METHODS AND COMPOSITIONS FOR RAPID FUNCTIONAL ANALYSIS OF GENE VARIANTS

20220357319

17/871402

July 22, 2022

Methods and compositions are disclosed for rapid functional analysis of gene variants based on analysis of protein-protein and protein-nucleic acid interactions.

ALBERT EINSTEIN COLLEGE OF MEDICINE, INC. (Bronx, NY, US)

1. A method of multiplex detecting a first protein-second protein interaction, in a sample, for up to at least four distinct first proteins, first proteins A, B, C and D respectively, the method comprising: contacting the sample with a (i) a first agent attached to a surface of a magnetic bead that is not labeled with a first primary optically-active label, and (ii) a second primary agent attached to a surface of a magnetic bead that is labeled with a first primary optically-active label, and (iii) a third primary agent attached to a surface of a non-magnetic bead that is not labeled with a second primary optically-active label, and (iv) a fourth primary agent attached to the surface of a non-magnetic bead that is labeled with a second primary optically-active label, wherein the first, second, third and fourth primary agents are different agents each capable of capturing the distinct first proteins A, B, C and D, respectively; contacting captured first protein-second protein complex(es) with a plurality of secondary agents, each of the plurality being specific for a distinct second protein, and each labeled with a separate secondary optically-active label wherein the secondary optically-active labels are not the same as the primary optically-active labels of the primary agents and are each distinct from the secondary optically-active label of every other of the optically-active labeled secondary agents; recovering magnetic beads complexes from the sample by applying a magnetic field; recovering non-magnetic bead complexes from the sample based on a non-magnetic physical property of the non-magnetic beads; passing the recovered magnetic bead complexes through a flow cytometer or optical plate reader; passing the recovered non-magnetic bead complexes through a flow cytometer or optical plate reader; detecting the optical signal(s) of the recovered magnetic bead complexes; and detecting the optical signal(s) of the recovered non-magnetic bead complexes; wherein the presence on a magnetic bead complex of only a secondary optically-active label indicates the interaction between the first protein A and a second protein corresponding to the secondary optically-active labeled secondary agent, and wherein the presence on a magnetic bead complex of both (i) a first primary optically-active label and (ii) a secondary optically-active label indicates the interaction of the first protein B and a second protein corresponding to the secondary optically-active labeled secondary agent, and wherein the presence on a non-magnetic bead complex of only a secondary optically-active label indicates the interaction of the first protein C and a second protein corresponding to the secondary optically-active secondary labeled agent, and wherein the presence on a non-magnetic bead complex of both (i) a second primary optically-active label and (ii) a secondary optically-active label indicates the interaction of the first protein D and a second protein corresponding to the secondary optically-active labeled secondary agent.

2. A method of multiplex detecting protein-nucleic acid interactions in a sample for up to at least four distinct proteins, proteins A, B, C and D respectively, the method comprising: a) contacting the sample with a (i) a first agent attached to a surface of a magnetic bead that is not labeled with a first primary optically-active label, and (ii) a second primary agent attached to a surface of a magnetic bead that is labeled with a first primary optically-active label, and (iii) a third primary agent attached to a surface of a non-magnetic bead that is not labeled with a second primary optically-active label, and (iv) a fourth primary agent attached to the surface of a non-magnetic bead that is labeled with a second primary optically-active label, wherein the first, second, third and fourth primary agents are different agents each capable of capturing the distinct proteins A, B, C and D, respectively, under conditions which permit capturing to the primary agents a first protein-nucleic acid complex from the sample; b) recovering magnetic beads complexes from the sample by applying a magnetic field and recovering non-magnetic bead complexes from the sample based on a non-magnetic physical property of the non-magnetic beads; c) contacting one or more of (i) the magnetic bead complexes not having a first primary optically-active label; (ii) the magnetic bead complexes having a first primary optically-active label; (iii) the non-magnetic bead complexes not having a first primary optically-active label; (iv) the non-magnetic bead complexes having a first primary optically-active label, with a Proteinase K so as to digest the proteins thereon and release any nucleic acids bound thereto; d) sequencing nucleic acid(s) released in step c)(i) so as to thereby identify the nucleic acids that have interacted with distinct protein A; in step c)(ii) so as to thereby identify the nucleic acids that have interacted with distinct protein B; in step c)(iii) so as to thereby identify the nucleic acids that have interacted with distinct protein C; and/or in step c)(iv) so as to thereby identify the nucleic acids that have interacted with distinct protein D.

3. The method of claim 2, further comprising probing the protein-nucleic acid complex(es) with one or more optically active secondary agents each specific for one of distinct proteins A, B, C and D, so as to identify bead complexes comprising a bead, a distinct protein and a primary agent, and recovering such bead complexes.

4. The method of claim 3, further comprising after step c) and before step d) passing the recovered magnetic bead complexes through a flow cytometer or optical plate reader and passing the recovered non-magnetic bead complexes through a flow cytometer or optical plate reader; and detecting the optical signal(s) of the recovered magnetic bead complexes and detecting the optical signal(s) of the recovered non-magnetic bead complexes and, optionally, quantifying the optical signal(s) detected so as to thereby quantify the amount of protein-nucleic acid interaction on the bead.

5. The method of claim 2, 3 or 4, further comprising amplifying the nucleic acids released after contacting with a Proteinase K, but prior to sequencing.

6. The method of any of claims 1-5, wherein the presence on a bead complex of a first primary optically-active label and/or a secondary optically-active label is determined by quantifying the optical signal thereof.

7. The method of claim 5, wherein the optical signal is collected with one or more photomultipliers.

8. The method of any of claims 1, 6 or 7, further comprising quantifying the optical signal(s) detected so as to thereby quantify the amount of first protein-second protein interaction on the bead and, optionally, comparing the quantified amount against a control amount or control curve.

9. The method of any of claims 1-7, wherein each primary agent comprises an antibody or comprises an antigen-binding fragment of an antibody.

10. The method of any of claims 1 or 6-9, wherein each secondary agent comprises an antibody or comprises an antigen-binding fragment of an antibody.

11. The method of any of claims 1-10, wherein the sample is a cell or tissue lysate.

12. The method of any of claims 1-11, wherein the sample is a cell, and the cell is fixed and permeabilized so as to permit primary and second agent entry into the cell prior to performing the method.

13. The method of any of claims 1 or 6-12, wherein the first protein-second protein complex comprises a protein of that is a product of a gene variant.

14. The method of any of claims 1 or 6-12, wherein the first proteins A, B, C and D are distinct variant forms of a single protein.

15. The method of any of claims 2-5, wherein the proteins A, B, C and D are distinct variant forms of a single protein.

16. The method of any of claims 1-15, wherein FSC and/or SSC are adjusted with a control un-complexed bead population prior to initiating the method so as to permit complexed beads to be detected.

17. The method of any of claims 1-16, wherein the ratio of primary agent to primary optically-active labels on primary agents so-labeled is 1:1.

18. The method of any of claims 1 or 6-17, wherein the ratio of secondary agent to secondary optically-active labels on secondary agents so-labeled is 1:1.

19. The method of any of claims 9-10, wherein the antibodies are monoclonal antibodies.

20. The method of any of claims 9-10 or 19, wherein the antibody fragments are F(ab′)2 fragments, Fab′ fragments or ScFvs.

21. The method of any of claims 1-20, wherein the magnetic beads are epoxy-coated magnetic beads.

22. The method of any of claims 1-21, wherein the non-magnetic beads are carboxyl modified beads.

23. The method of any of claims 1-22, wherein the agents are attached to the beads by covalent binding.

24. The method of any of claims 1-23, wherein optically-active labels are chosen from the group of fluorophores and nanocrystals.

25. The method of any of claims 1-24, wherein the sample is a lysate and magnetic beads are separated from the lysate using a magnetic field.

26. The method of any of claims 1-25, wherein the sample is a lysate and the non-magnetic beads are separated from the lysate by centrifugation.

27. The method of any of claims 1-26, wherein quantitatively measured optical activity of labeled agents bound to the complex is converted into a relative or an absolute quantitation number of co-binding molecules in each complex.

28. The method of any of claims 1-27, wherein forward scatter amplitude gain and side scatter voltage on a flow cytometer are set to register populations of bead events to on scale, followed by applying an inclusion gate where selected linear populations of beads form collective clusters containing interrogation targets can be analyzed in their entirety by flow cytometry.

29. The method of any of claims 1-28, wherein the cell or tissue lysate is from primary isolated cells, lymphoblasts, fibroblasts, cancer cells, a cell line, transfected cells, tissue or blood.

30. A kit for detecting changes in protein expression in cells and for analysis of gene variants, the kit comprising: magnetic beads for immunoprecipitation, non-magnetic beads for immunoprecipitation, a lysis formulation, one or more Proteinase K inhibitors, one or more phosphatase inhibitors, a coupling buffer, nucleic acids recovery elution buffer, one or more functional variant assay (FVA) buffers, a Western loading buffer, one or more optically active labels, and instructions for use of the kit.

31. The kit of claim 30, further comprising one or more of: a nucleic acid recovery buffer, a proteinase inhibitor, a fix-permeabilization buffer, and one or more primary agents for capturing protein-protein complexes or protein-nucleic acid complexes.

32. The kit of claim 30 or 31, wherein the optically active labels are nanoparticles and/or fluorescent dyes.

33. The kit of any of claims 30-32, wherein primary agents are attached to the beads.

34. The kit of any of claims 30-33, wherein a portion and/or subset of the beads are labeled with an optically active agent.

35. A method of detecting and analyzing a gene variant based on a protein-protein interaction, the method comprising: attaching a first primary agent to the surface of magnetic beads that are not labeled with an optically active label and attaching a second primary agent to the surface of magnetic beads that are labeled with an optically active label; attaching a third primary agent to the surface of non-magnetic beads that are not labeled with an optically active label and attaching a fourth primary agent to the surface of non-magnetic beads that are labeled with an optically active label, wherein the first, second, third and fourth primary agents are different agents and wherein the first, second, third and fourth primary agents are each capable of capturing a distinct protein complex from a cell or tissue lysate; capturing to the primary agents a protein complex from a cell or tissue lysate, where the protein complex comprises a protein of interest that is a product of a gene or a gene variant and where the protein of interest forms part of a complex with another protein; probing the protein-protein complex with one or more optically active secondary agents specific for a member of the complex; wherein the same one or more optically active labels can be used to label secondary agents on any of i) the magnetic beads that are not labeled with an optically active label, ii) the magnetic beads that are labeled with an optically active label, iii) the non-magnetic beads that are not labeled with an optically active label, and iv) the non-magnetic beads that are labeled with an optically active label; separating protein-magnetic bead complexes from the lysate based on magnetic properties of the magnetic beads; separating protein-non-magnetic bead complexes from the lysate based on a physical property of the non-magnetic beads; and measuring optical activity of optically active-labeled agents on the protein-bead complexes, wherein the absence or presence of the optically active label on the magnetic beads is used to distinguish optically active protein complexes captured by the first and second primary agents, respectively, and wherein the absence or presence of the optically active label on the non-magnetic beads is used to distinguish optically active protein complexes captured by the third and fourth primary agents, respectively.

36. A method of detecting and/or analyzing a gene variant based on a protein-nucleic acid interaction, the method comprising: attaching a first primary agent to the surface of magnetic beads that are not labeled with an optically active label and attaching a second primary agent to the surface of magnetic beads that are labeled with an optically active label; attaching a third primary agent to the surface of non-magnetic beads that are not labeled with an optically active label and attaching a fourth primary agent to the surface of non-magnetic beads that are labeled with an optically active label, wherein the first, second, third and fourth primary agents are different agents and wherein the first, second, third and fourth primary agents are each capable of capturing a distinct protein-nucleic acid complex from a cell or tissue lysate; capturing to the primary agents a protein-nucleic acid complex from a cell or tissue lysate, where the protein-nucleic acid complex comprises a gene or a gene variant nucleic acid sequence; separating protein-nucleic acid-magnetic bead complexes from the lysate based on magnetic properties of the magnetic beads; separating protein-nucleic acid-non-magnetic beads complexes from the lysate based on a physical property of the non-magnetic beads; digesting proteins on the protein-nucleic acid bead complexes to release nucleic acids; and amplifying the released nucleic acids; wherein the absence or presence of the optically active label on the magnetic beads is used to distinguish optically active protein-nucleic complexes captured by the first and second primary agents, respectively, and wherein the absence or presence of the optically active label on the non-magnetic beads is used to distinguish optically active protein-nucleic acid complexes captured by the third and fourth primary agents, respectively.

37. A kit for obtaining nuclear, cytoplasmic or whole-cell extract from cells or from tissue, the kit comprising: cell lysis buffer 1 M Dithiothreitol (DTT) one or more phosphatase inhibitors 10×PBS one or more phosphatase inhibitors 10× hypotonic buffer detergent written instructions for use of the kit.

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