title: Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry.
abstract: The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest--of either synthetic or biological origin--is modified at its 3'-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide-alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2'-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2'-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, Heidelberg 69120, Germany.
authors.forename: Marie-Luise
authors.initials: ML
authors.lastname: Winz
authors.forename: Ayan
authors.initials: A
authors.lastname: Samanta
authors.forename: Dirk
authors.initials: D
authors.lastname: Benzinger
authors.forename: Andres
authors.initials: A
authors.lastname: Jäschke
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry; D002384:Catalysis; D057930:Click Chemistry / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology
chemical_list: D001386:Azides; D002241:Carbohydrates; D005456:Fluorescent Dyes; D009711:Nucleotides; C017040:8-azidoadenosine 5'-triphosphate; D012313:RNA; D003300:Copper; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D011062:Polynucleotide Adenylyltransferase