Methods and kits using nucleic acid encoding and/or label

Abstract

Methods and Kits for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding are disclosed. The sample analysis kits employ nucleic acid encoding and/or nucleic acid recording of a molecular interaction and/or reaction, such as recognition events (e.g., between an antigen and an antibody, between a modified terminal amino acid residue, or between a small molecule or peptide therapeutic and a target, etc.). Assays that do not require the cyclic transfer of information between a coding tag and a recording tag are also disclosed, including single cycle assays.

1 . A method, comprising:
 (a) contacting:
 (i) a set of biological targets (e.g., proteins or polypeptides), wherein each biological target is associated directly or indirectly with a recording tag, which optionally comprises an encoding barcode that identifies the biological target, with 
 (ii) a library of agents, wherein each agent is immobilized on at least one separate support, and each separate support further comprises a coding tag comprising identifying information regarding the agent immobilized on the support; 
   (b) allowing transfer of information between:
 (i) the recording tag associated with each biological target that binds (directly or indirectly) and/or reacts (directly or indirectly) with one or more of the agents, and 
 (ii) the coding tag of the one or more agents, 
 wherein the transfer of information generates an extended recording tag and/or an extended coding tag; and 
   (c) analyzing the extended recording tag and/or the extended coding tag,
 thereby assaying the interaction(s) between the set of biological targets and the library of agents. 
   
     
     
         2 . The method of  claim 1 , wherein on each separate support, multiple molecules of the same agent and/or multiple molecules of the same coding tags are immobilized. 
     
     
         3 - 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the library of agents comprises a small molecule, a peptide or peptide mimetic, a peptidomimetic (e.g., a peptoid, a β-peptide, or a D-peptide peptidomimetic), a polysaccharide, or an aptamer (e.g., a nucleic acid aptamer, such as a DNA aptamer, or a peptide aptamer), or any combination thereof. 
     
     
         8 . The method of  claim 7 , wherein the agents are synthesized combinatorially on the support as one bead one compound. 
     
     
         9 . The method of  claim 7 , wherein the library of agents comprises individually produced protein targets or individually produced small molecule/peptide targets. 
     
     
         10 . The method of  claim 1 , wherein information is transferred from:
 at least one coding tag to at least one recording tag, thereby generating at least one extended recording tag; or   at least one recording tag to at least one coding tag, thereby generating at least one extended coding tag.   
     
     
         11 - 16 . (canceled) 
     
     
         17 . The method of  claim 1 , wherein the transfer of information is accomplished by ligation, a polymerase-mediated reaction, or any combination thereof. 
     
     
         18 . The method of  claim 1 , wherein the set of biological targets comprises a proteome or subset thereof. 
     
     
         19 . The method of  claim 18 , wherein the subset of the proteome comprises a kinome; a secretome; a receptome (e.g., GPCRome); an immunoproteome; a nutriproteome; a proteome subset defined by a post-translational modification (e.g., phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, lipidation, and/or nitrosylation), such as a phosphoproteome (e.g., phosphotyrosine-proteome, tyrosine-kinome, and tyrosine-phosphatome), a glycoproteome, etc.; a proteome subset associated with a tissue or organ, a developmental stage, or a physiological or pathological condition; a proteome subset associated a cellular process, such as cell cycle, differentiation (or de-differentiation), cell death, senescence, cell migration, transformation, or metastasis; or any combination thereof. 
     
     
         20 . The method of  claim 19 , wherein the method is for analysis of a molecular interaction between an antigen and an antibody. 
     
     
         21 . The method of  claim 20 , wherein the antibody comprises immunoglobulin A, immunoglobulin G, immunoglobulin D, immunoglobulin E, immunoglobulin M, or any immunoreactive component(s) of an antibody molecule. 
     
     
         22 . The method of  claim 1 , wherein the recording tag and/or the coding tag comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, an RNA molecule, a BNA molecule, an XNA molecule, a LNA molecule, a PNA molecule, a γPNA molecule, or a morpholino, or a combination thereof. 
     
     
         23 . The method of  claim 22 , wherein the recording tag comprises:
 a universal priming site;   a unique molecule identifier (UMI);   a barcode, such as an encoding barcode that identifies the biological target, a sample barcode, a compartment barcode, a partition barcode, an error correction barcode, or any combination thereof; and/or   an optional spacer at its 3′-terminus.   
     
     
         24 - 30 . (canceled) 
     
     
         31 . The method of  claim 1 , which is for parallel analysis of the interaction between the set of biological targets and the library of agents, in order to create a biological target-agent binding matrix. 
     
     
         32 . The method of  claim 1 , wherein the attachment of the recording tag to each biological target and/or the attachment of the coding tag to each agent occurs via ribosome or mRNA/cDNA display. 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 22 , wherein the coding tag comprises:
 an encoder sequence that identifies the agent; and/or   a spacer, a unique molecular identifier (UMI), a universal priming site, or any combination thereof.   
     
     
         35 - 38 . (canceled) 
     
     
         39 . A method, comprising:
 (a) contacting:
 (i) a set of biological targets (e.g., proteins or polypeptides) immobilized on a support, wherein each biological target is associated directly or indirectly with a recording tag, which optionally comprises an encoding barcode that identifies the biological target, with 
 (ii) a library of agents, wherein each agent is associated directly or indirectly with a coding tag comprising identifying information regarding the agent; 
   (b) allowing transfer of information between:
 (i) the recording tag associated with each biological target that binds (directly or indirectly) and/or reacts (directly or indirectly) with one or more of the agents, and 
 (ii) the coding tag of the one or more agents, 
 wherein the transfer of information generates an extended recording tag and/or an extended coding tag; and 
   (c) analyzing the extended recording tag and/or the extended coding tag,   thereby assaying the interaction(s) between the set of biological targets and the library of agents.   
     
     
         40 . The method of  claim 39 , wherein on each separate support, multiple molecules of the same biological target and/or multiple molecules of the same recording tags are immobilized. 
     
     
         41 . The method of  claim 39 , wherein the biological targets are obtained from a biological sample derived from the same sample or different samples, and/or obtained from the same subject or different subjects. 
     
     
         42 . The method of  claim 41 , wherein the biological targets on each separate support share the same barcode. 
     
     
         43 . The method of  claim 41 , wherein the biological targets from different samples are pooled and immobilized on the support. 
     
     
         44 . The method of  claim 41 , wherein the biological targets obtained from the same sample share the same sample barcode. 
     
     
         45 . The method of  claim 39 , wherein the library of agents comprises a small molecule, a peptide or peptide mimetic, a peptidomimetic (e.g., a peptoid, a β-peptide, or a D-peptide peptidomimetic), a polysaccharide, or an aptamer (e.g., a nucleic acid aptamer, such as a DNA aptamer, or a peptide aptamer), or any combination thereof. 
     
     
         46 - 47 . (canceled) 
     
     
         48 . The method of  claim 39 , wherein information is transferred:
 from at least one coding tag to at least one recording tag, thereby generating at least one extended recording tag, or   from at least one recording tag to at least one coding tag, thereby generating at least one extended coding tag.   
     
     
         49 - 54 . (canceled) 
     
     
         55 . The method of  claim 39 , wherein the transfer of information is accomplished by ligation, or any combination thereof. 
     
     
         56 . The method of  claim 39 , wherein the set of biological targets comprises a proteome or subset thereof. 
     
     
         57 . The method of  claim 56 , wherein the subset of the proteome comprises a kinome; a secretome; a receptome (e.g., GPCRome); an immunoproteome; a nutriproteome; a proteome subset defined by a post-translational modification (e.g., phosphorylation, ubiquitination, methylation, acetylation, glycosylation, oxidation, lipidation, and/or nitrosylation), such as a phosphoproteome (e.g., phosphotyrosine-proteome, tyrosine-kinome, and tyrosine-phosphatome), a glycoproteome, etc.; a proteome subset associated with a tissue or organ, a developmental stage, or a physiological or pathological condition; a proteome subset associated a cellular process, such as cell cycle, differentiation (or de-differentiation), cell death, senescence, cell migration, transformation, or metastasis; or any combination thereof. 
     
     
         58 . The method of  claim 39 , wherein the method is for analysis of a molecular interaction between an antigen and an antibody. 
     
     
         59 . The method of  claim 58 , wherein the antibody comprises immunoglobulin A, immunoglobulin G, immunoglobulin D, immunoglobulin E, immunoglobulin M, or any immunoreactive component(s) of an antibody molecule. 
     
     
         60 . The method of  claim 39 , wherein the recording tag and/or the coding tag comprises a nucleic acid, an oligonucleotide, a modified oligonucleotide, a DNA molecule, a DNA with pseudo-complementary bases, an RNA molecule, a BNA molecule, an XNA molecule, a LNA molecule, a PNA molecule, a γPNA molecule, or a morpholino, or a combination thereof. 
     
     
         61 . The method of  claim 60 , wherein the recording tag comprises:
 a universal priming site;   a priming site for amplification, sequencing, or both, for example, the universal priming site comprises a priming site for amplification, sequencing, or both;   a unique molecule identifier (UMI);   a barcode, such as an encoding barcode that identifies the biological target, a sample barcode, a compartment barcode, a partition barcode, an error correction barcode, or any combination thereof; and/or
 an optional spacer at its 3′-terminus. 
   
     
     
         62 - 68 . (canceled) 
     
     
         69 . The method of  claim 39 , which is for parallel analysis of the interaction between the set of biological targets and the library of agents, in order to create a biological target-agent binding matrix. 
     
     
         70 - 71 . (canceled) 
     
     
         72 . The method of  claim 60 , wherein the coding tag comprises:
 an encoder sequence that identifies the agent; and/or   a spacer, a unique molecular identifier (UMI), a universal priming site, or any combination thereof.   
     
     
         73 - 79 . (canceled) 
     
     
         80 . A kit, comprising any molecule, molecular complex or conjugate, reagent (e.g., chemical or biological), agent, structure (e.g., support, surface, particle, or bead), reaction intermediate, reaction product, binding complex, or any other article of manufacture disclosed and/or used in the method of  claim 1 , or any combination thereof. 
     
     
         81 . The method of  claim 1 , wherein the biological targets are obtained from a biological sample derived from the same sample or different samples, and/or obtained from the same subject or different subjects. 
     
     
         82 . The method of  claim 1 , wherein the encoding barcode comprises a sample barcode to distinguish biological targets from different samples. 
     
     
         83 . The method of  claim 23 , further comprising pooling the biological targets associated with recording tags comprising the sample barcodes. 
     
     
         84 . The method of  claim 39 , wherein the encoding barcode comprises a sample barcode to distinguish biological targets from different samples. 
     
     
         85 . The method of  claim 39 , wherein the attachment of the recording tag to each biological target and/or the attachment of the coding tag to each agent occurs via ribosome or mRNA/cDNA display. 
     
     
         86 . A kit, comprising any molecule, molecular complex or conjugate, reagent (e.g., chemical or biological), agent, structure (e.g., support, surface, particle, or bead), reaction intermediate, reaction product, binding complex, or any other article of manufacture disclosed and/or used in the method of  claim 39 , or any combination thereof.