Site-specific chemical labeling of long RNA molecules.
Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method is nonenzymatic and based on the formation of a four-way junction, where a donor strand is chemically coupled to an acceptor strand at a specific position via an activated chemical group. A disulfide bond in the linker is subsequently cleaved under mild conditions leaving a thiol group attached to the acceptor-RNA strand. The site-specific thiol-modified target RNA can then be chemically labeled with an optional group, here demonstrated by coupling of a maleimide-functionalized fluorophore. The method is rapid and allows site specific labeling of both in vitro and in vivo synthesized RNA with a broad range of functional groups.
Danish National Research Foundation, Center for DNA Nanotechnology at Interdisciplinary Nanoscience Center, Aarhus University, DK-8000 Aarhus, Denmark. jk@mb.au.dk
Kasper
K
Jahn
Eva Maria
EM
Olsen
Morten Muhlig
MM
Nielsen
Thomas
T
Tørring
Reza
R
MohammadZadegan
Ebbe Sloth
ES
Andersen
Kurt Vesterager
KV
Gothelf
Jørgen
J
Kjems
D001483:Base Sequence; D001665:Binding Sites; D015497:HIV-1; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity
D012367:RNA, Viral; D012313:RNA