Selective RNase H cleavage of target RNAs from a tRNA scaffold

Luc Ponchon; Geneviève Beauvais; Sylvie Nonin-Lecomte; Frédéric Dardel

doi:10.1007/978-1-62703-113-4_2

Selective RNase H cleavage of target RNAs from a tRNA scaffold

Methods Mol Biol. 2012:941:9-18. doi: 10.1007/978-1-62703-113-4_2.

Authors

Luc Ponchon¹, Geneviève Beauvais, Sylvie Nonin-Lecomte, Frédéric Dardel

Affiliation

¹ Laboratoire de Cristallographie et RMN biologiques, Université Paris Descartes, CNRS, Paris, France. luc.ponchon@parisdescartes.fr

PMID: 23065550
DOI: 10.1007/978-1-62703-113-4_2

Abstract

In vivo overproduction of tRNA chimeras yields an RNA insert within a tRNA scaffold. For some applications, it may be necessary to discard the scaffold. Here we present a protocol for selective cleavage of the RNA of interest from the tRNA scaffold, using RNase H and two DNA oligonucleotides. After cleavage, we show that the RNA of interest can be isolated in a one-step purification. This method has, in particular, applications in structural investigations of RNA.

MeSH terms

Electrophoresis, Polyacrylamide Gel
RNA Cleavage*
RNA, Ribosomal, 16S / isolation & purification
RNA, Ribosomal, 16S / metabolism*
RNA, Transfer / metabolism*
Ribonuclease H / biosynthesis
Ribonuclease H / isolation & purification
Ribonuclease H / metabolism*
Staining and Labeling

Substances

RNA, Ribosomal, 16S
RNA, Transfer
Ribonuclease H