Deoxyribozyme-mediated ligation for incorporating EPR spin labels and reporter groups into RNA.

Abstract

Preparation of site-specifically labeled RNA for spectroscopic studies is a multistep process and requires handling of delicate samples. This chapter is focused on the synthesis of spin-labeled RNA using convertible nucleosides and the application of the deoxyribozyme 9DB1* for the ligation of RNA fragments. The convertible nucleoside approach enables the attachment of nitroxyls as paramagnetic reporters at the exocyclic amino groups of cytidine, adenosine, and guanosine nucleobases in synthetic RNA. The deoxyribozyme 9DB1* is a synthetic single-stranded DNA with RNA ligase activity that can be used as an alternative to protein enzymes (T4 RNA/DNA ligases) for covalently joining RNA fragments via native 3'-5' phosphodiester bonds. The combination of solid-phase synthesis and DNA-catalyzed RNA ligation provides reliable access to site-specifically labeled functional RNAs for spectroscopic studies. A particular advantage of using deoxyribozymes for RNA ligation lies in the mild reaction conditions that prevent chemical damage to sensitive labels. As an example, we describe a detailed protocol for the synthesis of TEMPO-labeled SAM-I riboswitch RNA.