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

Abstract

Produced as linear biopolymers from four major types of building blocks, DNA and RNA are further furnished with a range of covalent modifications. Despite the impressive specificity of natural enzymes, the transferred groups are often poor reporters and not amenable to further derivatization. Therefore, strategies based on repurposing some of these enzymatic reactions to accept derivatized versions of the transferrable groups have been exploited. By far the most widely used are S-adenosylmethionine-dependent methyltransferases, which along with several other nucleic acids modifying enzymes offer a broad selection of tagging chemistries and molecular features on DNA and RNA that can be targeted in vitro and in vivo. Engineered enzymatic reactions have been implemented in validated DNA sequencing-based protocols for epigenome analysis. The utility of chemo-enzymatic labeling is further enhanced with recent advances in physical detection of individual reporter groups on DNA using super-resolution microscopy and nanopore sensing enabling single-molecule multiplex analysis of genetic and epigenetic marks in minute samples. Altogether, a number of new powerful techniques are currently in use or on the brink of real benchtop applications as research tools or next generation diagnostics.

Biocatalytic Alkylation Cascades: Recent Advances and Future Opportunities for Late‐Stage Functionalization

“…A pertinent exemplar of this promiscuity is DNA/RNA MTases which accept analogues of SAM and can be used as a tagging tool for biotechnologybased applications. [19,[25][26][41][42][43] Cofactor promiscuity is also a hallmark of a number of small molecule C-MTases such as NovO and CouO, which accept a variety of S-alkylated SAM analogues prepared separately by chemical synthesis. [44] A distinct limitation of preparing SAM analogues by chemical synthesis is the formation of R/S epimers at the S centre.…”

“…[53][54] The efficiency of (m)ethyl transfer to coumarin substrates in this tandem process ( Figure 6) was far higher using modified cofactors 16-17 a-b generated in situ relative to naturally occurring SAM. It enabled (m)ethylation of high-value coumarin analogues (18)(19)(20) such as the Hsp90 inhibitor (19) and the warfarin metabolite (20). This concept of exploiting modified cofactors generated in situ followed by MTase-mediated alkyl transfer was also recently extended to the MAT enzyme.…”

“…This diversity provides an excellent foundation to exploit these enzymes for biocatalytic applications. For over a decade, intensive efforts in the biocatalytic arena have focused on understanding the scope and limitations of enzymatic alkylation, with a view to developing the approach into a generalized and sustainable biocatalytic process for late‐stage functionalization . In this article, we highlight contemporary new discoveries in the area which potentially take us closer towards establishing a biocatalytic alkylation platform.…”

S‐Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C‐Alkylation

“…[3] In contrast, MTases catalyze regiospecific Cmethylation of biomolecules using the SAM cofactor as the corresponding methyl donor. [14][15][16][17] The repertoire of C-methylation extends to small molecules, which opens up opportunities to tailor these enzymes as a general platform for biocatalytic C-C bond formation (Figure 1a). A representative example is NovO, which catalyzes the Calkylation of coumarins (e.g.…”

S‐Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C‐Alkylation

“…[11][12][13] However,o btaining regiospecificity,p articularly when this is required at alate-stage in asynthetic workflow,is an enduring challenge. [14][15][16][17] Ther epertoire of C-methylation extends to small molecules,which opens up opportunities to tailor these enzymes as ag eneral platform for biocatalytic C À Cb ond formation (Figure 1a). [14][15][16][17] Ther epertoire of C-methylation extends to small molecules,which opens up opportunities to tailor these enzymes as ag eneral platform for biocatalytic C À Cb ond formation (Figure 1a).…”