NUCLEIC ACID-TAGGED COMPOSITIONS AND METHODS FOR MULTIPLEXED PROTEIN-PROTEIN INTERACTION PROFILING
First Claim
1. A multiplexed polypeptide affinity assay (MPA) composition comprising a population of prey polypeptide targets (PPTs), whereineach PPT in the population is chemically linked to a prey nucleic acid, wherein the prey nucleic acid is an RNA encoding the amino acid sequence of the chemically linked PPT,a largest difference between a first PPT representation in the library and a second PPT representation in the library is no greater than ten-fold;
- andthe PPT population comprises fusion polypeptides comprising the amino acid sequence of a haloalkane dehalogenase tag polypeptide fused at the N-terminus or C-terminus of the fusion polypeptides, and wherein the prey nucleic acid is chemically linked to a ligand that reacts specifically with and becomes covalently linked to the haloalkane dehalogenase tag polypeptide.
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Abstract
Methods and compositions for multiplexed protein-protein interaction profiling (e.g., immunoprofiling), based on nucleic acid tagging of polypeptides (e.g., by RNA display) are described. In some embodiments the described compositions and methods utilize a library of prey polypeptide targets linked to prey RNAs encoding them, and a population of bait polypeptides, e.g., a mixture of antibodies, that bind to one or more of the prey polypeptide targets and are used to isolate and identify the bound prey polypeptide targets by amplification of their associated prey RNAs and sequencing of the corresponding cDNAs. In other embodiments the prey polypeptide targets are linked to DNA Bar Codes, which serve as unique identifiers of the tagged polypeptide.
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Citations
43 Claims
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1. A multiplexed polypeptide affinity assay (MPA) composition comprising a population of prey polypeptide targets (PPTs), wherein
each PPT in the population is chemically linked to a prey nucleic acid, wherein the prey nucleic acid is an RNA encoding the amino acid sequence of the chemically linked PPT, a largest difference between a first PPT representation in the library and a second PPT representation in the library is no greater than ten-fold; - and
the PPT population comprises fusion polypeptides comprising the amino acid sequence of a haloalkane dehalogenase tag polypeptide fused at the N-terminus or C-terminus of the fusion polypeptides, and wherein the prey nucleic acid is chemically linked to a ligand that reacts specifically with and becomes covalently linked to the haloalkane dehalogenase tag polypeptide. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18)
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2. (canceled)
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13. (canceled)
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19. A kit, comprising:
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(i) a PPT library, wherein each PPT in the library is chemically linked to a prey nucleic acid, and wherein the relative abundance of PPTs in the population falls within about a ten fold range; and (ii) forward and reverse oligonucleotide primers to amplify the prey nucleic acid. - View Dependent Claims (20, 21, 23, 25)
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22. (canceled)
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24. (canceled)
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26-31. -31. (canceled)
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32. A method for antigen immunoprofiling a biological sample, comprising:
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(i) providing a biological sample, to be immunoprofiled, comprising a plurality of antibodies with diverse antigen specificities; (ii) contacting the biological sample with a population of PPTs, wherein each PPT in the population is chemically linked to a prey nucleic acid, and wherein the contacting conditions permit binding of one or more of the antibodies in the plurality of antibodies in the biological sample to one or more of the PPTs in the population of PPTs to obtain one or more antibody-PPT complexes; (iii) immunoprecipitating the one or more antibody-PPT complexes; (iv) amplifying prey nucleic acid sequences associated with the one or more immunoprecipitated antibody-PPT complexes to obtain a plurality of cDNAs comprising nucleic acid sequences corresponding to the amplified prey nucleic acid sequences; and (v) sequencing the plurality of cDNAs to obtain an antigen immunoprofile of the biological sample.
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33-41. -41. (canceled)
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42. A method for identifying a disease-associated antigen, comprising:
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(i) providing a first biological sample, comprising a plurality of antibodies from one or more control subjects; (ii) contacting the first biological sample with a population of PPTs, wherein each PPT in the population of PPTs is chemically linked to a prey nucleic acid, and wherein the contacting conditions permit binding of one or more of the PPTs with one or more of the antibodies in the plurality of antibodies from the one or more control subjects to obtain one or more control subject antibody-PPT complexes; (iii) immunoprecipitating the one or more control antibody-PPT complexes to obtain a counter-selected PPT population comprising a population of PPTs depleted of PPTS recognized by antibodies present in the first biological sample; (iv) contacting the counter-selected PPT population with a second biological sample comprising a plurality of antibodies from one or more patients suffering from the same health condition or diagnosed as being at risk of suffering from the same health condition to obtain one or more patient antibody-PPT complexes; (v) immunoprecipitating the one or more patient antibody-PPT complexes; (vi) amplifying prey nucleic acid sequences associated with the one or more immunoprecipitated patient antibody-PPT complexes to obtain a plurality of disease antigen-associated cDNAs comprising nucleic acid sequences corresponding to the amplified prey nucleic acid sequences; and (vii) sequencing the plurality of cDNAs to identify the disease-associated antigen.
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43-48. -48. (canceled)
Specification