Coincidence detection

Abstract

The invention relates to a method of detecting the coincidence of two biomolecular structures in a solid phase sample, said method comprising (i) providing a first and a second fusion protein, each fusion protein comprising (a) a detection domain, said detection domain comprising a DNA binding domain; said detection domain capable of binding a cognate specific nucleotide sequence in co-operation with a further detection domain; (b) a recognition domain, said recognition domain capable of binding a target biomolecular structure; and (c) a connector domain; said connector domain being fused at one end to the detection domain and being fused at the other end to the recognition domain; wherein at least two of (a), (b) and (c) are heterologous to one another; wherein the recognition domains of said first and said second fusion proteins are capable of binding to first and second biomolecular structures; (ii) contacting the sample with said first and second fusion proteins (iii) incubating to allow binding (iv) removing unbound fusion protein (v) contacting the sample with nucleic acid comprising said cognate specific nucleotide sequence (vi) incubating to allow heterotrimeric binding of the nucleic acid (vii) detecting nucleic acid bound to the sample wherein detection of nucleic acid in step (vii) indicates that the two biomolecular structures are present coincidentally in said sample.

1 . A method of detecting the coincidence of two biomolecular structures in a solid phase sample, said method comprising
 (i) providing a first and a second fusion protein, each fusion protein comprising   (a) a detection domain, said detection domain comprising a DNA binding domain; said detection domain capable of binding a cognate specific nucleotide sequence in co-operation with a further detection domain;   (b) a recognition domain, said recognition domain capable of binding a target biomolecular structure; and   (c) a connector domain; said connector domain being fused at one end to the detection domain and being fused at the other end to the recognition domain;   wherein at least two of (a), (b) and (c) are heterologous to one another;   wherein the recognition domains of said first and said second fusion proteins are capable of binding to first and second biomolecular structures;   (ii) contacting the sample with said first and second fusion proteins   (iii) incubating to allow binding   (iv) removing unbound fusion protein   (v) contacting the sample with nucleic acid comprising said cognate specific nucleotide sequence   (vi) incubating to allow heterotrimeric binding of the nucleic acid   (vii) detecting nucleic acid bound to the sample   wherein detection of nucleic acid in step (vii) indicates that the two biomolecular structures are present coincidentally in said sample.   
     
     
         2 . A method according to  claim 1  wherein the step of detecting nucleic acid bound to the sample comprises detection by total internal reflection (TIRF) analysis, by fluorescent polarisation (FP), by scintillation proximity technology or by fluorescence resonance energy transfer (FRET). 
     
     
         3 . A method according to  claim 1  wherein the step of detecting nucleic acid bound to the sample comprises
 (a) removing unbound nucleic acid; and 
 (b) detecting nucleic acid bound to the sample. 
 
     
     
         4 . A method according to  claim 3  wherein detection of the nucleic acid bound to the sample is by polymerase chain reaction (PCR), by hybridisation to an oligonucleotide probe, by binding to a quantum dot nanocrystal, or by rolling circle amplification. 
     
     
         5 . A method according to any preceding claim wherein the sample is first contacted with an antibody capable of binding to a first molecular structure and wherein the recognition domain of one of said first or second fusion proteins is capable of binding to said antibody. 
     
     
         6 . A method according to  claim 5  wherein the recognition domain of said first or second fusion protein capable of binding to said antibody comprises amino acid sequence of an antigen binding fragment or scFv of a secondary antibody capable of binding to said antibody. 
     
     
         7 . A method according to  claim 5  wherein the recognition domain of said first or second fusion protein capable of binding to said antibody comprises amino acid sequence of protein A or protein G or protein L. 
     
     
         8 . A method according to any preceding claim wherein said DNA binding domain comprises amino acid sequence derived from a nuclear receptor and said specific nucleotide sequence comprises a hormone response element. 
     
     
         9 . A method according to any preceding claim wherein said connector domain comprises a flexible amino acid sequence of approximately 5 to 15 nm in length. 
     
     
         10 . A fusion protein, said fusion protein comprising:
 i) a detection domain, said detection domain comprising a DNA binding domain; said detection domain capable of binding a cognate specific nucleotide sequence in co-operation with a further detection domain:   ii) a recognition domain, said recognition domain capable of binding a target biological molecule;   iii) a connector domain; said connector domain being fused at one end to the detection domain and being fused at the other end to the recognition domain;   wherein at least two of (i), (ii) and (iii) are heterologous to one another;   wherein said recognition domain comprises one or more of:   (a) a polypeptide capable of binding a mammalian immunoglobulin;   (b) a polypeptide which specifically binds to a target polypeptide sequence;   (c) a polypeptide which specifically binds a post translationally modified amino acid residue;   (d) an antibody variable heavy or light chain;   (e) a single chain variable fragment (ScFv); or   (f) a polypeptide which specifically binds a backbone protein sequence.   
     
     
         11 . A fusion protein according to  claim 10  wherein the recognition domain comprises (a) a polypeptide capable of binding a mammalian immunoglobulin n, and wherein said polypeptide is
 (ei) a polypeptide capable of binding the constant (Fc) region of a mammalian immunoglobulin; or 
 (eii) a polypeptide capable of binding the light chain of a mammalian immunoglobulin. 
 
     
     
         12 . A fusion protein according to  claim 10  wherein the recognition domain comprises (b) a polypeptide which specifically binds to a target polypeptide sequence, and wherein said polypeptide comprises one or more of: an aptamer; an SH3 domain; a PTB domain; a PH domain; a LIM domain; a WD40 domain; a PDZ domain; a WW domain; an EVH1 domain; an EH domain or an Ankyrin repeat domain. 
     
     
         13 . A kit comprising
 (a) a first fusion protein according to any of  claims 10  to  12 ; and   (b) a second fusion protein according to any of  claims 10  to  12 ; and   (c) a nucleic acid comprising a cognate specific nucleotide sequence according to  claim 10 ;   wherein said first and second recognition domains bind different target biological molecules, or bind different epitopes within a single biological molecule;   wherein binding of the first and second detection domains to the specific nucleotide sequence is by formation of heterotrimer.   
     
     
         14 . A kit according to  claim 13  wherein said specific nucleotide sequence comprises a hormone response element. 
     
     
         15 . A kit according to  claim 13  or  claim 14  wherein the detection domain of the first fusion protein comprises nucleic acid binding domain of a 9-cis retinoid acid receptor (retinoid X receptor RXR). 
     
     
         16 . A kit according to any of  claims 13  to  15  wherein the detection domain of the second fusion protein comprises nucleic acid binding domain selected from the group consisting of all-trans retinoic acid receptor (RLAR), thyroid hormone receptor (TR), the vitamin D3 receptor (VDR), the peroxisome proliferator activated receptor (PPAR) and the nerve growth factor induced-B receptor (NGFI-B). 
     
     
         17 . A kit according to any of  claims 13  to  16  wherein the recognition domain of one of said first or second fusion proteins comprises amino acid sequence of protein A or protein G or protein L. 
     
     
         18 . A fusion protein, said fusion protein comprising:
 i) a detection domain, said detection domain comprising a DNA binding domain; said detection domain capable of binding a cognate specific nucleotide sequence;   ii) a recognition domain, said recognition domain capable of binding a target biological molecule; the detection domain and the recognition domain being fused;   wherein (i) and (ii) are heterologous to one another; and   wherein said recognition domain comprises protein G or protein A or protein L.   
     
     
         19 . A fusion protein according to  claim 18  wherein the DNA binding domain comprises a Cys2His2 type zinc finger. 
     
     
         20 . A fusion protein according to  claim 19  which comprises the DNA binding domain of Zif268 (SEQ ID NO:23). 
     
     
         21 . A kit comprising
 (a) a fusion protein according to any of  claims 18  to  20 ; and   (b) a nucleic acid comprising a cognate specific nucleotide sequence according to  claim 18 ;   wherein binding of the detection domain to the specific nucleotide sequence is by formation of dimer.   
     
     
         22 . A nucleic acid comprising a nucleotide sequence encoding a fusion protein according to any of  claims 10  to  12 ,  18 ,  19  or  20 .