Identifying ribozyme-accessible sites using NUH triplet-targeting gapmers.

ABSTRACT: Accurately identifying accessible sites in RNA is a critical prerequisite for optimising the cleavage efficiency of hammerhead ribozymes and other small nucleozymes. Here we describe a simple RNase H-based procedure to rapidly identify hammerhead ribozyme-accessible sites in gene length RNAS: Twelve semi-randomised RNA-DNA-RNA chimeric oligonucleotide probes, known as 'gapmers', were used to direct RNase H cleavage of transcripts with the specificity expected for hammerhead ribozymes, i.e. after NUH sites (where H is A, C or U). Cleavage sites were identified simply by the mobility of RNase H cleavage products relative to RNA markers in denaturing polyacrylamide gels. Sites were identified in transcripts encoding human interleukin-2 and platelet-derived growth factor. Thirteen minimised ha

SUBMITTER: Mir AA 

PROVIDER: S-EPMC37256 | biostudies-literature | 2001 May

REPOSITORIES: biostudies-literature