Identifying ribozyme-accessible sites using NUH triplet-targeting gapmers

Nucleic Acids Research
A A MirP Hendry

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 hammerhead ribozymes, miniribozymes (Mrz), were synthesised and in vitro cleavage efficiency (37 degrees C, pH 7.6 and 1 mM MgCl2) at each site was analysed. Of the 13 Mrz, five were highly effective, demonstrating good initial rate constants and extents of cleavage. The speed and accuracy of this method commends its use in screening for hammerhead-accessible sites.

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Citations

Jan 16, 2003·Nucleic Acids Research·Mohammed AmarzguiouiHans Prydz
Jan 22, 2004·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Isabelle Gautherot, Regís Sodoyer
May 30, 2009·Drug Discovery Today·Lorena TedeschiLorenzo Citti
Jan 28, 2006·Journal of Cellular Biochemistry·Wei-Hua Pan, Gary A Clawson
Nov 22, 2005·Journal of Microbiological Methods·Dirk MuellerVera Meyer
Sep 12, 2002·Expert Opinion on Therapeutic Targets·John Goodchild
Feb 8, 2003·Molecular Therapy : the Journal of the American Society of Gene Therapy·Wei-Hua PanGary A Clawson

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