DNA Labeling Using DNA Methyltransferases

Abstract

DNA methyltransferases (MTases) uniquely combine the ability to recognize and covalently modify specific target sequences in DNA using the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet). Although DNA methylation plays important roles in biological signaling, the transferred methyl group is a poor reporter and is highly inert to further biocompatible derivatization. To unlock the biotechnological power of these enzymes, two major types of cofactor AdoMet analogs were developed that permit targeted MTase-directed attachment of larger moieties containing functional or reporter groups onto DNA. One such approach (named sequence-specific methyltransferase-induced labeling, SMILing) uses reactive aziridine or N-mustard mimics of the cofactor AdoMet, which render targeted coupling of a whole cofactor molecule to the target DNA. The second approach (methyltransferase-directed transfer of activated groups, mTAG) uses AdoMet analogs with a sulfonium-bound extended side chain replacing the methyl group, which permits MTase-directed covalent transfer of the activated side chain alone. As the enlarged cofactors are not always compatible with the active sites of native MTases, steric engineering of the active site has been employed to optimize their alkyltransferase activity. In addition to the described cofactor analogs, recently discovered atypical reactions of DNA cytosine-5 MTases involving non-cofactor-like compounds can also be exploited for targeted derivatization and labeling of DNA. Altogether, these approaches offer new powerful tools for sequence-specific covalent DNA labeling, which not only pave the way to developing a variety of useful techniques in DNA research, diagnostics, and nanotechnologies but have already proven practical utility for optical DNA mapping and epigenome studies.

Covalent labeling of nucleic acids

“…Examples of MTase-directed labeling via click reactions have been reviewed previously. 141,142 The scope of available AdoMet analogs with transferable groups was continuously expanded over the past few years and comprises terminal alkyne, alkene, [143][144][145] azido and amino functionalities 146 that can be modified in a second step via click chemistry or N-hydroxysuccinimidyl-esterification (NHS-esterification). An important recent addition to this toolbox were AdoMet analogs with a norbornene moiety (Fig.…”

Repurposing enzymatic transferase reactions for targeted labeling and analysis of DNA and RNA

“…DNA MTases generally target a specific nucleobase residue within a defined 2-8 bp recognition sequence producing m 6 A, m 4 C or m 5 C. Enzymes with over 350 distinct recognition sequences (REBASE, http://rebase.neb.com) have been identified suggesting that a broad repertoire of DNA sequences can potentially be targeted. However, a rather small fraction of representative enzymes have been isolated and characterized to different degrees in vitro [1] and subsequently a handful of those (based on their target specificity, stability and catalytic performance) have been recruited for DNA tagging applications [7] (Figures 3A and 4A). Following numerous demonstrations of targeted labeling of DNA and RNA in vitro or ex vivo, the potential of mTAG labeling is further unlocked with advances in instrumentation and techniques enabling optical detection of individual fluorophore molecules in biological samples.…”

“…A substantial progress in the mRNA cap modification has been made by exploiting two enzymes: the GlaTgs2 MTase, which methylates the N 2 position of the m 7 G cap [15] and the Ecm1 cap guanine-N7 methyltransferase [37]. Ecm1 and an engineered variant of GlaTgs2 were used to deposit extended side chains from a range of AdoMet analogs onto in vitro transcribed G or m 7 G capped RNAs, respectively, [13,15,17,19,32,37,67] as well as dinucleotide cap variants m 7 GpppA or GpppA in bacterial [15] or eukaryotic [13,15,17,32,67] cell lysates. The tagged substrates were further analyzed after subsequent conjugation to functional reporters [13,[15][16][17]19,32,37,67,68].…”

“…The broadest spectrum of engineered reactions has been achieved with AdoMet-dependent MTases. The latest version of the technology, termed methyltransferase-directed Transfer of Activated Groups, or mTAG [4], is widely exploited for targeted covalent tagging of DNA and RNA with demonstrated applications in (epi)genome and (epi)transcriptome analysis (please refer to recent review articles for comprehensive reading [5][6][7][8]). Several other DNA and RNA transferase reactions have been similarly redesigned and employed to varied degrees.…”

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4

“…In the present study, a C5-DNA-MTase from P. arcticus 273-4, ParI, was characterized on the basis of its potential to possess features relevant for biotechnological applications, such as labeling of biopolymers, DNA mapping or epigenetic analysis [22][23][24].…”