title: Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2'-O-methyl-modified RNA.
abstract: T7 RNA polymerase is an important biocatalyst that is used in diverse biotechnological applications such as in vitro transcription or protein expression. The enzyme displays high substrate specificity which is payed by significant limitations regarding incorporation of synthetic nucleotide analogs. Of specific interest is enzymatic synthesis of 2'-O-methyl-modified RNA as these nucleic acids exhibit improved biochemical and pharmacokinetic properties that make them attractive for diagnostic and therapeutic purposes. We report here on the development of an activity-based selection/screening approach for assessing polymerase activities in the presence of 2'-O-methyl-modified nucleotides, and on the identification of one variant T7 RNA polymerase which is capable of synthesizing all-2'-O-methyl RNA.
authors.affiliation: Department of Systemic Cell Biology, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.
authors.forename: Jenny
authors.initials: J
authors.lastname: Ibach
authors.forename: Laura
authors.initials: L
authors.lastname: Dietrich
authors.forename: Kyra R M
authors.initials: KR
authors.lastname: Koopmans
authors.forename: Nico
authors.initials: N
authors.lastname: Nöbel
authors.forename: Marc
authors.initials: M
authors.lastname: Skoupi
authors.forename: Susanne
authors.initials: S
authors.lastname: Brakmann
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009705:Nucleosides / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D009705:Nucleosides; D012329:RNA, Bacterial; D014764:Viral Proteins; D066246:ErbB Receptors; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: Aptamer; Modified nucleotide; Polymerase; RNA