Methods and reagents for detecting target binding by nucleic acid ligands

Abstract

The present invention provides novel methods and reagents for detecting the binding of protein targets to nucleic acid ligands. Using Universal Protein Stains (UPS), proteins bound by nucleic acid ligands may be labeled with a detectable moiety. The methods and reagents are particularly useful for the detection of protein targets bound to multiplexed arrays of nucleic acid ligands. The present invention also provides novel methods for the multiplexed evaluation of photocrosslinking nucleic acid ligands. The methods allow one simultaneously to: (1) evaluate the performance (dynamic range) of a plurality of photocrosslinking nucleic acid ligands; and (2) assess the specificity of each photocrosslinking nucleic acid ligand for its cognate target protein. Photocrosslinking nucleic acid ligands with the most desirable properties can then be selected for use in diagnostic and prognostic medical assays. The present invention also provides a photocrosslinking nucleic acid ligand that binds specifically to HIV gp120MN.

1 . A method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule is a protein, the method comprising; 
 a) providing a solid support, said solid support comprising a photoreactive nucleic acid ligand having specific affinity for said target protein, said photoreactive nucleic acid ligand binding specifically to said target molecule through non-Watson-Crick interactions;    b) contacting said solid support with said test mixture suspected of containing said target molecule, wherein a nucleic acid ligand-target molecule complex is formed if said target molecule is present;    c) irradiating said solid support, wherein said nucleic acid ligand-target molecule complex photocrosslinks;    d) removing non-specifically bound material from said solid support;    e) contacting said solid support with a Universal Protein Stain (UPS), said UPS comprising one or more reagents that label proteins with a detectable moiety; and    f) detecting the presence of said target molecule by detecting the presence of said detectable moiety on said solid support.    
     
     
         2 . The method of  claim 1  wherein said step d) is accomplished by exposing said biochip to conditions that denature nucleic acids.  
     
     
         3 . The method of  claim 1  wherein step d) is accomplished by exposing said biochip to conditions that denature proteins.  
     
     
         4 . The method of  claim 1  wherein said detectable moiety is a dye.  
     
     
         5 . The method of  claim 4  wherein said dye is a fluorophore.  
     
     
         6 . The method of  claim 1  wherein said detectable moiety is an enzyme.  
     
     
         7 . The method of  claim 6  wherein said enzyme is alkaline phosphatase.  
     
     
         8 . The method of  claim 6  wherein said enzyme is horseradish peroxidase.  
     
     
         9 . The method of  claim 1  wherein said detectable moiety is an enzyme substrate.  
     
     
         10 . The method of  claim 1  wherein said detectable moiety is a radiolabel.  
     
     
         11 . The method of  claim 1  wherein at least one of said UPS reagents reacts with primary amines.  
     
     
         12 . The method of  claim 11  wherein primary amine is present on a lysine residue.  
     
     
         13 . The method of  claim 11  wherein the reaction of at least one of said UPS reagents with said primary amine occurs in the presence of an organic solvent.  
     
     
         14 . The method of  claim 1  wherein at least one of said UPS reagents reacts with thiols.  
     
     
         15 . The method of  claim 1  wherein at least one of said UPS reagents reacts with alcohols.  
     
     
         16 . The method of  claim 1  wherein at least one of said UPS reagents reacts with carboxylates.  
     
     
         17 . The method of  claim 1  wherein said UPS comprises an N-hydroxysuccinimide-activated dye.  
     
     
         18 . The method of  claim 17  wherein said UPS comprises an N-hydroxysuccinimide-activated fluorophore.  
     
     
         19 . The method of  claim 1  wherein said UPS comprises CBQCA (3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde).  
     
     
         20 . The method of  claim 1  wherein said UPS comprises a reagent that bears an amine reactive group selected from the list consisting of isocyanates, isothiocyanates, acyl azides, sulfonyl chlorides, aldehydes, 4-sulfo-2,3,5,6-tetrafluorophenol (STP) esters, NBD (7-nitrobenz-2-oxa-1,3-diazole) chloride, NBD fluoride, and dichlorotriazines.  
     
     
         21 . The method of  claim 1  wherein said UPS comprises: 
 a) a biotin derivative capable of reacting with primary amines; and    b) a streptavidin conjugated to said detectable moiety.    
     
     
         22 . The method of  claim 1  wherein said UPS comprises: 
 a) a first biotin derivative capable of reacting with primary amines;    b) streptavidin; and    c) a second biotin derivative conjugated to said detectable moiety.    
     
     
         23 . The method of  claim 1  wherein said UPS comprises: 
 a) 2-iminothiolane; and    b) a thiol-reactive derivative of a dye.    
     
     
         24 . The method of  claim 23  wherein said thiol-reactive derivative of said dye comprises a maleimide group.  
     
     
         25 . The method of  claim 1  wherein said UPS comprises: 
 a) a hapten derivative capable of reacting with primary amines; and    b) an anti-hapten antibody conjugated to said detectable moiety.    
     
     
         26 . The method of  claim 1  wherein said UPS comprises: 
 a) a hapten derivative capable of reacting with primary amines;    b) an anti-hapten antibody; and    c) a secondary antibody conjugated to said detectable moiety, wherein said secondary antibody binds to said anti-hapten antibody.    
     
     
         27 . The method of  claim 1  wherein said UPS comprises: 
 a) a reagent that modifies amino acid side chains;    b) an antibody that specifically recognizes said modified amino acid side chain.    
     
     
         28 . The method of  claim 27  wherein said antibody is conjugated to said detectable moiety.  
     
     
         29 . The method of  claim 27  wherein said reagent that modifies amino acid side chains is a nitrosylating agent and wherein said antibody is an anti-nitro tyrosine antibody.  
     
     
         30 . The method of  claim 29  wherein said nitrosylating agent is tetranitromethane.  
     
     
         31 . The method of  claim 27  wherein said reagent that modifies amino acid side chains is a sulfo-N-hydroxysuccinimide acetate and wherein said antibody is an anti-acetylated lysine antibody.  
     
     
         32 . A method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule to be detected is a protein, the method comprising; 
 a) providing a biochip comprising a solid support, said solid support comprising a plurality of spatially defined addresses, each said address comprising at least one copy of a single species of nucleic acid ligand attached thereto, each said species of nucleic acid ligand having specific affinity for one of said target molecules suspected of being contained in said test mixture, and each said species of nucleic acid ligand binding specifically to said target molecule through non-Watson-Crick interactions;    b) contacting said biochip with said test mixture suspected of containing said target molecule;    c) removing non-specifically bound material from said biochip;    d) contacting said solid support with a Universal Protein Stain (UPS), said UPS comprising one or more reagents that label proteins with a detectable moiety; and    e) detecting the presence of said target molecule by detecting the presence of said detectable moiety at the appropriate address on said biochip.    
     
     
         33 . A method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule is a protein, the method comprising; 
 a) providing a solid support, said solid support comprising a nucleic acid ligand having specific affinity for said target protein, said nucleic acid ligand binding specifically to said target protein through non-Watson-Crick interactions;    b) contacting said solid support with said test mixture suspected of containing said target molecule;    c) removing non-specifically bound material from said solid support;    d) contacting said solid support with a Universal Protein Stain (UPS), said UPS comprising one or more reagents that label proteins with a detectable moiety; and    e) detecting the presence of said target molecule by detecting the presence of said detectable moiety at the appropriate address on said biochip.    
     
     
         34 . A method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule be to detected is a protein, the method comprising; 
 a) providing a biochip comprising a solid support, said solid support comprising a plurality of spatially defined addresses, each said address comprising at least one copy of a single species of nucleic acid ligand attached thereto, each said species of nucleic acid ligand having specific affinity for one of said target molecules suspected of being contained in said test mixture, each said species of nucleic acid ligand binding specifically to said target molecule through non-Watson-Crick interactions, and wherein said nucleic acid ligand having specific affinity for said target molecule to be detected is a photoreactive nucleic acid ligand;    b) contacting said biochip with said test mixture suspected of containing said target molecule, wherein a nucleic acid ligand-target molecule complex is formed if said target molecule is present;    c) irradiating said biochip, wherein said nucleic acid ligand-target molecule complex photocrosslinks;    d) removing non-specifically bound material from said biochip;    e) contacting said biochip with a reagent that reacts covalently with proteins and not with nucleic acids; and    f) detecting the presence of said target molecule by detecting the presence of said detectable moiety at the appropriate address on said biochip.    
     
     
         35 . A biochip comprising an array of a plurality of nucleic acid ligands attached to a solid support, wherein a plurality of said nucleic acid ligands are specifically associated with a target molecule through non-Watson-Crick interactions, and wherein said target molecules are labeled with a detectable moiety.  
     
     
         36 . A method for attaching a nucleic acid ligand to a solid support comprising: 
 a) derivatizing said nucleic acid ligand with a poly(ethylene glycol) (PEG);    b) attaching said PEG to said solid support.    
     
     
         37 . The method of  claim 36  wherein said PEG is vinyl sulfone-PEG, and wherein said solid support comprises thiol groups.  
     
     
         38 . A method for simultaneously measuring the dose-response characteristics of a plurality of species of photocrosslinking nucleic acid ligands, each said species of photocrosslinking nucleic acid ligands having specific affinity for a cognate target protein, the method comprising: 
 a) providing a plurality of arrays, each said array comprising a plurality of spatially defined addresses, each said address having at least one copy of a single species of photocrosslinking nucleic acid ligand attached thereto;    b) providing a plurality of target protein mixtures, wherein each mixture comprises a unique target protein concentration profile;    c) contacting each said array with a different one of said mixtures; and    d) measuring the amount of target protein bound to each said address on each said array;    whereby the dose-response characteristics of each said species of photocrosslinking nucleic acid ligands are measured simultaneously.    
     
     
         39 . The method of  claim 38  wherein each said target protein concentration profile comprises a concentration value of about 0 M for a different one of said target proteins.  
     
     
         40 . The method of  claim 38  wherein said target protein concentration profiles are configured such that for each pairwise combination of said cognate target proteins, at least one target protein mixture comprises the first member of the pairwise combination at a concentration that is at least one order of magnitude higher than the second member of the pairwise combination and at least one target protein mixture comprises the first member of the pairwise combination at a concentration that is at least one order of magnitude lower than the second member of the pairwise combination.  
     
     
         41 . The method of  claim 38  wherein said target protein concentration profiles are configured such that for each pairwise combination of said cognate target proteins, at least one target protein mixture comprises the first member of the pairwise combination at a concentration that is at least two orders of magnitude higher than the second member of the pairwise combination and at least one target protein mixture comprises the first member of the pairwise combination at a concentration that is at least two orders of magnitude lower than the second member of the pairwise combination.  
     
     
         42 - 45 . (canceled)