Modified Nucleic Acid Probes

Abstract

Oligonucleotide analogue arrays attached to solid substrates and methods related to the use thereof are provided. The oligonucleotide analogues hybridize to nucleic acids with either higher or lower specificity than corresponding unmodified oligonucleotides. Target nucleic acids which comprise nucleotide analogues are bound to oligonucleotide and oligonucleotide analogue arrays.

1 . A composition comprising an array of oligonucleotide analogues attached to a solid substrate. 
     
     
         2 . The composition of  claim 1 , wherein said solid substrate is selected from the group consisting of beads, chips, and slides. 
     
     
         3 . The composition of  claim 1 , wherein said composition comprises an array of oligonucleotide analogue probes 5 to 20 nucleotides in length. 
     
     
         4 . The composition of  claim 1 , wherein said array of oligonucleotide analogues comprises a nucleoside analogue with the formula 
       
         
           
           
               
               
           
         
         wherein: 
         R 1  is selected from the group consisting of hydrogen, methyl, hydroxy, alkoxy, alkylthio, halogen, cyano, and azido; 
         R 2  is selected from the group consisting of hydrogen, methyl, hydroxy, alkoxy, alkylthio, halogen, cyano, and azido; and 
         Y is a heterocyclic moiety. 
       
     
     
         5 . The composition of  claim 1 , wherein said array of oligonucleotide analogues comprises a nucleoside analogue with the formula 
       
         
           
           
               
               
           
         
       
       wherein:
 R 1  is selected from the group consisting of hydrogen, hydroxyl, methyl, methoxy, ethoxy, propoxy, allyloxy, propargyloxy, Fluorine, Chlorine, and Bromine; 
 R 2  is selected from the group consisting of hydrogen, hydroxyl, methyl, methoxy, ethoxy, propoxy, allyloxy, propargyloxy, Fluorine, Chlorine, and Bromine; and 
 Y is a base selected from the group consisting of purines, purine analogues, pyrimidines, pyrimidine analogues, 3-nitropyrrole and 5-nitroindole. 
 
     
     
         6 . The composition of  claim 1 , wherein said array of oligonucleotide analogues comprises a peptide nucleic acid. 
     
     
         7 . The composition of  claim 1 , wherein said array of oligonucleotide analogues is resistant to RNAase A. 
     
     
         8 . The composition of  claim 1 , wherein said solid substrate is attached to over 1000 different oligonucleotide analogues. 
     
     
         9 . The composition of  claim 1 , wherein said array of oligonucleotide analogues comprises 2′-O-methyl nucleotides. 
     
     
         10 . The composition of  claim 1 , wherein said array of oligonucleotide analogues and said solid substrate comprises a plurality of different members, each member having the formula:
   Y-L 1 -X 1 -L 2 -X 2      wherein,
 Y is a solid substrate; 
 X 1  and X 2  are complementary oligonucleotides containing at least one nucleotide analogue; 
 L 1  is a spacer; 
 L 2  is a linking group having sufficient length such that X 1  and X 2  form a double-stranded oligonucleotide. 
   
     
     
         11 . The composition of  claim 10 , wherein said composition comprises a library of unimolecular double-stranded oligonucleotide analogues. 
     
     
         12 . The composition of  claim 1 , wherein said array of oligonucleotide analogues comprises a conformationally restricted array of oligonucleotide analogues with the formula:
   —X 11 —Z—X 12  
   
       wherein X 11  and X 12  are complementary oligonucleotides or oligonucleotide analogues and Z is a presented moiety. 
     
     
         13 . The composition of  claim 1 , wherein said array of oligonucleotide analogues comprises a nucleotide with a base selected from the group of bases consisting of 7-deazaguanosine, 2-aminopurine, 8-aza-7-deazaguanosine, 1H-purine, and hypoxanthine. 
     
     
         14 . A composition comprising an oligonucleotide analogue array synthesized on a solid substrate, wherein said synthesis is performed by light-directed chemical coupling. 
     
     
         15 . The composition of  claim 14 , wherein said solid substrate is derivitized with a silane reagent prior to synthesis of said oligonucleotide analogue. 
     
     
         16 . A composition comprising an oligonucleotide analogue array synthesized on a solid substrate, wherein said synthesis is performed by flowing oligonucleotide analogue reagents over predetermined regions of the solid substrate. 
     
     
         17 . The composition of  claim 16 , wherein said solid substrate is derivitized with a silane reagent prior to synthesis of said oligonucleotide analogue. 
     
     
         18 . A method of improving the hybridization of a nucleic acid to an oligonucleotide array, comprising incorporating a base selected from the group consisting of 7-deazaguanosine, 2-aminopurine, 8-aza-7-deazaguanosine, 1H-purine, and hypoxanthine into the oligonucleotides of the array. 
     
     
         19 . The method of  claim 18 , wherein the oligonucleotide is a homopolymer. 
     
     
         20 . A method of determining if a target molecule is complementary to a probe, comprising the steps of:
 (a) synthesizing an oligonucleotide analogue array on a solid substrate;   (b) exposing said oligonucleotide analogue array on said solid substrate to a target nucleic acid; and   (c) determining whether an oligonucleotide analogue member of said oligonucleotide analogue array binds to said target oligonucleotide.   
     
     
         21 . The method of  claim 20 , wherein said target nucleic acid is selected from the group comprising genomic DNA, cDNA, unspliced RNA, mRNA, and rRNA. 
     
     
         22 . The method of  claim 20 , wherein said target nucleic acid is amplified prior to said hybridization step. 
     
     
         23 . The method of  claim 20 , wherein said oligonucleotide analogue array is synthesized on said solid support by light-directed very large scale immobilized polymer synthesis. 
     
     
         24 . The method of  claim 20 , wherein said oligonucleotide analogue array is synthesized on said solid support by causing oligonucleotide analogue synthetic reagents to flow over selected regions of said solid support. 
     
     
         25 . The method of  claim 20 , wherein said step (a) comprises the steps of:
 i) forming a plurality of channels adjacent to the surface of said substrate;   ii) placing selected monomers in said channels to synthesize oligonucleotide analogues at predetermined portions of selected regions, said portion of said selected regions comprising oligonucleotide analogues different from oligonucleotide analogues in at least one other of said selected regions; and   iii) repeating steps i) and ii) with said channels formed along a second portion of said selected regions, thereby forming an array of oligonucleotide analogues.   
     
     
         26 . The method of  claim 20 , wherein said solid substrate is selected from the group consisting of beads, slides, and chips. 
     
     
         27 . The method of  claim 20 , wherein said solid substrate is comprised of materials selected from the group consisting of silica, polymers and glass. 
     
     
         28 . The method of  claim 20 , wherein the oligonucleotide analogue members of said array are synthesized using photoremovable protecting groups. 
     
     
         29 . The method of  claim 20 , wherein said oligonucleotide analogue array is synthesized by a process comprising selective incorporation of MeNPoc onto the 5′ hydroxyl of a nucleoside analogue, which nucleoside analogue is incorporated into said oligonucleotide analogue array. 
     
     
         30 . The method of  claim 20 , wherein said oligonucleotide analogue array is synthesized from phosphoramidite nucleoside reagents. 
     
     
         31 . A composition comprising an array of oligonucleotide probes hybridized to a target nucleic acid, which target nucleic acid comprises a nucleotide analogue. 
     
     
         32 . The composition of  claim 31 , wherein the target nucleic acid is a PCR amplicon. 
     
     
         33 . The composition of  claim 31 , wherein the oligonucleotide probes comprise oligonucleotide analogues. 
     
     
         34 . The composition of  claim 31 , wherein the target nucleic acid is an RNA nucleic acid. 
     
     
         35 . A method of detecting a target nucleic acid, comprising enzymatically copying the target nucleic acid using nucleotides which comprise a nucleotide analogue, thereby producing a nucleic acid analogue amplicon, and hybridizing the nucleic acid amplicon to an oligonucleotide array. 
     
     
         36 . The method of  claim 35 , wherein the oligonucleotide array comprises an oligonucleotide analogue probe which is complementary to the nucleic acid analogue amplicon.