Patent ID: 6423493
Filing Date: 2002-07-23
Classification: A61K,C07H,C12N,C40B

Abstract:
A method of identifying an aptamer comprising the steps of:(a) synthesizing a random phosphodiester oligonucleotide combinatorial library wherein constituent oligonucleotides comprise at least a set of 5â€² and 3â€² PCR primer nucleotide sequences flanking a randomized nucleotide sequence; (b) amplifying the library enzymatically using a mix of four nucleotides, wherein one or more of the nucleotides comprises a mix of unmodified and thiophosphate-modified nucleotides, to form a partially thiophosphate-modified oligonucleotide combinatorial library; (c) contacting the partially thiophosphate-modified oligonucleotide combinatorial library with a target molecule and isolating a subset of oligonucleotides binding to the target molecule; (d) amplifying the subset of binding oligonucleotides enzymatically using a mix of four nucleotides, wherein at least a portion of at least one nucleotide is thiophosphate-modified, to form a thiophosphate-modified oligonucleotide sub-library; and (e) repeating steps (c)-(d) iteratively with increased stringency of the contacting step between each iteration until at least one aptamer comprising a thiophosphate-modified oligonucleotide population of defined sequence is obtained.