Biophysical and mechanistic investigation of RNA function requires site-specific incorporation of spectroscopic and chemical probes, which is difficult to achieve using current technologies. We have in vitro reconstituted a functional box C/D small ribonucleoprotein RNA 2′-O-methyltransferase (C/D RNP) from the thermophilic archaeon Pyrococcus abyssi and demonstrated its ability to transfer a prop-2-ynyl group from a synthetic cofactor analog to a series of preselected target sites in model tRNA and pre-mRNA molecules. Target selection of the RNP was programmed by changing a dodecanucleotide guide sequence in a 64-nt C/D guide RNA leading to efficient derivatization of three out of four new targets in each RNA substrate. We also show that the transferred terminal alkyne can be further appended with a fluorophore using a bioorthogonal azide-alkyne 1,3-cycloaddition (click) reaction. The described approach for the first time permits synthetically tunable sequence-specific labeling of RNA with single-nucleotide precision.

Methyltransferases catalyze specific transfers of methyl groups from the ubiquitous cofactor S-adenosyl-l-methionine (AdoMet) to various nucleophilic positions in biopolymers like DNA, RNA, and proteins. We had previously described synthesis and application of AdoMet analogues carrying sulfonium-bound 4-substituted but-2-ynyl side chains for transfer by methyltransferases. Although useful in certain applications, these cofactor analogues exhibited short lifetimes in physiological buffers. Examination of the reaction kinetics and products showed that their fast inactivation followed a different pathway than observed for AdoMet and rather involved a pH-dependent addition of a water molecule to the side chain. This side reaction was eradicated by synthesis of a series of cofactor analogues in which the separation between an electronegative group and the triple bond was increased from one to three carbon units. The designed hex-2-ynyl moiety-based cofactor analogues with terminal amino, azide, or alkyne groups showed a markedly improved enzymatic transalkylation activity and proved well suitable for methyltransferase-directed sequence-specific labeling of DNA in vitro and in bacterial cell lysates.