe are different.

[0076] In certain embodiments, the oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

C. Conjugation/Linker

[0077] When a probe contains a binding agent and an oligonucleotide, they may be conjugated (e.g., covalently coupled) using any method known in the art. For example, in certain embodiments, a binding agent comprises a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated to each respective binding agent via the UAA. A given conjugation strategy will depend upon which UAA is selected as the UAA preferably contains a chemical group or moiety that facilitates conjugation not present in a naturally occurring amino acid. In certain embodiments, the UAA contains an azide functional group and the oligonucleotide contains an amine functional group. The UAA can be first reacted with a DBCO-NHS ester and subsequently reacted with the oligonucleotide to form a final conjugate. Exemplary methods of UAA incorporation and oligonucleotide conjugation are described in Synakewi etal. (2019) SCIENTIFIC REPORTS 9:13820, Italia etal. (2017) NAT. CHEM. BIOL. 13(4):446-450, Liu etal. (2010) ANNU. REVBIOCHEM. 79:413-44, Dumas etal. (2015) CHEM. SCI. 6(l):50-69, and Zheng etal. (2018) BIOCHEMISTRY 57(4):441-445.

D. Lock Probes

[0078] Certain contemplated methods comprise the use of a lock probe. The lock probe comprises (a) a first region that is capable of hybridizing to a first probe ( e.g ., to the complementary region of the first probe) and (b) a second region that is capable of hybridizing to a second probe (e.g., the complementary region of the second probe). The lock probe may be of any format that allows for simultaneous hybridization to the first and second probe if the first and second probe are in sufficient proximity to each other (e.g, when the first and second probe are both bound to a target analyte).

[0079] The lock probe may be modified or designed to ensure that it does not serve as a primer in a downstream amplification reaction (e.g., a PCR reaction). For example, in certain embodiments, the lock probe is designed to contain a 3’ dideoxy nucleotide, which does not permit chain extension via polymerase and amplification. In certain embodiments, the lock probe comprises one, two or more 3’ mismatches to prevent chain extension via a polymerase and amplification.

[0080] In certain embodiments, the lock probe comprises the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3.

III. Unnatural Amino Acids

[0081] It is contemplated that a probe may comprise a protein sequence comprising an unnatural amino acid (UAA). In certain embodiments, an oligonucleotide is conjugated to the protein sequence via the UAA.

[0082] As used herein, an unnatural amino acid refers to any amino acid, modified amino acid, or amino acid analogue other than the following twenty genetically encoded alpha- amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine. See, e.g ., Biochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York, for structures of the twenty natural amino acids. The term unnatural amino acid also includes amino acids that occur by modification (e.g. post- translational modifications) of a natural amino acid but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex.

[0083] Because unnatural amino acids typically differ from natural amino acids only in the structure of the side chain, unnatural amino acids may, for example, form amide bonds with other amino acids in the same manner in which they are formed in naturally occurring proteins. However, the unnatural amino acids have side chain groups that distinguish them from the natural amino acids. For example, the side chain may comprise an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkyl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amine, and the like, or any combination thereof. Other non-naturally occurring amino acids include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon -linked sugar-containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moiety.

[0084] In addition to unnatural amino acids that contain novel side chains, unnatural amino acids also optionally comprise modified backbone structures. [0085] Many unnatural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and the like. Tyrosine analogs include para-substituted tyrosines, ortho-substituted tyrosines, and meta substituted tyrosines, wherein the substituted tyrosine comprises a keto group (including but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C6-C20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a nitro group, or the like. In addition, multiply substituted aryl rings are also contemplated. Glutamine analogs include, but are not limited to, a-hydroxy derivatives, g-substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives. Exemplary phenylalanine analogs include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenylalanines, and meta- substituted phenylalanines, wherein the substituent comprises a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), or the like. Specific examples of unnatural amino acids include, but are not limited to, a p-acetyl-L-phenylalanine, a p-propargyl- phenylalanine, O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3 -methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcP-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p- acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L- phenylalanine, an isopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and the like.

[0086] Examples of structures of a variety of unnatural amino acids are provided in U.S. Patent Application Publication Nos. 2003/0082575 and 2003/0108885, PCT Publication No. WO 2002/085923, and Kiick et al. (2002) PROC. NATL. ACAD. SCI. USA 99: 19-24.

[0087] Any suitable unnatural amino acid can be used with the methods described herein.

[0088] The unnatural amino acid may be a leucine analog. The invention provides a leucine analog depicted in FIGURE 8A, or a composition comprising the leucine analog. For example, Formula A in FIGURE 8A depicts an amino acid analog containing a side chain including a carbon containing chain n units (0-20 units) long. An O, S, CFh, or NH is present in at position X, and another carbon containing chain of n units (0-20 units) long can follow. A functional group Y is attached to the terminal carbon of second carbon containing chain (for example, functional groups 1-12 as depicted in FIGURE 8A, where R represents a linkage to the terminal carbon atom the second carbon containing side chain). In one example, these functional groups can be used for bioconjugation of any amenable ligand to any protein of interest that is amenable to site-specific UAA incorporation. Formula B in FIGURE 8A depicts a similar amino acid analog containing an side chains denoted as either Z-Y2 or Z-Y3 attached to the second carbon containing chain or the first carbon containing chain, respectively. Z represents a carbon chain comprising (CFhjn units, where n is any integer from 0-20. Y2 or Y3, independently, can be the same or different groups as those of Yi. The invention also provides a leucine analog depicted in FIGURE 8B (LCA, LKET, or ACA), or a composition comprising the leucine analog depicted in FIGURE 8B. Additional exemplary leucine analogs include those selected from linear alkyl halides and linear aliphatic chains comprising a functional group, for example, an alkyne, azide, cyclopropene, alkene, ketone, aldehyde, diazirine, or tetrazine functional group, as well as structures 1-6 shown in FIGURE 8C. However, it is contemplated that the amino and carboxylate groups both attached to the first carbon of any amino acid shown in FIGURES 8A-8C would constitute portions of peptide bonds when the leucine analog is incorporated into a protein or polypeptide chain.

[0089] In certain embodiments, the unnatural amino acid is a tryptophan analog (also referred to herein as a derivative). Exemplary tryptophan analogs include 5-azidotryptophan, 5-propargyloxytryptophan, 5-aminotryptophan, 5-methoxytryptophan, 5-O-allyltryptophan or 5-bromotryptophan. Additional exemplary tryptophan analogs are depicted in FIGURE 9. However, it is contemplated that the amino and carboxylate groups both attached to the first carbon of the tryptophan analogs in FIGURE 9 would constitute portions of peptide bonds when the tryptophan analog is incorporated into a protein or polypeptide chain.

[0090] Many unnatural amino acids are commercially available, e.g ., from Sigma-Aldrich (St. Louis, Mo., USA), Novabiochem (Darmstadt, Germany), or Peptech (Burlington, Mass., USA). Those that are not commercially available can be synthesized using standard methods known to those of ordinary skill in the art. For organic synthesis techniques, see, e.g. , Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). Additional exemplary publications describing the synthesis of unnatural amino acids appear in PCT Publication No. W02002/085923, U.S. Patent Application Publication No. 2004/0198637, Matsoukas etal. (1995) J. MED. CHEM. 38:4660-4669, King etal. (1949) J. CHEM. SOC. 3315-3319, Friedman etal. (1959) J. AM. CHEM. SOC. 81:3750-3752, Craig etal. (1988) J. ORG. CHEM. 53:1167- 1170, Azoulay etal. (1991) EUR. J. MED. CHEM. 26:201-5, Koskinen et al. (1989) J. ORG. CHEM. 54:1859-1866, Christie et al. (1985) J. ORG. CHEM. 50:1239-1246, Barton etal.

(1987) TETRAHEDRON 43:4297-4308, and Subasinghe etal. (1992) J. MED. CHEM. 35:4602-7.

[0091] The core elements required for the site-specific incorporation of one or more unnatural amino acids (UAAs) into a protein of interest include: an engineered tRNA, an engineered aminoacyl-tRNA synthetase (aaRS) that charges the tRNA with a UAA, and a unique codon, e.g., a stop codon, directing the incorporation of the UAA into the protein as it is being synthesized (see FIGURE 7). Central to this approach is the use of an engineered tRNA/aaRS pair in which the aaRS charges the tRNA with the UAA of interest without cross-reacting with the tRNAs and amino acids normally present in the expression host cell. This has been accomplished by using an engineered tRNA/aaRS pair derived from an organism in different domain of life as the expression host cell so as to maximize the orthogonality between the engineered tRNA/aaRS pair (e.g., an engineered bacterial tRNA/aaRS pair) and the tRNA/aaRS pairs naturally found in the expression host cell (e.g., mammalian cell). The engineered tRNA, which is charged with the UAA via the aaRS, binds or hybridizes to the unique codon, such as a premature stop codon (UAG, UGA, UAA) present in the mRNA encoding the protein to be expressed. To date, a variety of orthogonal tRNA/aaRS pairs have been produced for certain of the naturally occurring amino acids (see, e.g., U.S. Patent Publication US2017/0349891, and Zheng etal. (2018) BlOCHEM. 57:441- 445).

[0092] A protein may have at least one, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more UAAs. The UAAs can be the same or different. For example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different UAAs. A protein may have at least one, but fewer than all, of a particular amino acid present in the protein substituted with the UAA. For a given protein with more than one UAA, the UAA can be identical or different (for example, the protein can include two or more different types of UAAs, or can include two of the same UAA). For a given protein with more than two UAAs, the UAAs can be the same, different or a combination of a multiple unnatural amino acid of the same kind with at least one different UAA.

[0093] UAAs may be incorporated into a protein of interest using any appropriate translation system. The term “translation system” refers to a system including components necessary to incorporate an amino acid into a growing polypeptide chain (protein). Components of a translation system can include, e.g ., ribosomes, tRNA's, synthetases, mRNA and the like. Translation systems may be cellular or cell-free, and may be prokaryotic or eukaryotic. For example, translation systems may include, or be derived from, a non-eukaryotic cell, e.g. , a bacterium (such as E. coli ), a eukaryotic cell, e.g. , a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, or an insect cell.

[0094] Translation systems include host cells or cell lines. The nucleic acid encoding the necessary components for UAA incorporation can be expressed in an expression host cell either as an autonomously replicating vector within the expression host cell (e.g., a plasmid, or viral particle) or via a stable integrated element or series of stable integrated elements in the genome of the expression host cell, e.g., a mammalian host cell. Host cells are genetically engineered (including but not limited to, transformed, transduced or transfected), for example, using nucleic acids or vectors. For example, in certain embodiments, one or more vectors include coding regions for an orthogonal tRNA, an orthogonal aminoacyl- tRNA synthetase, and, optionally, a protein to be modified by the inclusion of one or more UAAs, which are operably linked to gene expression control elements that are functional in the desired host cell or cell line. The vectors are transfected into cells and/or microorganisms by standard methods including electroporation or infection by viral vectors, and clones can selected via expression of the selectable marker (for example, by antibiotic resistance).

[0095] Exemplary prokaryotic host cells or cell lines include cells derived from a bacteria, e.g., Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida. Exemplary eukaryotic host cells or cell lines include cells derived from a plant (e.g, a complex plant such as a monocot or dicot), an algae, a protist, a fungus, a yeast (including Saccharomyces cerevisiae ), or an animal (including a mammal, an insect, an arthropod, etc.). Additional exemplary host cells or cell lines include HEK293, HEK293T, Expi293, CHO, CHOK1, Sf9, Sf21, HeLa, U20S, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-RB50, HepG2, DUKX- XI 1, J558L, BHK, COS, Vero, NS0, or ESCs. It is understood that a host cell or cell line can include individual colonies, isolated populations (monoclonal), or a heterogeneous mixture of cells.

[0096] A contemplated cell or cell line includes, for example, one or multiple copies of an orthogonal tRNA/aminoacyl-tRNA synthetase pair, optionally stably maintained in the cell’s genome or another piece of DNA maintained by the cell.

[0097] Methods to introduce a nucleic acid encoding a tRNA and/or an aminoacyl-tRNA synthetase into the genome of a cell of interest, or to stably maintain the nucleic acid in DNA replicated by the cell that is outside of the genome, are well known in the art. The nucleic acid encoding the tRNA and/or an aminoacyl-tRNA synthetase can be provided to the cell in an expression vector, transfer vector, or DNA cassette. The expression vector transfer vector, or DNA cassette encoding the tRNA and/or aminoacyl-tRNA synthetase can contain one or more copies of the tRNA and/or aminoacyl-tRNA synthetase optionally under the control of an inducible or constitutively active promoter. The expression vector, transfer vector, or DNA cassette may, for example, contain other standard components (enhancers, terminators, etc.). It is contemplated that the nucleic acid encoding the tRNA and the nucleic acid encoding the aminoacyl-tRNA synthetase may be on the same or different vector, may be present in the same or different ratios, and may be introduced into the cell, or stably integrated in the cellular genome, at the same time or sequentially.

[0098] Vectors ( e.g ., expression vectors or transfer vectors) include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), retrotransposons (e.g. piggyback, sleeping beauty), and viruses (e.g, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide of interest.

[0099] Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both (including but not limited to, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.

[00100] In certain embodiments, the vector comprises a regulatory sequence or promoter operably linked to the nucleotide sequence encoding the suppressor tRNA and/or the tRNA synthetase. The term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene. Operably linked nucleotide sequences are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.

[00101] The production of an exemplary cell line capable of producing antibodies incorporating a UAA is described in Roy et al. (2020) MABS 12(1), el684749).

[00102] Translation systems also include whole cell preparations such as permeabilized cells or cell cultures wherein a desired nucleic acid sequence can be transcribed to mRNA and the mRNA translated. Cell-free translation systems are commercially available and many different types and systems are well-known. Examples of cell-free systems include, but are not limited to, prokaryotic lysates such as Escherichia coli lysates, and eukaryotic lysates such as wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates and human cell lysates. Reconstituted translation systems may also be used. Reconstituted translation systems may include mixtures of purified translation factors as well as combinations of lysates or lysates supplemented with purified translation factors such as initiation factor-1 (IF-1), IF-2, IF-3 (a or b), elongation factor T (EF-Tu), or termination factors. Cell-free systems may also be coupled transcription/translation systems wherein DNA is introduced to the system, transcribed into mRNA and the mRNA is translated.

[00103] In certain embodiments, the protein is expressed from a nucleic acid sequence comprising a premature stop codon. The translation system ( e.g host cell or cell line) may, for example, contain a tRNA synthetase capable of charging a suppressor tRNA with an unnatural amino acid which is incorporated into the protein at a position corresponding to the premature stop codon. In certain embodiments, the suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the protein at the position corresponding to the premature stop codon.

IV. Amplification Reactions [00104] The terms "amplify," "amplifying," "amplification reaction” and their variants, refer generally to any action or process whereby at least a portion of a nucleic acid molecule (also referred to as a template nucleic acid molecule) is replicated or copied into at least one additional nucleic acid molecule. The additional nucleic acid molecule optionally includes sequence that is substantially identical or substantially complementary to at least some portion of the template nucleic acid molecule. The template nucleic acid molecule can be single-stranded or double-stranded and the additional nucleic acid molecule can independently be single-stranded or double- stranded. In some embodiments, amplification includes a template-dependent in vitro enzyme-catalyzed reaction for the production of at least one copy of at least some portion of the nucleic acid molecule or the production of at least one copy of a nucleic acid sequence that is complementary to at least some portion of the nucleic acid molecule. Amplification optionally includes linear or exponential replication of a nucleic acid molecule. In some embodiments, such amplification is performed using isothermal conditions; in other embodiments, such amplification can include thermocycling. In some embodiments, the amplification is a multiplex amplification that includes the simultaneous amplification of a plurality of target sequences in a single amplification reaction. At least some of the target sequences can be situated, on the same nucleic acid molecule or on different target nucleic acid molecules included in the single amplification reaction. In some embodiments, "amplification" includes amplification of at least some portion of DNA- and RNA-based nucleic acids alone, or in combination. The amplification reaction can include single or double-stranded nucleic acid substrates and can further include any of the amplification processes known to one of ordinary skill in the art.

[00105] The synthesis of nucleic acid in the present invention means the elongation or extension of nucleic acid from an oligonucleotide serving as the origin of synthesis. If not only this synthesis but also the formation of other nucleic acid and the elongation or extension reaction of this formed nucleic acid occur continuously, a series of these reactions is comprehensively called amplification. The polynucleic acid produced by the amplification technology employed is generically referred to as an "amplicon" or "amplification product." [00106] A number of nucleic acid polymerases can be used in the amplification reactions utilized in certain embodiments provided herein, including any enzyme that can catalyze the polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Such nucleotide polymerization can occur in a template-dependent fashion. Such polymerases can include without limitation naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase can be a mutant polymerase comprising one or more mutations involving the replacement of one or more amino acids with other amino acids, the insertion or deletion of one or more amino acids from the polymerase, or the linkage of parts of two or more polymerases. Typically, the polymerase comprises one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization can occur. Some exemplary polymerases include without limitation DNA polymerases and RNA polymerases. The term "polymerase" and its variants, as used herein, also includes fusion proteins comprising at least two portions linked to each other, where the first portion comprises a peptide that can catalyze the polymerization of nucleotides into a nucleic acid strand and is linked to a second portion that comprises a second polypeptide. In some embodiments, the second polypeptide can include a reporter enzyme or a processivity-enhancing domain. Optionally, the polymerase can possess 5' exonuclease activity or terminal transferase activity. In some embodiments, the polymerase can be optionally reactivated, for example through the use of heat, chemicals or re-addition of new amounts of polymerase into a reaction mixture. In some embodiments, the polymerase can include a hot-start polymerase or an aptamer-based polymerase that optionally can be reactivated.

[00107] "Forward primer binding site" and "reverse primer binding site" refer to the regions on the template DNA and/or the amplicon to which the forward and reverse primers bind. The primers act to delimit the region of the original template polynucleotide which is exponentially amplified during amplification. In some embodiments, additional primers may bind to the region 5' of the forward primer and/or reverse primers. Where such additional primers are used, the forward primer binding site and/or the reverse primer binding site may encompass the binding regions of these additional primers as well as the binding regions of the primers themselves. For example, in some embodiments, the method may use one or more additional primers which bind to a region that lies 5' of the forward and/or reverse primer binding region. Such a method was disclosed, for example, in W00028082 which discloses the use of "displacement primers" or "outer primers".

[00108] A primer set for the amplification of a target nucleic acid typically includes a forward primer and a reverse primer that are complementary to a target nucleic acid or the complement thereof. In some embodiments, amplification can be performed using multiple target-specific primer pairs in a single amplification reaction, wherein each primer pair includes a forward target-specific primer and a reverse target-specific primer, where each includes at least one sequence that substantially complementary or substantially identical to a corresponding target sequence in the sample, and each primer pair having a different corresponding target sequence.

[00109] In some embodiments, the amplification reaction includes a PCR-based assay, e.g., quantitative PCR (qPCR). PCR is described, for example, in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188. PCR is typically carried out using a thermostable DNA-dependent DNA polymerase. The polymerase most commonly used in PCR systems is a Thermus aquaticus (Taq) polymerase. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification. Commercial technologies such as SYBR^® green or TaqMan^® (Applied Biosystems, Foster City, CA) can be used in accordance with the vendor’s instructions. Suitable primer sets for PCR analysis can be designed and synthesized by one of skill in the art, without undue experimentation. Alternatively, PCR primer sets for practicing the present invention can be purchased from commercial sources, e.g., Applied Biosystems. PCR primers preferably are about 17 to 25 nucleotides in length. Primers can be designed to have a particular melting temperature (Tm), using conventional algorithms for Tm estimation. Software for primer design and Tm estimation are available commercially, e.g, Primer Express™ (Applied Biosystems), and also are available on the internet, e.g, Primer3 (Massachusetts Institute of Technology).

[00110] Additional amplification methods include loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification, etc. (See, e.g., Fakruddin etal. (2013) J Pharm Bioallied Sci. 5(4):245-252.) Other suitable amplification methods include rolling circle amplification (RCA), multiple displacement amplification (MDA), ligation extension, ligase chain reaction (Wu and Wallace (1988) GENOMICS 4:560-569); the strand displacement assay (Walker et al. (1992) PROC. NATL. ACAD. SCI. USA 89:392-396, Walker et al. (1992) NUCL. ACIDS RES. 20: 1691-1696, and U.S. Patent No. 5,455,166); and several transcription-based amplification systems, including the methods described in U.S. Patent Nos. 5,437,990, 5,409,818, and 5,399,491, the transcription amplification system (TAS) (Kwoh etal. (1989) PROC. NATL. ACAD. SCI. USA 86:1173-1177), and self-sustained sequence replication (3SR) (Guatelli etal. (1990) PROC. NATL. ACAD. SCI. USA 87:1874-1878 and WO 92/08800). A review of known amplification methods is provided in Abramson etal. (1993) CURRENT OPINION IN BIOTECHNOLOGY 4:41-47.

[00111] Amplification products generated using any of the above methods can be analyzed by the use of denaturing gradient gel electrophoresis. Different products can be identified based on sequence-dependent melting properties and electrophoretic migration in solution. See Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, Chapter 7 (W.H. Freeman and Co, New York, 1992).

[00112] Amplification products may also be identified by DNA sequencing methods, such as the chain termination method (Sanger et al. (1977) PROC. NATL. ACAD. SCI. 74:5463- 5467) or PCR-based sequencing. See Sambrook et al. , MOLECULAR CLONING, A LABORATORY MANUAL (2nd Ed., CSHP, New York 1989) and Zyskind et al. , RECOMBINANT DNA LABORATORY MANUAL (Acad. Press, 1988).

[00113] Sequence reads can also be achieved with commercially available next generation sequencing (NGS) platforms, including platforms that perform any of sequencing by synthesis, sequencing by ligation, pyrosequencing, using reversible terminator chemistry, using phospholinked fluorescent nucleotides, or real-time sequencing. As an example, amplified nucleic acids may be sequenced on an Illumina MiSeq platform.

[00114] When pyrosequencing, libraries of NGS fragments are cloned in-situ amplified by capture of one matrix molecule using granules coated with oligonucleotides complementary to adapters. Each granule containing a matrix of the same type is placed in a microbubble of the “water in oil” type and the matrix is cloned amplified using a method called emulsion PCR. After amplification, the emulsion is destroyed and the granules are stacked in separate wells of a titration picoplate acting as a flow cell during sequencing reactions. The ordered multiple administration of each of the four dNTP reagents into the flow cell occurs in the presence of sequencing enzymes and a luminescent reporter, such as luciferase. In the case where a suitable dNTP is added to the 3 ' end of the sequencing primer, the resulting ATP produces a flash of luminescence within the well, which is recorded using a CCD camera. It is possible to achieve a read length of more than or equal to 400 bases, and it is possible to obtain 10⁶ readings of the sequence, resulting in up to 500 million base pairs (megabytes) of the sequence. Additional details for pyrosequencing is described in Voelkerding et al., CLIN. CHEM., 55: 641-658, 2009; MacLean et al., NATURE REV. MlCRBIOL., 7: 287-296; U.S. Patent No. 6,210,891; and U.S. Patent No. 6,258,568.

[00115] On the Solexa / Illumina platform, sequencing data is produced in the form of short readings. In this method, fragments of a library of NGS fragments are captured on the surface of a flow cell that is coated with oligonucleotide anchor molecules. An anchor molecule is used as a PCR primer, but due to the length of the matrix and its proximity to other nearby anchor oligonucleotides, elongation by PCR leads to the formation of a “vault” of the molecule with its hybridization with the neighboring anchor oligonucleotide and the formation of a bridging structure on the surface of the flow cell. These DNA loops are denatured and cleaved. Straight chains are then sequenced using reversibly stained terminators. The nucleotides included in the sequence are determined by detecting fluorescence after inclusion, where each fluorescent and blocking agent is removed prior to the next dNTP addition cycle. A dditional details for sequencing using the Illumina platform is found in Voelkerding et al., CLIN. CHEM., 55: 641-658, 2009; MacLean et al., NATURE REV. MlCRBIOL., 7: 287-296; U.S. Patent No. 6,833,246; U.S. Patent No. 7,115,400; and U.S. Patent No. 6,969,488.

[00116] Sequencing of nucleic acid molecules using SOLiD technology includes clonal amplification of the library of NGS fragments using emulsion PCR. After that, the granules containing the matrix are immobilized on the derivatized surface of the glass flow cell and annealed with a primer complementary to the adapter oligonucleotide. However, instead of using the indicated primer for 3 'extension, it is used to obtain a 5' phosphate group for ligation for test probes containing two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels. In the SOLiD system, test probes have 16 possible combinations of two bases at the 3 'end of each probe and one of four fluorescent dyes at the 5' end. The color of the fluorescent dye and, thus, the identity of each probe, corresponds to a certain color space coding scheme. After many cycles of alignment of the probe, ligation of the probe and detection of a fluorescent signal, denaturation followed by a second sequencing cycle using a primer that is shifted by one base compared to the original primer. In this way, the sequence of the matrix can be reconstructed by calculation; matrix bases are checked twice, which leads to increased accuracy. Additional details for sequencing using SOLiD technology is found in Voelkerding etal. , (2009) supra ; MacLean etal ., supra ; U.S. Patent No. 5,912,148; and U.S. Patent No. 6,130,073.

[00117] Sequencing reads obtained from the NGS methods can be filtered by quality and grouped by barcode sequence using any algorithms known in the art.

VI. Kits

[00118] In addition, the invention provides kits comprising, for example, (i) a first probe comprising a first binding agent capable of binding an analyte conjugated to a first oligonucleotide, and a second probe comprising a second binding agent capable of binding the analyte conjugated to a second oligonucleotide, wherein the first binding agent and the second binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated (e.g., covalently coupled) to each respective binding agent via the UAA, and the first and second oligonucleotide each comprise (a) a cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region; (ii) a lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe; (iii) a DNA ligase; and (iv) optionally, a cleavage agent capable of cleaving the cleavage site of the first and second probe. In certain embodiments, the kit further comprises any components necessary to perform an amplification reaction disclosed herein. For example, the kit may comprise a first PCR primer that binds to a first PCR primer binding site on the first probe and/or a second PCR primer that binds to a second PCR primer binding site on the second probe.

[00119] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[00120] In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[00121] Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and/or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[00122] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[00123] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[00124] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[00125] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[00126] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

EXAMPLES

[00127] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

Example 1

[00128] This Example describes the testing of a set of oligonucleotides for use as probes in the detection of an analyte.

[00129] Mixtures containing combinations of a right oligo (RO; SEQ ID NO: 1), a left oligo (LO; SEQ ID NO: 2), and a lock probe (LP; SEQ ID NO: 3), each as shown in FIGURE 3 (but without conjugation to antibody) were generated. Mixtures were incubated with T4 DNA Ligase (NEB) according to manufacturer’s protocol (~2 mM final LO/RO, ~6 mM LP, 1 :3 molar ratio). Ligations were incubated for 2 hours at room temperature (RT), or 16°C overnight. To see if residual UDG would inhibit the PCR reaction, UDG was added according to manufacturer’s protocol (NEB). The resulting DNA was used as a template for a PCR reaction. Results are shown in FIGURE 4. As depicted, the target 202bp band is only amplified in the presence of all required components (LO, RO, LP, and ligase) and amplification is uninhibited in the presence of UDG. The ligation can also be completed within 2 hours.

Example 2

[00130] This example describes the site-specific conjugation of an oligonucleotide to an antibody to generate a conjugate for use as a probe in the detection of an analyte. [00131] A conjugation scheme was followed to generate homogenous monoclonal antibody (mAh) products mAb-left oligo (mAb-LO) and mAb-right oligo (mAb-RO). A schematic depiction of the conjugation scheme is shown in FIGURE 5A. In this conjugation scheme, a naked antibody (trastuzumab, targeting HER2, and comprising a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 5) containing a site-specifically introduced azide containing amino acid was generated using previously reported genetic code expansion methodologies (see, e.g., Addy etal. (2017) J. AM. CHEM. SOC. 139:11670-11673) and identified by HPLC-HIC (FIGURE 5B). 6 mM azide was labeled with excess DBCO-PEG4- NHS Ester linker molecule by incubating for 3 hours at room temperature (RT). The conjugation mixture was buffer exchanged into PBS using a 100K MWCO Amicon filter and normalized to 6 pM and analyzed by HPLC-HIC (FIGURE 5C). Successful labeling with DBCO-PEG4-NHS Ester is expected to produce a shift to the right in the HPLC-HIC trace, and the expected hydrophobic shift to the right was observed. Next, 6 pM mAb-NHS protein was labeled with excess of 10 pM RO or LO containing an amine conjugation handle (5’ on LO, 3’ on RO) overnight at 4°C. The sample was buffer exchanged as previously described and analyzed by HPLC-HIC (FIGURE 5D and FIGURE 5E). All labeled proteins were analyzed by SDS-PAGE in reducing conditions (FIGURE 5F). SDS-PAGE lanes 1-3 correspond to FIGURES 5C-E respectively.

[00132] Together, these results show that the anti-HER2 antibody was site-specifically modified to contain an exposed LO or RO.

Example 3

[00133] This Example describes the testing of a set of antibody-oligonucleotide conjugates for use as probes in the detection of an analyte.

[00134] mAb-RO and mAb-LO, as described in Example 2, were tested in an assay similar to that described in Example 1. DNA controls were run as above. As outlined in the table of FIGURE 6, each mAb-Oligo was introduced in a 20 pL T4 DNA ligation according to manufacturer’s protocol (NEB) with 5 pM lock probe. mAb-RO and mAb-LO were added at 33 nM final concentration each. Ligation proceeded for 2 hours at room temperature (RT), followed by 65°C heat denaturation. Subsequently, 1 pL of ligation mixture containing the LO-RO hybrid was used as a template for PCR amplification. Results are shown in FIGURE 6. Only in the presence of both mAb-RO and mAb-LO or the pairing of the mAb-oligo with its partner DNA (e.g., mAb-LO with RO-amine) resulted in the target 202bp PCR product.

[00135] If a UDG cleavage step is required, 1 pL UDG (NEB) is added to the 20 pL ligation mixture and incubated overnight (ON) at 37°C according to manufacturer’s protocol. [00136] Together, these results show that the antibody-oligonucleotide conjugates can be used as probes for the specific detection of an analyte, e.g., HER2. This detection can be extended to a standard ELISA format, as described in FIGURE 1 and FIGURE 2, or to the detection of multiple analytes in a multiplexed assay. INCORPORATION BY REFERENCE

[00137] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[00138] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

SEQUENCE LISTING

[00139] SEQ ID NO: 1

GCGCTcgacgacAGGAGGCAATTGTATAAGAACGTGAAGTGTCTATCACCCCTAGGCCCTAC

GTCTCCATCGCTTGCCCAAGTTGAagtcatgcaCGCnCCCttaacgtcaGTTTCCCGCATAT

TAACGCCTGATTGTATCCGCATTTGATGCTACCGTGG

[00140] SEQ ID NO: 2

GTTTCCCGCATATTAACGCCTGATTGTATCCGCATTTGATGCTACCGTGGAATAATACGCnC CCAATAGCAAGTAgcTAGCTACGTACGTACG TACGTACGTACGTaATATGAATGCGACCTCG AAGAGGCCaGTGCATcgacggACTGCATCGATACATAAAAC

[00141] SEQ ID NO: 3

GTTCTTATACAATTGCCTCCTgtcgtcgAGCGCGTTTTATGTATCGATGCAGTccgtcgATG

CAC

[00142] SEQ ID NO: 4

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI YPTNGYTRYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW SVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

[00143] SEQ ID NO: 5

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFI FPPSDEQ LKSGTASW CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

WHAT IS CLAIMED IS:

1. A method for detecting the presence or amount of an analyte in a sample, the method comprising:

(i) contacting the sample with a first probe comprising a first binding agent capable of binding the analyte conjugated to a first oligonucleotide and a second probe comprising a second binding agent capable of binding the analyte conjugated to a second oligonucleotide, under conditions to permit the first probe and the second probe bind the analyte if the analyte is present in the sample, wherein the first binding agent and the second binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated to each respective binding agent via the UAA, and the first and second oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region;

(ii) contacting the sample with a lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe, under conditions to permit the lock probe to simultaneously hybridize to the first and second probe to form a duplex if the first and second probe are in sufficient proximity to each other;

(iii) ligating the first probe and the second probe to form a ligation product;

(iv) optionally cleaving the ligation product with a cleavage agent capable of cleaving the cleavage site of the first and second probe thereby removing the first and second binding agents to form a cleaved ligation product;

(v) using the ligation product or the cleaved ligation product, if produced, as a template for an amplification reaction; and

(vi) detecting a product from the amplification reaction, which, if present, is indicative of the presence and/or amount of the analyte in the sample.

2. The method of claim 1, wherein the first binding agent and the second binding agent bind the same epitope on the analyte.

3. The method of claim 2, wherein the first binding agent and the second binding agent are substantially identical.

4. The method of claim 1, wherein the first binding agent and the second binding agent bind different epitopes on the analyte.

5. The method of any one of claims 1-4, wherein the UAA comprises an azide or alkyne functional group.

6. The method of any one of claims 1-4, wherein the UAA comprises hydroxytryptophan.

7. The method of any one of claims 1-6, wherein the oligonucleotide is conjugated to the UAA via a linker (e.g., by a DBCO linker).

8. The method of any one of claims 1-7, wherein the first and second binding agents are antibodies.

9. The method of claim 8, wherein the first and second binding agents are anti-HER2 antibodies.

10. The method of claim 9, wherein the first and second binding agents are a trastuzumab antibody comprising the UAA.

11. The method of any one of claims 1-10, wherein the spacer sequence of the first probe and the spacer sequence of the second probe are substantially the same length.

12. The method of any of claims 1-11, wherein the spacer sequence of the first probe and the spacer sequence of the second probe have different lengths.

13. The method of any one of claims 1-12, wherein the spacer sequence of the first probe is from about 50 to about 500, about 50 to about 400, about 50 to about 300, about 50 to about 300, about 50 to about 200, about 50 to about 100, about 100 to about 500, about 100 to about 400, about 100 to about 300, about 100 to about 300, about 100 to about 200, about 200 to about 500, about 200 to about 400, about 200 to about 300, about 200 to about 300, about 300 to about 500, about 300 to about 400, or about 400 to about 500 nucleotides in length and/or the spacer sequence of the second probe is from about 50 to about 500, about 50 to about 400, about 50 to about 300, about 50 to about 300, about 50 to about 200, about 50 to about 100, about 100 to about 500, about 100 to about 400, about 100 to about 300, about 100 to about 300, about 100 to about 200, about 200 to about 500, about 200 to about 400, about 200 to about 300, about 200 to about 300, about 300 to about 500, about 300 to about 400, or about 400 to about 500 nucleotides in length.

14. The method of any one of claims 1-13, wherein the complementary region of the first probe and the complementary region of the second probe are substantially identical.

15. The method of any one of claims 1-14, wherein the complementary region of the first probe and the complementary region of the second probe are different.

16. The method of any one of claims 1-15, wherein the lock probe comprises a 3’ dideoxy nucleotide.

17. The method of any one of claims 1-16, wherein the first a second probe are ligated by a DNA ligase (e.g.,_ T4 DNA ligase).

18. The method of any one of claims 1-17, wherein the cleavage site is a uracil and/or the cleavage agent is a uracil-DNA glycosylase.

19. The method of any one of claims 1-18, wherein the amplification reaction is a polymerase chain reaction (PCR) or rolling chain amplification (RCA).

20. The method of claim 19, wherein the amplification reaction is PCR.

21. The method of claim 20, wherein the first probe comprises a first PCR primer binding site.

22. The method of claim 21, wherein the first PCR primer binding site is 5’ to the spacer sequence of the first probe, within the spacer sequence of the first probe, or partially within the spacer sequence of the first probe.

23. The method of any one of claims 20-22, wherein the second probe comprises a second PCR primer binding site.

24. The method of claim 23, wherein the second PCR primer binding site is 3’ to the spacer sequence of the second probe, within the spacer sequence of the second probe, or partially within the spacer sequence of the second probe.

25. The method of any one of claims 20-24, wherein the PCR reaction comprises incubating the ligation product or the cleaved ligation product with a first primer capable of binding the first PCR primer binding and a second primer capable of binding the second PCR primer binding site.

26. The method of any one of claims 1-25, wherein the first probe comprises the cleavage site, the spacer sequence, the unique identifying sequence, and the complementary region in a 5’ to 3’ orientation.

27. The method of any one of claims 1-26, wherein the second probe comprises the cleavage site, the spacer sequence, the unique identifying sequence, and the complementary region in a 3’ to 5’ orientation.

28. A method for detecting the presence or amount of multiple analytes in a sample, the method comprising, for each analyte:

(i) contacting the sample with a first probe comprising a first binding agent capable of binding the analyte conjugated to a first oligonucleotide and a second probe comprising a second binding agent capable of binding the analyte conjugated to a second oligonucleotide, under conditions to permit the first probe and the second probe to bind the analyte if the analyte is present in the sample, wherein the first binding agent and the second binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated to each respective binding agent via the UAA, and the first and second oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region;

(ii) contacting the sample with a lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe, under conditions to permit the lock probe to simultaneously hybridize to the first and second probe to form a duplex if the first and second probe are in sufficient proximity to each other;

(iii) ligating the first probe and the second probe to form a ligation product;

(iv) optionally cleaving the ligation product with a cleavage agent capable of cleaving the cleavage site of the first and second probe thereby removing the first and second binding agents to form a cleaved ligation product;

(v) using the ligation product or the cleaved ligation product as a template for an amplification reaction; and

(vi) detecting a product form the amplification reaction, which, if present, is indicative of the presence and/or amount of the analyte in the sample.

29. A method for detecting the presence or amount of a first analyte and a second analyte in a sample, the method comprising:

(i) contacting the sample with a first probe comprising a first binding agent capable of binding the first analyte conjugated to a first oligonucleotide, a second probe comprising a second binding agent capable of binding the first analyte conjugated to a second oligonucleotide, a third probe comprising a third binding agent capable of binding the second analyte conjugated to a third oligonucleotide, and a fourth probe comprising a fourth binding agent capable of binding the second analyte conjugated to a fourth oligonucleotide, under conditions to permit the first probe and the second probe to bind the first analyte if the first analyte is present in the sample and to permit the third probe and the fourth probe to bind the second analyte if the second analyte is present in the sample, wherein the first, second, third, and fourth binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated to each respective binding agent via the UAA, and the first, second, third, and fourth oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region;

(ii) contacting the sample with a first lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe, and a second lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the third probe and (b) a second region that is capable of hybridizing to the complementary region of the fourth probe, under conditions to permit the first lock probe to simultaneously hybridize to the first and second probe to form a duplex if the first and second probe are in sufficient proximity to each other, and to permit the second lock probe to simultaneously hybridize to the third and fourth probe to form a duplex if the third and fourth probe are in sufficient proximity to each other;

(iii) ligating the first probe and the second probe to form a first ligation product and the third and fourth probe to form a second ligation product;

(iv) optionally cleaving the first and second ligation product with a cleavage agent capable of cleaving the cleavage site of the first, second, third, and fourth probes thereby removing the first, second, third, and fourth binding agents to form a first and second cleaved ligation product;

(v) using the first and second ligation product or cleaved ligation product as a template for an amplification reaction; and

(vi) detecting a product form the amplification reaction, which, if present, is indicative of the presence and/or amount of the first and second analyte in the sample.

30. A kit compri sing :

(i) a first probe comprising a first binding agent capable of binding an analyte conjugated to a first oligonucleotide, and a second probe comprising a second binding agent capable of binding the analyte conjugated to a second oligonucleotide, wherein the first binding agent and the second binding agent each comprise a protein sequence comprising an unnatural amino acid (UAA) and each oligonucleotide is conjugated to each respective binding agent via the UAA, and the first and second oligonucleotide each comprise (a) an optional cleavage site, (b) a spacer sequence, (c) a unique identifying sequence, and (d) a complementary region;

(ii) a lock probe comprising (a) a first region that is capable of hybridizing to the complementary region of the first probe and (b) a second region that is capable of hybridizing to the complementary region of the second probe; (iii) a DNA ligase; and

(iv) optionally, a cleavage agent capable of cleaving the optional cleavage site of the first and second probe

31. The kit of claim 29, further comprising: (v) a first PCR primer that binds to a first PCR primer binding site on the first probe;

(vi) a second PCR primer that binds to a second PCR primer binding site on the second probe.