Nucleic acid ligand diagnostic biochip

Abstract

A nucleic acid ligand “biochip” is disclosed, consisting of a solid support to which one or more specific nucleic acid ligands is attached in a spatially defined manner. Each nucleic acid ligand binds specifically and avidly to a particular target molecule contained within a test mixture, such as a bodily fluid. The target molecules include, but are not limited to, proteins (cellular, viral, bacterial, etc.) hormones, sugars, metabolic byproducts, cofactor, and intermediates, drugs, and toxins. Contacting the test mixture with the biochip leads to the binding of a target molecule to its cognate nucleic acid ligand. The biochip may then be contacted with a reagent(s) that reacts covalently with proteins and not with nucleic acids. Each protein target in the test mixture may then detected by detecting the presence of the reagent at the appropriate address on the biochip.

1 . A method for detecting a target molecule that may be present in a test mixture, the method comprising: 
 (a) contacting a test mixture with a nucleic acid ligand comprising a first probe and having a specific affinity for a target molecule, wherein a nucleic acid ligand-target complex is formed if said target molecule is present in said test mixture;    (b) contacting a surface of a solid support comprising a second probe with said nucleic acid ligand-target complex, such that said first probe is permitted to interact with said second probe;    (c) at any point prior to (d), contacting said nucleic acid ligand-target complex with a detectable molecule; and    (d) detecting said target molecule on said surface by detecting said detectable molecule.    
   
   
       2 . The method of  claim 1 , wherein said nucleic acid ligand is a single-stranded nucleic acid or a double-stranded nucleic acid.  
   
   
       3 . The method of  claim 2 , wherein said nucleic acid ligand comprises DNA or RNA  
   
   
       4 . The method of  claim 3 , wherein said nucleic acid ligand comprises at least one chemical modification.  
   
   
       5 . The method of  claim 4 , wherein said at least one chemical modification is a chemical substitution at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.  
   
   
       6 . The method of  claim 4 , wherein said at least one chemical modification is independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH2), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-OMe), a 5-position pyrimidine modification, an 8-position purine modification, a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap.  
   
   
       7 . The method of  claim 2  wherein said first probe is included on the 5′-end of the nucleic acid ligand.  
   
   
       8 . The method of  claim 2  wherein said first probe is included on the 3′-end of the nucleic acid ligand.  
   
   
       9 . The method of  claim 1 , wherein said target molecule is a protein.  
   
   
       10 . The method of  claim 1 , wherein said test mixture is a bodily fluid.  
   
   
       11 . The method  claim 10 , wherein said bodily fluid is selected from the group consisting of blood plasma, urine, semen, saliva, lymph fluid, meningial fluid, amniotic fluid, glandular fluid, cerebrospinal fluid, feces, tissues, and biopsy samples.  
   
   
       12 . The method of  claim 1 , wherein said first probe comprises a first nucleotide sequence and said second probe comprises a second nucleotide sequence, and wherein said first nucleotide sequence is complementary to said second nucleotide sequence.  
   
   
       13 . The method of  claim 12  wherein said first probe interacts with said second probe through hybridization.  
   
   
       14 . The method of  claim 1 , wherein said surface comprises a plurality of spatially defined addresses, and wherein each of a plurality of said addresses comprises at least one probe disposed thereon.  
   
   
       15 . The method of  claim 14 , wherein detecting said target molecule comprises detecting said detectable molecule at an address on said surface.  
   
   
       16 . The method of  claim 1 , wherein said solid support is a biochip.  
   
   
       17 . A method for detecting a target molecule that may be present in a test mixture, the method comprising: 
 (a) contacting a test mixture with a nucleic acid ligand comprising a first probe and having a specific affinity for a target molecule, wherein a nucleic acid ligand-target complex is formed if said target molecule is present in said test mixture;    (b) irradiating said nucleic acid ligand-target complex, wherein said nucleic acid ligand-target complex photocrosslinks;    (c) contacting a surface of a solid support comprising a second probe with a photocrosslinked nucleic acid ligand-target complex, such that said first probe is permitted to interact with said second probe;    (d) at any point prior to (e), contacting said photocrosslinked nucleic acid ligand-target complex with a detectable molecule; and    (e) detecting said target molecule on said surface by detecting said detectable molecule.    
   
   
       18 . The method of  claim 17 , wherein said nucleic acid ligand is a single-stranded nucleic acid or a double-stranded nucleic acid.  
   
   
       19 . The method of  claim 18 , wherein said nucleic acid ligand comprises DNA or RNA  
   
   
       20 . The  claim 18 , wherein said nucleic acid ligand comprises at least one chemical modification.  
   
   
       21 . The method of  claim 20 , wherein said at least one chemical modification is a chemical substitution at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.  
   
   
       22 . The method of  claim 20 , wherein said at least one chemical modification is independently selected from a 2′-position sugar modification, a 2′-amino (2′-NH2), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-OMe), a 5-position pyrimidine modification, an 8-position purine modification, a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap.  
   
   
       23 . The method of  claim 17  wherein said first probe is included on the 5′-end of the nucleic acid ligand.  
   
   
       24 . The method of  claim 17  wherein said first probe is included on the 3′-end of the nucleic acid ligand.  
   
   
       25 . The method of  claim 17 , wherein said target molecule is a protein.  
   
   
       26 . The method of  claim 17 , wherein said test mixture is a bodily fluid.  
   
   
       27 . The method of  claim 26 , wherein said bodily fluid is selected from the group consisting of blood plasma, urine, semen, saliva, lymph fluid, meningial fluid, amniotic fluid, glandular fluid, cerebrospinal fluid, feces, tissues, and biopsy samples.  
   
   
       28 . The method of  claim 17 , wherein said first probe comprises a first nucleotide sequence and said second probe comprises a second nucleotide sequence, and wherein said first nucleotide sequence is complementary to said second nucleotide sequence.  
   
   
       29 . The method of  claim 28  wherein said first probe interacts with said second probe through hybridization.  
   
   
       30 . The method of  claim 17 , wherein said surface comprises a plurality of spatially defined addresses, and wherein each of a plurality of said addresses comprises at least one probe disposed thereon.  
   
   
       31 . The method of  claim 30 , wherein detecting said target molecule comprises detecting said detectable molecule at an address on said surface.  
   
   
       32 . The method of  claim 17 , wherein said solid support is a biochip.