Oligonucleotides containing pyrazolo[3,4-d] pyrimidines for hybridization and mismatch discrimination

Abstract

Oligonucleotides in which one or more purine residues are substituted by pyrazolo[3,4-d]pyrimidines exhibit improved hybridization properties. Oligonucleotides containing pyrazolo[3,4-d]pyrimidine base analogues have higher melting temperatures than unsubstituted oligonucleotides of identical sequence. Thus, in assays involving hybridization of an oligonucleotide probe to a target polynucleotide sequence, higher signals are obtained. In addition, mismatch discrimination is enhanced when pyrazolo[3,4-d]pyrimidine-containing oligonucleotides are used as hybridization probes, making them useful as probes and primers for hybridization, amplification and sequencing procedures, particularly those in which single- or multiple-nucleotide mismatch discrimination is required.

1 - 47 . (canceled) 
     
     
         48 . An oligonucleotide probe capable of hybridizing with a target sequence comprising a modified target binding sequence wherein one or more purine residues of said target binding sequence are substituted by a pyrazolo[3,4-d]pyrimidine residue, and further comprising an attached minor groove binder, said modified target binding sequence corresponding to and capable of binding with a target sequence containing no pyrazolo[3,4-d]pyrimidine residues. 
     
     
         49 . An oligonucleotide probe of  claim 48 , wherein the minor groove binder is selected from the group consisting of a trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC5). 
     
     
         50 . An oligonucleotide probe of  claim 48 , further comprising a detectable label. 
     
     
         51 . An oligonucleotide probe of  claim 50  wherein the detectable label is selected from radioactive isotopes, chromophores, fluorophores, chemiluminescent agents, electrochemiluminescent agents, magnetic labels, immunologic labels, ligands and enzymatic labels. 
     
     
         52 . An oligonucleotide probe of  claim 50 , wherein the label is located is located at the oligonucleotide 5′ end. 
     
     
         53 . An oligonucleotide probe of  claim 50 , wherein the label is located is located at the oligonucleotide 3′ end. 
     
     
         54 . An oligonucleotide probe of  claim 50 , wherein the detectable label is a fluorescent label. 
     
     
         55 . An oligonucleotide probe of  claim 54  further comprising a quenching agent which quenches the fluorescence emission of the fluorescent label. 
     
     
         56 . An oligonucleotide probe of  claim 50 , comprising multiple fluorescent labels. 
     
     
         57 . An oligonucleotide probe of  claim 56 , wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels. 
     
     
         58 . An oligonucleotide probe according to  claim 48 , wherein the purine residues are selected from guanine residues and adenine residues. 
     
     
         59 . An oligonucleotide probe according to  claim 48 , wherein the purine residues are guanine residues, and all of the guanine residues are substituted by a pyrazolo[3,4-d]pyrimidine residue. 
     
     
         60 . An oligonucleotide probe according to  claim 48 , comprising four guanine residues in sequence that are each substituted by a pyrazolo[3,4-d]pyrimidine residue. 
     
     
         61 . An oligonucleotide probe according to  claim 48 , comprising six guanine residues in sequence that are each substituted by a pyrazolo[3,4-d]pyrimidine residue. 
     
     
         62 . An oligonucleotide probe according to  claim 48  comprising at least 12 but less than 21 bases.