Patent ID: 6355418
Filing Date: 2002-03-12
Classification: C07H

Abstract:
A method of identifying an antisense binding site in a target mRNA, which comprises:(1) an incubation step, wherein a target mRNA is incubated with an oligonucleotide library and a duplex-cutting RNAase under conditions which provide for the target mRNA to be cleaved at an antisense binding site; and (2) an identification step, wherein the antisense binding site from the position of the cut in the mRNA is identified; wherein (a) all of the oligonucleotides in the oligonucleotide library are present simultaneously in the incubation step with the target mRNA; and (b) the oligonucleotide library comprises a plurality of distinct chimeric oligonucleotides capable of hybridizing to mRNA to form a duplex, the nucleotide sequences of which each have a common length ranging from 7 to 20 bases, which are generated randomly or generated based on the sequence of the target mRNA, wherein substantially all the nucleotide sequences of said common length which are present as sub-sequences in the target mRNA are represented in the library, and wherein each nucleotide sequence comprises: (a) a recognition region comprising a sequence of nucleotides that is recognized as a substrate by a duplex cutting RNAase when hybridized to the mRNA thereby permitting cleavage of said mRNA; and (b) a flanking region on one or both sides of said recognition region, wherein said flanking region is distinct from said recognition region and comprises a sequence of chemically-modified nucleotides which binds to the mRNA sufficiently tightly to stabilize the duplex for cutting of the mRNA in the duplex by the duplex cutting RNAase, wherein the nucleotides constituting the flanking region are different from those constituting the recognition region, and wherein each oligonucleotide is protected against exonuclease attack.