Multiplexed colocalization-by-linkage assays for the detection and analysis of analytes

Abstract

Provided herein are colocalization-by-linkage assay (CLA) compositions and methods for multiplexed analysis of an analyte or analytes. The CLA compositions and methods, as described herein, are engineered to detect multiple analytes using multiple detection reagents coupled to a single support. Readout or detection sensitivity is achieved through the use of release-dependent transduction (RDT) or displacer-dependent transduction. Further multiplexing can also be achieved through the use of barcoded elements used in the capture and detection of an analytes.

1 . A method of detecting and/or quantifying an analyte in a sample, the method comprising:
 (a) contacting the sample with a complex comprising: (i) a support, (ii) a capture reagent releasably coupled to the support, (iii) a detection reagent releasably coupled to the support, wherein the capture reagent and detection reagents are configured to simultaneously bind to the analyte;   (b) decoupling the detection reagent from the support;   (c) decoupling the capture reagent from the support; and   (d) detecting one or both of the released detection reagent or capture reagent.   
     
     
         2 . The method of  claim 1 , wherein the support further comprises a first anchor element and second anchor element coupled thereto, and wherein the capture reagent is releasably coupled to the support via the first anchor element and the detection reagent is releasably coupled to the support via the second anchor element. 
     
     
         3 . The method of  claim 2 , wherein the capture reagent comprises a first hook element coupled thereto, and wherein the first hook element is releasably coupled to the first anchor element. 
     
     
         4 . The method of  claim 2 , wherein the detection reagent comprises a second hook element coupled thereto, and wherein the second hook element is releasably coupled to the second anchor element. 
     
     
         5 . The method of  claim 2 , wherein (1) the capture reagent comprises a first hook element coupled thereto, and wherein the first hook element is releasably coupled to the first anchor element, and (2) the detection reagent comprises a second hook element coupled thereto, and wherein the second hook element is releasably coupled to the second anchor element. 
     
     
         6 . The method of  claim 1 , wherein the support further comprises an anchor element coupled thereto, and wherein the capture reagent is releasably coupled to the support via the anchor element and the detection reagent is releasably coupled to the support via the anchor element. 
     
     
         7 . The method of  claim 6 , wherein (1) the capture reagent comprises a first hook element coupled thereto, and wherein the first hook element is releasably coupled to the anchor element, and (2) the detection reagent comprises a second hook element coupled thereto, and wherein the second hook element is releasably coupled to the anchor element. 
     
     
         8 . The method of any one of  claims 1  to  7 , wherein (b), decoupling comprises applying a stimulus that decouples the detection reagent from the support. 
     
     
         9 . The method of any one of  claims 1  to  7 , wherein (c), decoupling comprises applying a stimulus that decouples the capture reagent from the support. 
     
     
         10 . The method of any one of  claims 8  to  9 , wherein the stimulus is a thermal stimulus, photo-stimulus, chemical stimulus, a mechanical stimulus, a radiation stimulus, a biological stimulus, or any combination thereof. 
     
     
         11 . The method of any one of  claims 1  to  10 , wherein (b), decoupling comprises providing a displacer agent that decouples the detection reagent from the support. 
     
     
         12 . The method of any one of  claims 1  to  11 , wherein (c), decoupling comprises providing a displacer agent that decouples the capture reagent from the support. 
     
     
         13 . The method of any one of  claims 1  to  12 , wherein (d), detecting comprises identifying a nucleic acid molecule corresponding to the detection reagent or capture reagent. 
     
     
         14 . The method of  claim 13 , wherein identifying the nucleic acid molecule comprises performing a sequencing reaction, a PCR, a qPCR, or nucleic acid probe-based assay. 
     
     
         15 . The method of  claim 13 , wherein the nucleic acid molecule comprises a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         16 . The method of any one of  claims 1  to  15 , wherein (b), decoupling comprises providing a detectable displacer agent that decouples the detection reagent from the support. 
     
     
         17 . The method of  claim 16 , wherein the detectable displacer agent is an oligonucleotide. 
     
     
         18 . The method of  claim 17 , wherein the oligonucleotide comprises a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         19 . The method of any one of  claims 1  to  18 , wherein (c), decoupling comprises providing a detectable displacer agent that decouples the capture reagent from the support. 
     
     
         20 . The method of  claim 19 , wherein the detectable displacer agent is an oligonucleotide. 
     
     
         21 . The method of  claim 20 , wherein the oligonucleotide comprises a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         22 . The method of any one of  claims 16 - 21 , wherein (e), detecting comprises detecting the displacer agent. 
     
     
         23 . The method of any one of  claims 1 - 22 , wherein subsequent to (d), the method comprises capturing the detection reagent and/or the capture reagent on a second support. 
     
     
         24 . A method of processing an analyte for the detection and quantification of the analyte, the method comprising:
 (a) contacting a sample comprising the analyte with a complex comprising: (i) a support, (ii) a capture reagent attached to the support, (iii) a detection reagent attached to the support, thereby generating an analyte bound complex comprising the analyte coupled to the capture reagent and the detection reagent;   (b) decoupling the detection reagent from the support; and   (c) decoupling the capture reagent from the support,   
       wherein the detection reagent comprises a detectable element. 
     
     
         25 . The method of  claim 24 , wherein the detectable element is a nucleic acid sequence configured to be detected by a sequencing reaction, a nucleic acid amplification reaction, or couple to a labeled probe (e.g. a detectable displacer agent). 
     
     
         26 . The method of any one of  claims 24  to  25 , wherein the support further comprises a first anchor element and second anchor element coupled thereto, and wherein the capture reagent is releasably coupled to the support via the first anchor element and the detection reagent is releasably coupled to the support via the second anchor element. 
     
     
         27 . The method of  claim 26 , wherein the first anchor element comprises a first anchor oligonucleotide and the second anchor element comprise a second anchor oligonucleotide. 
     
     
         28 . The method of any one of  claims 26  to  27 , wherein the capture reagent comprises a first hook element coupled thereto, and wherein the first hook element is releasably coupled to the first anchor element. 
     
     
         29 . The method of any one of  claims 26  to  27 , wherein the detection reagent comprises a second hook element coupled thereto, and wherein the second hook element is releasably coupled to the second anchor element. 
     
     
         30 . The method of any one of  claims 26  to  27 , wherein (1) the capture reagent comprises a first hook element coupled thereto, and wherein the first hook element is releasably coupled to the first anchor element, and (2) the detection reagent comprises a second hook element coupled thereto, and wherein the second hook element is releasably coupled to the second anchor element. 
     
     
         31 . The method of  claim 24 , wherein the support further comprises an anchor element coupled thereto, and wherein the capture reagent is releasably coupled to the support via the anchor element and the detection reagent is releasably coupled to the support via the anchor element. 
     
     
         32 . The method of  claim 31 , wherein (1) the capture reagent comprises a first hook element coupled thereto, and wherein the first hook element is releasably coupled to the anchor element, and (2) the detection reagent comprises a second hook element coupled thereto, and wherein the second hook element is releasably coupled to the anchor element. 
     
     
         33 . The method of any one of  claims 28 - 32 , wherein the first hook element comprises a first hook oligonucleotide and the second hook element comprises a second hook oligonucleotide. 
     
     
         34 . The method of  claim 33 , wherein detectable element comprises a barcode sequence, a unique molecular identifier sequence, a primer binding sequence, a sequence configured to hybridize to a displacer oligonucleotide, or a combination thereof. 
     
     
         35 . The method of any one of  claims 24 - 34 , wherein the method further comprises (d) detecting the detection reagent. 
     
     
         36 . The method of  claim 35 , wherein the detectable element comprises a nucleic acid molecule, and in (d), detecting comprises identifying a nucleic acid molecule corresponding to the detection reagent. 
     
     
         37 . The method of  claim 36 , wherein identifying the nucleic acid molecule comprises performing a sequencing reaction, a PCR, a qPCR, or nucleic acid probe-based assay. 
     
     
         38 . The method of  claim 36 , wherein the nucleic acid molecule comprises a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         39 . The method of any one of  claims 24  to  38 , wherein (b), decoupling comprises providing a displacer agent that decouples the detection reagent from the support. 
     
     
         40 . The method of  claim 39 , wherein the displacer agent is an oligonucleotide. 
     
     
         41 . The method of  claim 40 , wherein the oligonucleotide comprises a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         42 . The method of any one of  claims 24  to  41 , wherein (b) or (c), decoupling comprises providing a detectable displacer agent that decouples the capture reagent or detection reagent from the support. 
     
     
         43 . The method of  claim 42 , wherein the detectable displacer agent is an oligonucleotide. 
     
     
         44 . The method of  claim 43 , wherein the oligonucleotide comprises a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         45 . The method of any one of  claims 1 - 44 , wherein subsequent to (d), the method comprises capturing the detection reagent and/or the capture reagent on a second support. 
     
     
         46 . A method of detecting and/or quantifying an analyte in a sample, the method comprising:
 (a) contacting the sample with a complex comprising: (i) a support, and (ii) a capture reagent releasably coupled to the support;   (b) providing a detection reagent, wherein the capture reagent and detection reagent are configured to simultaneously bind to the analyte;   (b) decoupling the detection reagent from the support;   (c) decoupling the capture reagent from the support; and   (d) detecting one or both of the released detection reagent or capture reagent.   
     
     
         47 . A method of processing an analyte for the detection and quantification of the analyte, the method comprising:
 (a) contacting a sample comprising the analyte with a complex comprising: (i) a support, and (ii) a capture reagent attached to the support, thereby generating the analyte coupled to the capture reagent;   (b) contacting the analyte bound complex with a detection reagent configured to couple to the analyte and couple to the support, thereby generating an analyte bound complex comprising the analyte coupled to the capture reagent and the detection reagent; and   (c) decoupling at least one of the capture reagent and the detection reagent from the support,   
       wherein at least one of the detection reagent comprises a detectable element. 
     
     
         48 . The method of  claim 47 , wherein at least one of the capture reagent and the detection reagent are configured to be detected by a sequencing reaction, a nucleic acid amplification reaction (e.g. PCR), or coupling to labeling agent (e.g. a displacer reagent). 
     
     
         49 . The method of any one of  claims 47  to  48 , wherein the method further comprises (d) detecting at least one of the capture reagent and the detection reagent. 
     
     
         50 . The method of any one of  claims 46  to  49 , wherein the support further comprises a first anchor element and second anchor element coupled thereto, and wherein the capture reagent is releasably coupled to the support via the first anchor element and the detection reagent is releasably coupled to the support via the second anchor element. 
     
     
         51 . The method of  claim 50 , wherein the first anchor element comprises a first anchor oligonucleotide and the second anchor element comprise a second anchor oligonucleotide. 
     
     
         52 . The method of any one of  claims 46  to  51 , wherein the capture reagent comprises a first hook element coupled thereto, and wherein the first hook element is releasably coupled to the first anchor element. 
     
     
         53 . The method of any one of  claims 46  to  52 , wherein the detection reagent comprises a second hook element coupled thereto, and wherein the second hook element is configured to releasably couple to the second anchor element. 
     
     
         54 . The method of any one of  claims 46  to  53 , wherein (i) the capture reagent comprises a first hook element coupled thereto, and wherein the first hook element is releasably coupled to the first anchor element, and (ii) the detection reagent comprises a second hook element coupled thereto, and wherein the second hook element is configured to releasably couple to the second anchor element. 
     
     
         55 . The method of any one of  claims 46  to  51 , wherein the support further comprises an anchor element coupled thereto, and wherein the capture reagent is releasably coupled to the support via the anchor element is releasably coupled to the support via the anchor element and the detection reagent is configured to releasably couple to the support via the anchor element. 
     
     
         56 . The method of  claim 55 , wherein (i) the capture reagent comprises a first hook element coupled thereto, and wherein the first hook element is releasably coupled to the anchor element, and (ii) the detection reagent comprises a second hook element coupled thereto, and wherein the second hook element is releasably coupled to the anchor element. 
     
     
         57 . The method of any one of  claims 50 - 56 , wherein the first hook element comprises a first hook oligonucleotide and the second hook element comprises a second hook oligonucleotide. 
     
     
         58 . The method of  claim 57 , wherein one or both of the first hook oligonucleotide and the second hook oligonucleotide comprise a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         59 . The method of any one of  claims 46  to  58 , wherein (b) or (c), decoupling comprises providing a detectable displacer agent that decouples the detection reagent from the support. 
     
     
         60 . The method of  claim 59 , wherein the detectable displacer agent is an oligonucleotide. 
     
     
         61 . The method of  claim 60 , wherein the oligonucleotide comprises a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         62 . The method of any one of  claims 46  to  58 , wherein (b) or (c), decoupling comprises providing a detectable displacer agent that decouples the capture reagent from the support. 
     
     
         63 . The method of  claim 62 , wherein the detectable displacer agent is an oligonucleotide. 
     
     
         64 . The method of  claim 63 , wherein the oligonucleotide comprises a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         65 . The method of any one of  claims 46 - 64 , wherein (i) the analyte is an antibody molecule, (ii) the capture reagent is an antigen, and (iii) the detection reagent is specific for an immunoglobulin class IgG, IgM, IgA, IgD, or IgE. 
     
     
         66 . The method of  claim 65 , wherein detection reagent comprises an anti-IgG, -IgM, -IgA, -IgD, or -IgE antibody. 
     
     
         67 . The method of  claim 65 , wherein detection reagent comprises a protein A, protein G, or protein M. 
     
     
         68 . The method of any one of  claims 46 - 64 , wherein (i) the analyte is protein comprising posttranslational modification, (ii) the capture reagent binds the protein, and (iii) the detection reagent is specific for the posttranslational modification or the protein comprising the posttranslation modification. 
     
     
         69 . A method of detecting analytes in a sample, the method comprising:
 (a) contacting the sample with a the complex comprising: (i) a support, (ii) a first capture reagent and a first detection reagent coupled to the support, wherein the first capture reagent and first detection reagent are configured to simultaneously couple to a first analyte in said sample, and (iii) a second capture reagent and a second detection reagent coupled to the support, wherein the second capture reagent and second detection reagent are configured to simultaneously couple to the second analyte;   (b) providing a first displacer reagent configured to decouple the first detection reagent and/or second detection reagent from the support;   (c) providing a second displacer reagent configured to decouple the first capture reagent and/or second capture reagent from the support; and   (d) detecting at least one of the (i) decoupled first capture reagent and decoupled first detection reagent, and/or (ii) decoupled second capture reagent and decoupled second detection reagent.   
     
     
         70 . A method of processing an analyte for the detection and quantification of the analyte, the method comprising:
 (a) contacting the sample with a the complex comprising: (i) a support, (ii) a first capture reagent and a first detection reagent coupled to the support, wherein the first capture reagent and first detection reagent are configured to simultaneously couple to a first analyte in said sample, and (iii) a second capture reagent and a second detection reagent coupled to the support, wherein the second capture reagent and second detection reagent are configured to simultaneously couple to the second analyte;   (b) contacting the analyte bound complex with a detection reagent configured to couple to the analyte and couple to the support, thereby generating an analyte bound complex comprising the analyte coupled to the capture reagent and the detection reagent; and   (c) decoupling at least one of the capture reagent and the detection reagent from the support; and   
       wherein at least one of (i) the first capture reagent and second capture reagent, and/or (ii) the first detection reagent and second detection reagent comprise a detectable element. 
     
     
         71 . The method of  claim 70 , wherein at least one of (i) the first capture reagent and second capture reagent, and/or (ii) the first detection reagent and second detection reagent are configured to be detected by a sequencing reaction, a nucleic acid amplification reaction (e.g. PCR), or coupling to labeling agent (e.g. a displacer reagent). 
     
     
         72 . The method of any one of  claims 70  to  71 , wherein the method further comprises (d) detecting at least one of the (i) decoupled first capture reagent and decoupled first detection reagent, and/or (ii) decoupled second capture reagent and decoupled second detection reagent. 
     
     
         73 . The method of any one of  claims 69  to  72 , wherein the support further comprises a first anchor element, second anchor element, a third anchor element, and a fourth anchor element coupled thereto, and wherein (i) the first capture reagent is releasably coupled to the support via the first anchor element and the first detection reagent is releasably coupled to the support via the second anchor element, and (ii) the second capture reagent is releasably coupled to the support via the third anchor element and the second detection reagent is releasably coupled to the support via the fourth anchor element. 
     
     
         74 . The method of  claim 73 , wherein the first anchor element comprises a first anchor oligonucleotide, the second anchor element comprise a second anchor oligonucleotide, the second anchor element comprise a second anchor oligonucleotide. 
     
     
         75 . The method of any one of  claims 69  to  74 , wherein (i) the first capture reagent comprises a first hook element coupled thereto and the second capture reagent comprises a third hook element coupled thereto, and (ii) the first hook element is releasably coupled to the first anchor element and the second hook element is releasably coupled to the third anchor element. 
     
     
         76 . The method of any one of  claims 69  to  75 , wherein (i) the first detection reagent comprises a second hook element coupled thereto and the fourth detection hook element coupled thereto, and (ii) the second hook element is configured to releasably couple to the second anchor element and the fourth hook element is configured to releasably couple to the fourth anchor element. 
     
     
         77 . The method of any one of  claims 69  to  76 , wherein (i) the first capture reagent comprises a first hook element coupled thereto and the second capture reagent comprises a third hook element coupled thereto, and (ii) the first hook element is releasably coupled to the first anchor element and the second hook element is releasably coupled to the third anchor element; and
 wherein (iii) the first detection reagent comprises a second hook element coupled thereto and the fourth detection hook element coupled thereto, and (iv) the second hook element is configured to releasably couple to the second anchor element and the fourth hook element is configured to releasably couple to the fourth anchor element. 
 
     
     
         78 . The method of any one of  claims 69  to  74 , wherein the support further comprises a first anchor element coupled thereto and a second anchor element coupled thereto, and wherein (i) the first capture reagent is releasably coupled to the support via the first anchor element and the first detection reagent is configured to releasably couple to the support via the first anchor element, and (ii) the second capture reagent is releasably coupled to the support via the second anchor element and the second detection reagent is configured to releasably couple to the support via the second anchor element. 
     
     
         79 . The method of any one of  claims 73 - 78 , wherein the first hook element comprises a first hook oligonucleotide, the second hook element comprises a second hook oligonucleotide, the third hook element comprises a third hook oligonucleotide, and/or fourth hook element comprises a fourth hook oligonucleotide. 
     
     
         80 . The method of  claim 79 , at least one of the first hook oligonucleotide, the second hook oligonucleotide, the third hook oligonucleotide, and the fourth hook oligonucleotide comprise a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         81 . The method of any one of  claims 69  to  80 , wherein (b) or (c), decoupling comprises providing a displacer agent that decouples the capture reagent from the support. 
     
     
         82 . The method of  claim 81 , wherein the detectable displacer agent is an oligonucleotide. 
     
     
         83 . The method of  claim 82 , wherein the oligonucleotide is configured to be detected. 
     
     
         84 . The method of  claim 83 , wherein the oligonucleotide comprises a barcode sequence, unique molecular identifier sequence, a primer binding sequence, or a combination thereof. 
     
     
         85 . The method of  claim 83 , wherein the oligonucleotide comprises a fluorescent label. 
     
     
         86 . A co-localization by linkage assay composition, the composition comprising:
 a complex comprising (i) a support, (ii) a capture reagent releasably coupled to the support, (iii) a detection reagent releasably coupled to the support, wherein the capture reagent and detection reagent are configured to simultaneously bind to the analyte.   
     
     
         87 . The co-localization by linkage assay composition of  claim 86  for use in any one of the methods of  claims 1  to  45  or  69  to  85 . 
     
     
         88 . Use of a co-localization by linkage assay composition of  claim 86  in any one of the methods of  claims 1  to  45  or  69  to  85 , wherein the co-localization by linkage assay composition comprises a complex comprising (i) a support, (ii) a capture reagent releasably coupled to the support, (iii) a detection reagent releasably coupled to the support, wherein the capture reagent and detection reagent are configured to simultaneously bind to the analyte. 
     
     
         89 . A co-localization by linkage assay composition, the composition comprising a complex comprising: (i) a support, (ii) a first capture reagent and a first detection reagent coupled to the support, wherein the first capture reagent and first detection reagent are configured to simultaneously couple to a first analyte in said sample, and (iii) a second capture reagent and a second detection reagent coupled to the support, wherein the second capture reagent and second detection reagent are configured to simultaneously couple to the second analyte. 
     
     
         90 . The co-localization by linkage assay composition of  claim 89  for use in any one of the methods of  claims 1  to  45  or  69  to  85 . 
     
     
         91 . Use of a co-localization by linkage assay composition of  claim 89  in any one of the methods of  claims 1  to  45  or  69  to  85 , wherein the co-localization by linkage assay composition comprises a complex comprising (i) a support, (ii) a capture reagent releasably coupled to the support, (iii) a detection reagent releasably coupled to the support, wherein the capture reagent and detection reagent are configured to simultaneously bind to the analyte. 
     
     
         92 . The method of any one of  claims 1  to  85  or the compositions of any one of  claims 86  to  91 , where in the sample is a bodily fluid, an extract, a solution containing proteins and/or DNA, a cell extract, a cell lysate, a single cell lysate, or a tissue lysate.