NUCLEIC ACID-TAGGED COMPOSITIONS AND METHODS FOR MULTIPLEXED PROTEIN-PROTEIN INTERACTION PROFILING

• US 20180201923A1
• Filed: 02/26/2018
• Published: 07/19/2018
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: LaBaer, Joshua
• Granted Patent: US 10,717,977 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6854 Immunoglobulins

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.

Claims

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.

3. The MPA composition of claim 1, wherein the RNA comprises one or more modified ribonucleotides.

4. The MPA composition of claim 1, wherein the prey nucleic acid comprises a DNA bar code.

5. The MPA composition of claim 1, wherein 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 polypepide.

6. The MPA composition of claim 1, wherein the PPT population comprises a plurality of amino acid sequences from a plurality of pathogens.

7. The MPA composition of claim 6, wherein the plurality of amino acid sequences comprises amino acid sequences of viral pathogens, bacterial pathogens, eukaryotic pathogens, or a combination thereof.

8. The MPA composition of claim 1, wherein the PPT population comprises a plurality of amino acid sequences from a plurality of antigens associated with one or more autoimmune diseases.

9. The MPA composition of claim 1, wherein the PPT population comprises a plurality of polypeptides comprising random amino acid sequences.

10. The MPA composition of claim 1, further comprising a population of bait polypeptides comprising diverse amino acid sequences, wherein at least one of the bait polypeptides binds to at least one of the PPTs.

11. The MPA composition of claim 10, wherein the population of bait polypeptides comprises a plurality of antibodies with diverse antigen specificities.

12. The MPA composition of claim 11, wherein the population of bait polypeptides comprises serum.

14. The MPA composition of claim 11, further comprising a polypeptide that binds specifically to the Fc region of an immunoglobulin.

15. The MPA composition of claim 14, wherein the polypeptide that binds specifically to the Fc region of an immunoglobulin is Protein G, Protein A, Protein A/G, or an antibody that binds specifically to the Fc region of an immunoglobulin.

16. The MPA composition of claim 11, wherein the plurality of antibodies are biotinylated antibodies.

17. The MPA composition of claim 10, wherein the population of bait polypeptides comprises diverse amino acid sequences from at least one of transcription factors, G-proteins, receptors, protein kinases, protein phosphatases, proteases, or a combination thereof.

18. The MPA composition of claim 17, wherein the population of bait polypeptides consists essentially of a population of diverse amino acid sequences from one or more of G-proteins, receptors, protein kinases, protein phosphatases, proteases, or a combination thereof.

2. (canceled)

13. (canceled)

19. A kit, comprising:

(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.

20. The kit of claim 19, wherein the PPT library 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 polypepide.

21. The kit of claim 19, wherein the prey nucleic acid comprises a DNA bar code.

23. The kit of claim 19, wherein the PPT library comprises a plurality of amino acid sequences from a plurality of pathogens.

25. The kit of claim 19, wherein the PPT library comprises a plurality of amino acid sequences from a plurality of antigens associated with autoimmune diseases.

22. (canceled)

24. (canceled)

26-31. -31. (canceled)

32. A method for antigen immunoprofiling a biological sample, comprising:

(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.

33-41. -41. (canceled)

42. A method for identifying a disease-associated antigen, comprising:

(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.

43-48. -48. (canceled)