title: Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases.
abstract: Biophysical and mechanistic investigation of RNA function requires site-specific incorporation of spectroscopic and chemical probes, which is difficult to achieve using current technologies. We have in vitro reconstituted a functional box C/D small ribonucleoprotein RNA 2'-O-methyltransferase (C/D RNP) from the thermophilic archaeon Pyrococcus abyssi and demonstrated its ability to transfer a prop-2-ynyl group from a synthetic cofactor analog to a series of preselected target sites in model tRNA and pre-mRNA molecules. Target selection of the RNP was programmed by changing a dodecanucleotide guide sequence in a 64-nt C/D guide RNA leading to efficient derivatization of three out of four new targets in each RNA substrate. We also show that the transferred terminal alkyne can be further appended with a fluorophore using a bioorthogonal azide-alkyne 1,3-cycloaddition (click) reaction. The described approach for the first time permits synthetically tunable sequence-specific labeling of RNA with single-nucleotide precision.
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
authors.forename: Migle
authors.initials: M
authors.lastname: Tomkuviene
authors.forename: Béatrice
authors.initials: B
authors.lastname: Clouet-d'Orval
authors.forename: Ignas
authors.initials: I
authors.lastname: Cerniauskas
authors.forename: Elmar
authors.initials: E
authors.lastname: Weinhold
authors.forename: Saulius
authors.initials: S
authors.lastname: Klimasauskas
mesh_terms: D000478:Alkylation; D001483:Base Sequence; D057930:Click Chemistry; D005456:Fluorescent Dyes; D008780:Methyltransferases / Q000378:metabolism*; D016566:Organoselenium Compounds / Q000737:chemistry / Q000378:metabolism; D043903:Pyrococcus abyssi / Q000201:enzymology; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D016566:Organoselenium Compounds; D012322:RNA Precursors; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C576042:SeAdoYn; D012436:S-Adenosylmethionine; D012343:RNA, Transfer; D008780:Methyltransferases