DNAzyme-dependent Analysis of rRNA 2'-O-Methylation

Research output: Contribution to journalArticlepeer-review

Colleges, School and Institutes

External organisations

  • School of Biosciences, University of Birmingham

Abstract

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2'-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2'-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2'-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-'O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2'-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2'-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Details

Original languageEnglish
Article numbere59700
JournalJournal of Visualized Experiments
Issue number151
Publication statusPublished - 16 Sep 2019

Keywords

  • RNA modification, 2’-O-methylation, rRNA, DNAzyme, snoRNA, RNA analysis