title: Direct and site-specific quantification of RNA 2'-O-methylation by PCR with an engineered DNA polymerase.
abstract: Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR.
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, D-78457 Konstanz, Germany.
authors.forename: Joos
authors.initials: J
authors.lastname: Aschenbrenner
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, D-78457 Konstanz, Germany andreas.marx@uni-konstanz.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D016133:Polymerase Chain Reaction / Q000379:methods; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*
chemical_list: D012337:RNA, Ribosomal, 18S; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase