Method and kit for the screening, the detection and/or the quantification of transcriptional factors

Abstract

The present invention is related to a screening, detection and/or quantification method of one or more transcriptional factor(s) ( 1 ) possibly present in a biological sample, said method comprising the steps of: possibly extracting and isolating said transcriptional factor ( 1 ) from said biological sample, putting into contact the transcriptional factor ( 1 ) with a double-stranded DNA sequence ( 2 ) bound to an insoluble solid support ( 3 ), and detecting and/or quantifying said fixed transcriptional factor ( 1 ), said double-stranded DNA sequence having a specific sequence able to be fixed by the transcriptional factor ( 1 ) and being preferably located at a distance of at least about 6.8 nm from the surface of the solid support ( 3 ), and said double-stranded DNA sequence being bound to the surface of the insoluble solid support ( 3 ) at a concentration of at least 0.01 pmole/cm 2 of solid support surface ( 3 ). The present invention is also related to the kit comprising means and media for performing said method.

What is claimed is:  
     
         1 . A screening and/or quantification method of one or more activated transcriptional factor(s) ( 1 ) present in a cell or cell lysate, said method comprising the steps of: 
 binding to an insoluble solid support ( 3 ) double-stranded DNA sequence(s) ( 2 ) at the concentration of at least 0.01 μmole/cm2 of said solid support surface, said double-stranded DNA sequence(s) comprising a specific sequence able to bind said transcriptional factor and being linked to a spacer having a length of at least 6.8 nm,    putting into contact said transcriptional factor ( 1 ) with said bound double-stranded DNA sequence(s) ( 2 ); and    identifying and/or quantifying a signal resulting from the binding of said transcriptional factor(s) ( 1 ) upon said double-stranded DNA sequence(s) ( 2 ) and resulting from one characteristic specific of the transcriptional factor activation.    
     
     
         2 . The method according to the  claim 1 , wherein said characteristic specific of the transcriptional factor ( 1 ) activation is a phosphorylation or dephosphorylation of amino acids of the transcriptional factor ( 1 ).  
     
     
         3 . The screening and,/ or quantification method according to the  claim 1 , wherein the characteristic specific of the transcriptional factor ( 1 ) activation is a binding of the transcriptional factor ( 1 ) to a protein which is part of its active complex.  
     
     
         4 . The method according to the  claim 3 , wherein the protein is an activator or an inhibitor of the transcriptional factor ( 1 ) activation.  
     
     
         5 . The method according to the  claim 1 , wherein the signal resulting from the binding of the transcriptional factor ( 1 ) upon the double-stranded DNA sequence(s) ( 2 ) and from at least one characteristic specific of the transcriptional factor ( 1 ) activation is detected by using 
 firstly a primary antibody ( 4 ) or an hypervariable portion thereof specific for the activated form of the transcriptional factor, and by    a secondary labelled antibody ( 5 ) directed against the primary antibody ( 4 ) or the specific hypervariable portion thereof.    
     
     
         6 . The method of  claim 5 , wherein the secondary labelled antibody ( 5 ) is conjugated with an enzyme such as a peroxidase.  
     
     
         7 . The method according to the  claim 1 , wherein the spacer corresponds to a double-stranded DNA sequence of at least 20 base pairs.  
     
     
         8 . The method according to the  claim 1 , wherein the spacer corresponds to a chemical spacer of at least 10 atoms.  
     
     
         9 . The method according to the  claim 1 , wherein the binding of the double-stranded DNA sequence(s) ( 2 ) to the insoluble solid support ( 3 ) is a covalent binding.  
     
     
         10 . The method according to the  claim 1 , wherein the binding of the double-stranded DNA sequence(s) ( 2 ) to the insoluble solid support ( 3 ) is of non-covalent type and corresponds to a binding pair comprising a first element ( 6 ) and a second member ( 7 ), said first element being bound to the double-stranded DNA sequence, said second member ( 7 ) being bound to the surface of the solid support ( 3 ), said first element ( 6 ) being able to interact with said second member ( 7 ).  
     
     
         11 . The method according to  claim 10 , wherein the binding pair is selected from the group consisting of biotin/streptavidin, hapten/receptor and antigen/antibody.  
     
     
         12 . The method according to the  claim 1 , wherein the transcriptional factor is present in solution at a concentration lower than 20 nmolar (nM).  
     
     
         13 . The method according to the  claim 1 , wherein the signal resulting from the binding of the transcriptional factor ( 1 ) upon the double-stranded DNA sequence(s) ( 2 ) and resulting from one characteristic specific of the transcriptional factor activation is a non radioactive resulting signal.  
     
     
         14 . The method according to the  claim 1 , wherein the signal resulting from the binding of the transcriptional factor ( 1 ) upon the double-stranded DNA sequence(s) ( 2 ) and resulting from one characteristic specific of the transcriptional factor activation is obtained through an enzymatic reaction.  
     
     
         15 . The method according to the  claim 1 , wherein multiple different transcriptional factors ( 1 ) are present in a same biological sample.  
     
     
         16 . The method according to the  claim 1 , wherein the transcriptional factor ( 1 ) is selected from the group consisting of NF-KB, AP-1, CREB, SP-1, C/EBP, GR, HIF-1, Myc, NF-AT, Oct, TBP and CBF- 1 or factors listed in table 1.  
     
     
         17 . The method according to the  claim 1 , for the screening and/or quantification of multiple different transcriptional factors ( 1 ) upon a same support ( 3 ).  
     
     
         18 . The method according to the  claim 1 , wherein the solid support ( 3 ) is an array bearing upon at least 4 spots/cm 2  of solid support surface, each spot containing double-stranded DNA sequence(s) ( 2 ) for the binding of transcriptional factor(s) ( 1 ).  
     
     
         19 . The method according to the  claim 1 , wherein the double-stranded DNA sequence(s) fixed on the support surface contain in part or totally one or several of the consensus DNA sequences presented in the table 1.  
     
     
         20 . The method according to the  claim 1 , wherein said transcriptional factor is the HIV integrase.  
     
     
         21 . The method according to the  claim 1 , which further comprises the steps of screening, quantifying and/or recovering compounds able to bind to said transcriptional factor(s) or inhibit the binding of transcriptional factor(s) ( 1 ) to the specific sequence upon the double-stranded DNA sequence(s) ( 2 ) bound to said solid support ( 3 ).  
     
     
         22 . The method according to the preceding  claim 1 , which further comprises the step of screening and/or quantifying a compound able to modulate the activity of said transcriptional factor by a modification of the level of the characteristic specific of the transcriptional factor activation.  
     
     
         23 . The method according to the  claim 1 , which further comprises the steps of screening, quantifying and/or recovering compounds which modulate the binding and/or the activity of the said transcriptional factor(s) when they are put in contact with elements selected from the group consisting of cells, tissues or organisms.  
     
     
         24 . The method according to the  claim 1 , which further comprises the steps of screening, quantifying and/or recovering compounds which modulate the activity of enzyme(s) or protein(s) acting on transcriptional factor(s) and then assayed for the activity of said transcriptional factor(s).  
     
     
         25 . The method according to the  claim 1 , which further comprises the step of identification of transcriptional factor(s) and/or of peptides which are part of their active complex.  
     
     
         26 . The method according to the  claim 1 , which further comprises the step of adding in the cell lysate an externally added transcriptional factor or a compound which is able to bind to a consensus sequence.  
     
     
         27 . A kit for the screening and/or quantification of transcriptional factor(s) ( 1 ) or (a) compound(s) able to bind to said transcriptional factor(s) ( 1 ) or inhibit the binding of said transcriptional factor(s) to a specific nucleotide sequence, which comprises double-stranded DNA sequence(s) ( 2 ) bound to an insoluble solid support ( 3 ) via a spacer having a length of at least 6.8 nm, at a concentration of at least 0.01 pmole/cm 2  of solid support surface ( 3 ) and a primary antibody or a specific hypervariable portion thereof, both being specific for the activated form of the transcriptional factor(s) ( 1 ) and possibly a second labelled antibody ( 5 ) directed against the primary antibody ( 4 ) or the specific hypervariable portion thereof.  
     
     
         28 . The kit according to the  claim 27 , wherein said secondary labelled antibody ( 5 ) is conjugated with an enzyme such as a peroxidase.  
     
     
         29 . The kit according to the  claim 27 , comprising a solid support bearing on its surface one or several double-stranded DNA consensus sequences at a concentration of at least 0.01 pmole/cm 2  comprising in part or totally one or several of the consensus sequence(s) listed in table 1 allowing the binding of a transcriptional factor present in solution and its detection and/or quantification.  
     
     
         30 . The kit according to the  claim 27 , wherein the solid support is an array having at least 4 spot/cm 2  of solid support surface containing double-stranded DNA sequence(s) ( 2 ) for the binding of the transcriptional factor(s) ( 1 ).  
     
     
         31 . The kit according to the  claim 27 , wherein said spacer is a double-stranded DNA nucleotide sequence of at least 20 base pairs.  
     
     
         32 . The kit according to the  claim 27 , wherein said spacer is a double-stranded DNA nucleotide sequence of at least 40 base pairs.  
     
     
         33 . The kit according to the  claim 27 , wherein the double-stranded DNA sequence ( 2 ) is bound to a first member ( 6 ) of a binding pair, able to interact with a second member ( 7 ) of the binding pair bound to the surface of the solid support ( 3 ).  
     
     
         34 . The kit according to the  claim 27 , wherein the double-stranded DNA sequence ( 2 ) is covalently bound to the surface of the solid support.  
     
     
         35 . The kit according to the  claim 27 , being a high-throughput screening device.