Methods and compositions for rapid functional analysis of gene variants

Abstract

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

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 , further comprising amplifying the nucleic acids released after contacting with a Proteinase K, but prior to sequencing. 
     
     
         6 . The method of  claim 1 , 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  claim 1 , 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  claim 1 , wherein each primary agent comprises an antibody or comprises an antigen-binding fragment of an antibody. 
     
     
         10 . The method of  claim 1 , wherein each secondary agent comprises an antibody or comprises an antigen-binding fragment of an antibody. 
     
     
         11 . The method of  claim 1 , wherein the sample is a cell or tissue lysate. 
     
     
         12 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , 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 - 18 . (canceled) 
     
     
         19 . The method of  claim 9 , wherein the antibodies are monoclonal antibodies. 
     
     
         20 . The method of  claim 9 , wherein the antibody fragments are F(ab′) 2  fragments, Fab′ fragments or ScFvs. 
     
     
         21 . The method of  claim 1 , wherein the magnetic beads are epoxy-coated magnetic beads. 
     
     
         22 . The method of  claim 1 , wherein the non-magnetic beads are carboxyl modified beads. 
     
     
         23 - 27 . (canceled) 
     
     
         28 . The method of  claim 1 , 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 . (canceled) 
     
     
         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 - 36 . (canceled) 
     
     
         37 . A kit for obtaining nuclear, cytoplasmic or whole-cell extract from cells or from tissue, the kit comprising:
 cell lysis buffer   1M 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.