Site-specific isotope labeling of long RNA for structural and mechanistic studies

Abstract

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, ‘isotopically labeled guanosine 5′-monophosphate’ (5′-GMP), steadily gave a 5′-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5′-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with 15N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.