Viktoras Masevičius

Methyltransferase-Directed Derivatization of 5-Hydroxymethylcytosine in DNA†

Cytosine modification by AdoMet–dependent DNA methyltransferases is part of an epigenetic regulatory network in vertebrates. Here we show that, in the absence of AdoMet, bacterial cytosine-5 methyltransferases can catalyze condensation of aliphatic thiols and selenols to 5-hydroxymethylcytosine in DNA yielding 5-chalcogenomethyl derivatives. These new atypical reactions open new ways for sequence-specific derivatization and analysis of 5-hydroxymethylcytosine, a recently discovered nucleobase in mammalian DNA

Enhanced chemical stability of adomet analogues for improved methyltransferase-directed labeling of DNA.

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.

Synthesis of S-Adenosyl-L-Methionine Analogs with Extended Transferable Groups for Methyltransferase-Directed Labeling of DNA and RNA.

S‐Adenosyl‐L‐methionine (AdoMet) is a ubiquitous methyl donor for a variety of biological methylation reactions catalyzed by methyltransferases (MTases). AdoMet analogs with extended propargylic chains replacing the sulfonium‐bound methyl group can serve as surrogate cofactors for many DNA and RNA MTases enabling covalent deposition of these linear chains to their cognate targets sites in DNA or RNA. Here we describe synthetic procedures for the preparation of two representative examples of AdoMet analogs with a transferable hex‐2‐ynyl group carrying a terminal azide or amine functionality. Our approach is based on direct chemoselective alkylation of S‐adenosyl‐L‐homocysteine at sulfur with corresponding nosylates under acidic conditions. We also describe synthetic routes to 6‐substituted hex‐2‐yn‐1‐ols and their conversion to the corresponding nosylates. Using these protocols, synthetic AdoMet analogs can be prepared within 1 to 2 weeks.

Animal Hen1 2′-O-methyltransferases as tools for 3′-terminal functionalization and labelling of single-stranded RNAs

Abstract S-adenosyl-L-methionine-dependent 2′-O-methylati-on of the 3′-terminal nucleotide plays important roles in biogenesis of eukaryotic small non-coding RNAs, such as siRNAs, miRNAs and Piwi-interacting RNAs (piRNAs). Here we demonstrate that, in contrast to Mg2+/Mn2+-dependent plant and bacterial homologues, the Drosophila DmHen1 and human HsHEN1 piRNA methyltransferases require cobalt cations for their enzymatic activity in vitro. We also show for the first time the capacity of the animal Hen1 to catalyse the transfer of a variety of extended chemical groups from synthetic analogues of the AdoMet cofactor onto a wide range (22–80 nt) of single-stranded RNAs permitting their 3′-terminal functionalization and labelling. Moreover, we provide evidence that deletion of a small C-terminal region of the DmHen1 protein further increases its modification efficiency and abolishes a modest 3′-terminal nucleotide bias observed for the full-length protein. Finally, we show that fluorophore-tagged ssRNA molecules are successfully detected in fluorescence resonance energy transfer assays both individually and in a total RNA mixture. The presented DmHen1-assisted RNA labelling provides a solid basis for developing novel chemo-enzymatic approaches for in vitro studies and in vivo monitoring of single-stranded RNA pools.