Surface-bound, double-stranded DNA protein arrays

Abstract

The invention provides a synthetic array of surface-bound, bimolecular, double-stranded nucleic acid molecules, the array comprising a solid support and a plurality of bimolecular double-stranded nucleic acid molecule members, a member comprising a first nucleic acid strand linked to the solid support and a second nucleic acid strand which is substantially complementary to the first strand and complexed to the first strand by Watson-Crick base pairing, wherein for at least a portion of the members, each member comprises a recognition site within a nucleic acid sequence for a protein, wherein a recognition site within a nucleic acid sequence for a protein of a first member is different from a recognition site within a nucleic acid sequence for a protein of a second member and wherein a protein is bound to a member thereof.

What is claimed is:  
     
         1 . A synthetic array of surface-bound, bimolecular, double-stranded nucleic acid molecules, said array comprising 
 a solid support, and    a plurality of bimolecular double-stranded nucleic acid molecule members, a said member comprising a first nucleic acid strand linked to said solid support and a second nucleic acid strand which is substantially complementary to said first strand and complexed to said first strand by Watson-Crick base pairing, wherein for at least a portion of said members, each said member comprises a recognition site within a nucleic acid sequence for a protein, wherein a recognition site within a nucleic acid sequence for a protein of a first member is different from a recognition site within a nucleic acid sequence for a protein of a second member and wherein a said protein is bound to a said member thereof.    
     
     
         2 . The array of  claim 1 , wherein the 3′ end of said first strand is linked to said support.  
     
     
         3 . The array of  claim 1 , wherein the 5′ end of said first strand and the 3′ end of said second strand are not linked via a covalent bond.  
     
     
         4 . The array of  claim 1 , wherein the 5′ end of said second strand is not linked to said support.  
     
     
         5 . The array of  claim 1 , wherein said recognition site within a nucleic acid sequence for a protein is selected from the group that includes naturally-occurring recognition sites within a nucleic acid sequence for a protein or proteins, synthetic variants of naturally-occurring recognition sites within a nucleic acid sequence for a protein or proteins and randomized nucleic acid sequences.  
     
     
         6 . The array of  claim 5 , wherein said recognition site within a nucleic acid sequence for a protein comprises two half-sites, wherein either is recognized by a different protein than is the other.  
     
     
         7 . The array of  claim 1 , wherein said protein which is bound to a said member thereof comprises a detectable label.  
     
     
         8 . The array of  claim 1 , wherein said protein is a chimeric protein.  
     
     
         9 . The array of  claim 8 , wherein said chimeric protein comprises a DNA-binding domain fused in-frame with a protein:protein dimerization domain.  
     
     
         10 . The array of  claim 8 , wherein said chimeric protein comprises a DNA-binding domain fused in-frame to Green Fluorescent Protein.  
     
     
         11 . The array of  claim 1 , wherein said solid support is a silica support.  
     
     
         12 . The array of  claim 1 , wherein said first strand is produced by chemical synthesis and said second strand is produced by enzymatic synthesis.  
     
     
         13 . The array of  claim 12 , wherein said first strand is used as the template on which said second strand is enzymatically produced.  
     
     
         14 . The array of  claim 13 , wherein said first strand of each said member contains at its 3′ end a binding site for an oligonucleotide primer which is used to prime enzymatic synthesis of said second strand, and at its 5′ end a variable sequence.  
     
     
         15 . The array of  claim 12 , wherein said enzymatic synthesis is performed using an enzyme.  
     
     
         16 . The array of  claim 14 , wherein said oligonucleotide primer is between 10 and 30 nucleotides in length.  
     
     
         17 . The array of  claim 1 , wherein said first strand comprises DNA.  
     
     
         18 . The array of  claim 1 , wherein said second strand comprises DNA.  
     
     
         19 . The array of  claim 1 , wherein said first and second strands each comprise from 16 to 60 monomers selected from the group that includes ribonucleotides and deoxyribonucleotides.  
     
     
         20 . The array of  claim 1 , wherein said solid support is a silica support and said first and second strands (X) each comprise from 16 to 60 monomers selected from the group that includes ribonucleotides and deoxyribonucleotides.  
     
     
         21 . The array of  claim 1 , wherein at least a portion of said plurality have a second nucleic acid strand that is substantially complementary to- and base-paired with said first strand along the entire length of said first strand.  
     
     
         22 . A method for the construction of a synthetic array of surface-bound, bimolecular, double-stranded nucleic acid molecules, comprising the steps of 
 (a) providing an array of first nucleic acid strands linked to a solid support,    (b) hybridizing to said first strands of step (a) an oligonucleotide primer that is substantially complementary to a sequence comprised by a said first strand,    (c) performing enzymatic synthesis of a second nucleic acid strand that is complementary to a said first strand of step (a) so as to permit Watson-Crick base pairing and so as to form an array comprising a plurality of bimolecular, double-stranded nucleic acid molecule members, wherein for at least a portion of said members, each said member comprises a recognition site within a nucleic acid sequence for a protein and wherein a recognition site within a nucleic acid sequence for a protein of a first member is different from a recognition site within a nucleic acid sequence for a protein of a second member, and    (d) incubating said array with a protein sample comprising a protein under conditions that permit specific binding of said protein to a said member of said array, such that a said protein becomes bound to a said recognition site within a nucleic acid sequence for a protein on a said member to form a nucleic acid protein array.    
     
     
         23 . The method according to  claim 22 , wherein the 3′ end of said first strand is linked to said support.  
     
     
         24 . The method according to  claim 22 , wherein the 5′ end of said first strand and the 3′ end of said second strand are not linked via a covalent bond.  
     
     
         25 . The method according to  claim 22 , wherein the 5′ end of said second strand is not linked to said solid support.  
     
     
         26 . The method according to  claim 22 , wherein said recognition site within a nucleic acid sequence for a protein is selected from the group that includes naturally-occurring recognition sites within a nucleic acid sequence for a protein or proteins, synthetic variants of naturally-occurring recognition sites within a nucleic acid sequence for a protein or proteins and randomized nucleic acid sequences.  
     
     
         27 . The method according to  claim 26 , wherein said recognition site within a nucleic acid sequence for a protein comprises two half-sites, wherein either is recognized by a different protein than is the other.  
     
     
         28 . The method according to  claim 22 , wherein said protein which is bound to a said member of said array comprises a detectable label.  
     
     
         29 . The method according to  claim 22 , wherein said protein is a chimeric protein.  
     
     
         30 . The method according to  claim 29 , wherein said chimeric protein comprises a DNA-binding domain fused in-frame with a protein:protein dimerization domain.  
     
     
         31 . The method according to  claim 29 , wherein said chimeric protein comprises a DNA-binding domain fused in-frame to Green Fluorescent Protein.  
     
     
         32 . The method according to  claim 22 , wherein said solid support is a silica support.  
     
     
         33 . The method according to  claim 22 , wherein said first strand of each said member contains at its 3′ end a binding site for an oligonucleotide primer which is used to prime enzymatic synthesis of said second, and at its 5′ end a variable sequence, wherein said binding site is present in each said member of said array.  
     
     
         34 . The method according to  claim 33 , wherein said enzymatic synthesis is performed using an enzyme.  
     
     
         35 . The method according to  claim 22 , wherein said oligonucleotide primer of step (b) is between 10 and 30 nucleotides in length.  
     
     
         36 . The method according to  claim 22 , wherein said first strand of step (a) comprises DNA.  
     
     
         37 . The method according to  claim 22 , wherein said second strand of step (c) comprises DNA.  
     
     
         38 . The method according to  claim 22 , wherein said first and second strands each comprise from 16 to 60 monomers selected from the group that includes ribonucleotides and deoxyribonucleotides.  
     
     
         39 . The method according to  claim 22 , wherein said solid support is a silica support and said first and second strands each comprise from 16 to 60 monomers selected from the group that includes ribonucleotides and deoxyribonucleotides.  
     
     
         40 . The method according to  claim 28 , wherein said protein sample comprises a candidate inhibitor of binding of said protein to a said recognition site within a nucleic acid sequence for a protein on a said member of said array.  
     
     
         41 . The method according to  claim 28 , wherein said protein sample comprises a candidate inhibitor of binding of said protein to a second protein.  
     
     
         42 . A method of determining a consensus nucleic acid sequence for a recognition site within a nucleic acid sequence for a protein comprising the steps of 
 a) providing a nucleic acid protein array comprising a solid support and a plurality of bimolecular double-stranded nucleic acid molecule members, a said member comprising a first nucleic acid strand linked to said solid support and a second nucleic acid strand which is substantially complementary to said first strand and complexed to said first strand by Watson-Crick base pairing, wherein for at least a portion of said members, each said member comprises a recognition site within a nucleic acid sequence for a protein, wherein a recognition site within a nucleic acid sequence for a protein of a first member is different from a recognition site within a nucleic acid sequence for a protein of a second member and wherein a said protein comprising a detectable label is bound to a said member thereof, and    b) performing a detection step to detect the presence of said label on a feature of said array, wherein nucleotides that are shared among said recognition sites within a nucleic acid sequence for a protein present on said features on which said label is detected form a consensus nucleic acid sequence for a recognition site within a nucleic acid sequence for a protein specific for said protein.    
     
     
         43 . A method of identifying for a first protein which binds a nucleic acid as half of a protein:protein heterodimer complex one or a plurality of candidate second proteins with which it might dimerize and bind a nucleic acid molecule in vivo, comprising the steps of 
 a) providing a nucleic acid array comprising a solid support, and a plurality of bimolecular double-stranded nucleic acid molecule members, a said member comprising a first nucleic acid strand linked to said solid support and a second nucleic acid strand which is substantially complementary to said first strand and complexed to said first strand by Watson-Crick base pairing, wherein for at least a portion of said members, each said member comprises a recognition site within a nucleic acid sequence for a protein, wherein a recognition site within a nucleic acid sequence for a protein of a first member is different from a recognition site within a nucleic acid sequence for a protein of a second member, wherein a said recognition site within a nucleic acid sequence for a protein comprises two half-sites and wherein either of said half-sites of a said recognition site within a nucleic acid sequence for a protein is recognized by a different protein than is the other,    b) incubating said array with a protein sample comprising a first protein which recognizes a first half-site of a said recognition site within a nucleic acid sequence for a protein and one or a plurality of candidate second proteins under conditions which permit heterodimerization of a said first and candidate second protein and binding of a protein:protein heterodimer to a said recognition site within a nucleic acid sequence for a protein,    c) recovering a said protein:protein heterodimer complex from a said member of said array under conditions whereby said first protein and said candidate second protein dissociate from one another, and    d) identifying said candidate second protein, wherein each said candidate second protein so identified represents a protein with which said first protein may interact in vivo.    
     
     
         44 . The method of  claim 43 , wherein said identifying in step d) of said candidate second protein comprises sequencing thereof.  
     
     
         45 . The method of  claim 43 , wherein said identifying in step d) of said candidate second protein comprises binding of said candidate second protein to an antibody which is specific therefor.  
     
     
         46 . The method according to  claim 43 , wherein said first protein comprises a detectable label.  
     
     
         47 . The method according to  claim 47 , further comprising the step of performing a detection step to detect the presence of said label on a feature of said array, wherein the recognition site within a nucleic acid sequence for a protein present on a feature upon which said label is detected represents a candidate recognition site within a nucleic acid sequence for a protein which said heterodimer may bind in vivo.  
     
     
         48 . A method of identifying candidate members of a set of co-regulated genes, comprising the steps of 
 a) providing a nucleic acid protein array comprising a solid support and a plurality of bimolecular double-stranded nucleic acid molecule members, a said member comprising a first nucleic acid strand linked to said solid support and a second nucleic acid strand which is substantially complementary to said first strand and complexed to said first strand by Watson-Crick base pairing, wherein for at least a portion of said members, each said member comprises a recognition site within a nucleic acid sequence for a protein, wherein a recognition site within a nucleic acid sequence for a protein of a first member is different from a recognition site within a nucleic acid sequence for a protein of a second member and wherein a said protein comprising a detectable label is bound to a said member thereof, and    b) performing a detection step to detect the presence of said label on a feature of said array, wherein a gene having among its regulatory sequences one or more of said recognition sites within a nucleic acid sequence for a protein present on a said feature on which said label is detected is characterized as a candidate member of a set of co-regulated genes genes that are regulated by said protein.    
     
     
         49 . A method of assaying a candidate inhibitor of protein/nucleic acid interactions, comprising the steps of 
 a) providing a nucleic acid array comprising a solid support and a plurality of bimolecular double-stranded nucleic acid molecule members, a said member comprising a first nucleic acid strand linked to said solid support and a second nucleic acid strand which is substantially complementary to said first strand and complexed to said first strand by Watson-Crick base pairing, wherein for at least a portion of said members, each said member comprises a recognition site within a nucleic acid sequence for a protein, wherein a recognition site within a nucleic acid sequence for a protein of a first member is different from a recognition site within a nucleic acid sequence for a protein of a second member,    b) incubating said array with a protein sample comprising a protein comprising a detectable label and a candidate inhibitor of binding of said protein to a recognition site within a nucleic acid sequence for a protein on a said member of said array, under conditions which normally permit binding of said protein to said member, and    c) performing a detection step to detect the presence of said label on said member, wherein the presence of said label on said member corresponds with binding of said protein to said member and wherein the negation of- or reduction in binding of said protein to said member is indicative of efficacy of said candidate inhibitor of protein:nucleic acid interactions in inhibiting binding of said protein to said recognition site within a nucleic acid sequence for a protein.    
     
     
         50 . A method of assaying a candidate inhibitor of a protein/protein interaction, comprising the steps of 
 a) providing a nucleic acid array comprising a solid support and a plurality of bimolecular double-stranded nucleic acid molecule members, a said member comprising a first nucleic acid strand linked to said solid support and a second nucleic acid strand which is substantially complementary to said first strand and complexed to said first strand by Watson-Crick base pairing, wherein for at least a portion of said members, each said member comprises a recognition site within a nucleic acid sequence for a protein, wherein a recognition site within a nucleic acid sequence for a protein of a first member is different from a recognition site within a nucleic acid sequence for a protein of a second member,    b) incubating said array with a protein sample comprising a first comprising a detectable label, wherein binding of said first protein to a recognition site within a nucleic acid sequence for a protein on a said member of said array is dependent upon an interaction between said first protein and a second protein and wherein said protein sample further comprises said second protein and a candidate inhibitor of said interaction, under conditions which normally permit said interaction, and    c) performing a detection step to detect the presence of said label on a said member of said array, wherein the presence of said label on a said member corresponds with binding of said nucleic-acid-binding protein to said member and wherein the negation of- or reduction in binding of said protein to said member is indicative of efficacy of said candidate inhibitor in inhibiting said interaction between said first protein and said second protein.