ADVANTAGES OF 2'-O-METHYL OLIGORIBONUCLEOTIDE PROBES FOR DETECTING RNA TARGETS

We have compared various kinetic and melting properties of oligoribonu cleotide probes containing 2'-O-methylnucleotides or 2'-deoxynucleotid es with regard to their use in assays for the detection of nucleic aci d targets. 2'-O-Methyl oligoribonucleotide probes bound to RNA targets faster and with much higher melting temperatures (T-m values) than co rresponding 2'-deoxy oligoribonucleotide probes at all lengths tested (8-26 bases). T-m values of both probes increased with length up to -1 9 bases, with maximal differences in T-m between 2'-O-methyl and 2'-de oxy oligoribonucleotide probes observed at lengths of 16 bases or less . In contrast to RNA targets, 2'-O-methyl oligoribonucleotide probes b ound more slowly and with the same T-m to DNA targets as corresponding 2'-deoxy oligoribonucleotide probes. Because of their greatly enhance d T-m when bound to RNA, 2'-O-methyl oligoribonucleotide probes can ef ficiently bind to double-stranded regions of structured RNA molecules. A 17 base 2'-O-methyl oligoribonucleotide probe was able to bind a do uble-stranded region of rRNA whereas the same 17 base 2'-deoxy oligori bonucleotide probe did not. Due to their enhanced T-m when bound to RN A targets, shorter 2'-O-methyl oligoribonucleotide probes can be used in assays in place of longer 2'-deoxy oligoribonucleotide probes, resu lting in enhanced discrimination between matched and mismatched RNA ta rgets, A 12 base 2'-O-methyl oligoribonucleotide probe had the same T- m as a 19 base 2'-deoxy oligoribonucleotide probe when bound to a matc hed RNA target but exhibited a much larger decrease in T-m than the 2' -deoxy oligoribonucleotide probe when bound to an RNA target containin g either 1 or 2 mismatched bases, The increased T-m, faster kinetics o f hybridization, ability to bind to structured targets and increased s pecificity of 2'-O-methyl oligoribonucleotide probes render them super ior to corresponding 2'-deoxy oligoribonucleotides for use in assays t hat detect RNA targets.