PAPER: Site-specific chemical labeling of long RNA molecules

Jahn, K.; Olsen, E. M.; Nielsen, M. M.; Tørring, T.; Mohammad Zadegan, R.; Andersen, E. S.; Gothelf, K. V.; Kjems, J.

Bioconjugate Chem. 2011, 22, 95–100, doi: 10.1021/bc100422k

Danish National Research Foundation, Center for DNA Nanotechnology (CDNA) at Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology, and Department of Chemistry, Aarhus University, DK-8000 Aarhus, Denmark

Abstract

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.

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