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. Author manuscript; available in PMC: 2020 Feb 1.
Published in final edited form as: Curr Opin Biotechnol. 2018 Oct 6;55:114–123. doi: 10.1016/j.copbio.2018.09.008

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

Miglė Tomkuvienė 1, Milda Mickutė 1, Giedrius Vilkaitis 1, Saulius Klimašauskas 1,*
PMCID: PMC6513755  EMSID: EMS82798  PMID: 30296696

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.

Introduction

DNA and RNA serve key functions of storing, copying and transmitting the genetic information in all known organisms. Originally produced as linear biopolymers from four major types of building blocks, they are further processed by furnishing with a range of covalent modifications required for their functions. Among numerous enzymatic reactions involved in nucleic acid metabolism, methylation is the most simple and ubiquitous natural modification playing a variety of regulatory and structural roles in the cell (reviewed in [1–3]). Selective methylation of specific targets in DNA and RNA is performed by numerous dedicated methyltransferase enzymes (MTases), most of which use the cofactor S-adenosyl-L-methionine (AdoMet) as the source of a methyl group.

Although the biological modifications are deposited by corresponding enzymes in a highly specific manner, the transferred groups are usually poor reporters themselves and/or are not amenable to further derivatization. To unlock the biotechnological power of these enzymes, strategies based on repurposing enzymatic reactions to accept expanded (derivatized) versions of the transferrable groups have been exploited (Figure 1). 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 [5–8]). Several other DNA and RNA transferase reactions have been similarly redesigned and employed to varied degrees. Here we discuss the latest developments in this rapidly expanding field with a focus on i) engineering of suitable enzyme-cofactor pairs in vitro and in vivo, ii) analysis of the tagged DNA and RNA using next generation sequencing and advanced optical techniques and iii) applications of the newly created tools in biochemical and biomedical research including genomic/epigenomic as well as transcriptomic/epitranscriptomic studies.

Figure 1. Covalent derivatization (R) of nucleic acids using cofactor analogs and engineered transferase enzymes.

Figure 1

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Biological R moieties are shown in grey, transferred functional groups – blue, reporters – magenta. A, Top. AdoMet analog and methyltransferase (MTase)-dependent modification of DNA and RNA (for “R” variants see Figure 2). Bottom. Non-canonical modification of hmC residues in DNA with aliphatic thiols by DNA cytosine-5 methyltransferases. HisTag and StrepTag – peptidyl affinity tags. B. UDP-glc analog and beta-glucosyltransferase (BGT)-dependent modification of hmC residues in DNA. C. Agmatine analog and tRNAIle2-agmatidine synthetase (Tias)-dependent modification of RNA. D. PreQ1 derivative and tRNA guanine transglycosylase (TGT)-dependent modification of RNA.

Engineering AdoMet-dependent transmethylation reactions

Although initially the development of AdoMet analogs for MTase-directed labeling was based on coupling reactions [9], the most recent approach relies on synthetic cofactors in which the transferable sulfonium-bound methyl group of AdoMet is replaced with an activated extended side chain (Figure 1A Top and Figure 2) [4,10–13]. Typically, linear linkers are used to carry terminal amino or azide and alkyne functional groups, which permit subsequent selective labeling via amino-NHS ester or azide-alkyne cycloaddition (AAC) reactions, respectively [12,14]. In addition, groups carrying an alkene [15–18], a norbornene moiety (for bioorthogonal tetrazine ligation [13]), a photocrosslinker [19] and a photo-removable cage [20] have recently become available. An increasingly popular choice is one-step mTAG labeling with fluorophores or biotin [21–25].

Figure 2. Structure of synthetic AdoMet analogs containing activated side chains with functional or reporter groups.

Figure 2

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Incorporation of an activating π-electron system (a multiple C-C bond or an aromatic ring, shown in green) next to the transferable carbon atom facilitates MTase-directed transfer of the side chain. Besides the mandatory activating group, linear linkers (shown in black) are used to carry terminal alkyne, azide, amino, alkene or norbornene functional groups (shown in blue). Reporter groups such as fluorophores or biotin (shown in magenta) can be further added to a transferable group (via coupling chemistries mentioned above) to yield cofactors suitable for one-step mTAG labeling. Some transferrable side chains combine an activating and functional group (terminal alkene or alkyne, photo-reactive aryl-azide or diazirine groups, photo-removable 2-nitrobenzyl) in a single moiety (shown in brown).

To date, three routes for production of mTAG cofactors have been proposed (reviewed in [26]). Chemical synthesis is based on direct “recharging” of the reaction co-product AdoHcy with a desired side chain. This route affords multi-milligram amounts of cofactor analogs, usually as diastereomeric mixtures [12,27]. Cofactors with large reporter groups such as chromophores or biotin are often obtained by further appending the side chains of functionalized cofactors via suitable conjugation chemistries under mild conditions [24,25,28]. An alternative more recent strategy is based on the natural route of AdoMet production from methionine and ATP catalyzed by AdoMet synthetase (also known as methionine adenosyltransferase, MAT). Enzyme engineering and synthetic efforts in several laboratories led to an expanded repertoire of methionine analogs that can be converted to stereochemically pure AdoMet analogs, although the size of the transferable moiety is currently limited to 5–7 carbon units [29–31]. The MAT-directed conversion of respective methionine analogs bypasses the issues of cofactor shelf-life or cell impermeability inherent for most of the AdoMet analogs and is well suited for coupled one-pot production/tagging reactions in vitro [32]. Proof-of-principle demonstrations are also available for covalent in vivo tagging of proteins [30] and RNA [33]. Yet another approach exploits halogenases [34,35]; in particular, the SalL chlorinase naturally catalyzes chloride-dependent decomposition of AdoMet to methionine and 5’-chloroadenosine. In the reverse reaction, the methionine analogs can be converted to extended cofactors in vitro, which can be in situ used for mTAG labeling [35]. In vivo application of the system may be limited due to its intolerance to chloride ions present in cellular environments.

With respect to mTAG catalysts, both DNA and RNA MTases show a wide range of plasticity in accepting chemically expanded cofactors. On one end of the spectrum are highly promiscuous enzymes that require no engineering for the transfer of even the bulkiest groups (for example, the TaqI DNA adenine-N6 MTase (M.TaqI) [13,20–23], the HEN1 2’-O-RNA MTases from plants and animals [24,25,36] and the 5’-cap specific Ecm1 RNA MTase [13,19,37]). Meanwhile, structure-guided steric engineering of the cofactor pocket in DNA cytosine-5 MTases is required to achieve desired transalkylation activity [38,39] (Figure 3). On the other hand, the most compact functionalized groups such as allyl or propargyl are accepted by a broad range of natural enzymes [18,33,40]. However, the suitability of such cofactors for in vivo studies may be limited by their poor MTase selectivity (simultaneous acceptance by multiple cellular wt MTases).

Figure 3. Substrate specificity of AdoMet-dependent methyltransferases recruited for targeted covalent derivatization of DNA and RNA.

Figure 3

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“R” – extended transferrable group. Enzymes that require engineering for mTAG reactions are marked with an asterisk. A. DNA MTases with their respective sequence recognition sites (the target base underlined) and the positions of base modification. B. RNA MTases with their respective target sites in the mRNA cap, an internal sequence or a 3’-terminal nucleoside.

DNA modifying enzymes for genomic and epigenomic analysis

Applications of DNA MTases in genomic analysis

DNA MTases generally target a specific nucleobase residue within a defined 2–8 bp recognition sequence producing m6A, m4C or m5C. 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. The key idea here is to determine physical positions of fluorescently–labeled target sites along the DNA contour (in linearly stretched single molecules) and analyze the resulting pattern to reveal the distribution of the target sequences in the genome. Such optical mapping (OM) has been implemented as an automated process using a limited set of available sequence-specific nicking nucleases for DNA labeling (commercialized by Bionano Genomics Inc [41]). Introduction of MTase-directed labeling, which makes no nicks in DNA, can open a new page in OM analysis [42] by i) extending the size of genomic maps to 20-100 Mbp and ii) expanding the selection of potentially available target specificities [14,43]. At this point, OM of mTAG labeled DNA has progressed to demonstrate its potential applicability for virus or macrosatellite genotyping [21,44]. Similarly, after mTAG biotin labeling, DNA sequences can be monitored using nanopore sensing. Biotin-bound streptavidin gives rise to current blockade signals during DNA passage through a quartz nanopore, which allowed simultaneous detection of two short genomes in a mixture [23]. Although presently both approaches are at the stage of technical refinement, the MTase-aided single molecule genotyping technologies may offer new versatile tools for genome analysis on the submegabase scale.

Figure 4. Schematic survey of transferase-directed covalent derivatization of nucleic acids and its applications.

Figure 4

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A. Site-specific methyltransferase (MTase)-directed tagging of DNA and RNA: B. Beta-glucosyltransferase (BGT)-directed tagging of 5-hydroxymethylcytosine in DNA. C. tRNA-modifying enzyme (tRNA guanine transglycosylase, TGT, or tRNAIle2-agmatidine synthetase, Tias)-directed tagging of RNA.

Applications of DNA MTases in epigenomic research

Modifications of cytosine (largely to m5C and hmC) in CG dinucleotides act as key epigenetic signals affecting gene expression and cellular differentiation in higher eukaryotes. Typically, 65–80% of the total 28 million CG sites are methylated in human tissues with characteristic epigenetic profiles observed in each tissue, cell type and disease condition, including cancers. Since the activity of DNA cytosine-5 MTases is blocked when the target cytosine residue is already modified, a CG-specific DNA C5-MTase, M.SssI, was engineered for two-step biotin labeling of unmodified CG dinucleotides. Subsequent enrichment of the covalently tagged DNA fraction followed by next generation sequencing yielded so-called “unmethylome” (inverse of the canonical DNA methylome) [39]. This medium resolution approach has been subsequently advanced by attaching a DNA oligonucleotide (instead of biotin) in the second step of labeling. Remarkably, it was found that a covalently tethered oligonucleotide can promote non-homologous priming of a DNA polymerase at the tagged sites to directly produce adjoining regions for their sequencing and precise genomic mapping at single CpG resolution [45]. The method named Tethered Oligonucleotide-Primed sequencing (TOP-seq), was validated for whole-genome epigenetic profiling of human tissues and cancer cell lines.

Notably, the M.TaqI adenine MTase is also blocked by C methylation within its recognition sequence (TCGA), permitting DNA unmethylome analysis in a subset of genomic CG sites. Moreover, a combination of sequence-specific [46] and methylation-sensitive mTAG labeling and multicolor readout yields a hybrid genetic/epigenetic map of chromosome segments spanning hundreds of kilobases [47]. Similarly, Gilboa et al. [22] demonstrated a proof of principle of epigenetic analysis using nanopore sensing. Again, M.TaqI was used to attach fluorophores at unmethylated TCGA sequences, in a mono-chromic or bi-colour manner. Electrical and optical signals were then recorded simultaneously as individual labeled DNA molecules were passing through solid-state nanopores. The demonstrated ability to detect and quantify multiple colors/signals from single DNA molecules holds promise of a high throughput automated multiplex analysis of distinct (epi)genetic features in clinical samples in near future (see accompanying review by Heck et al.).

In addition to their proper activity, DNA C5-MTases, which use a covalent mechanism for nucleophilic activation of their target cytosine residues, can also promote reactions involving non-cofactor-like substrates. In particular, the C5-MTase-activated 5-hydroxymethylcytosine (hmC) residues in DNA can undergo derivatization with aliphatic thiols yielding corresponding 5-alkylthiomethyl derivatives (Figure 1A Bottom and 4A Bottom) [48,49]. M.SssI-directed tagging of hmC with cysteamine and subsequent amine-selective biotin labeling was exploited for selective covalent capture of 5-hydroxymethylated-CG sites [48] (commercialized as EpiJET 5hmC Enrichment Kit by Thermo Fisher Scientific) and found laboratory applications [50,51]. Similar M.HhaI-directed labeling has been used for a proof-of-principle demonstration of a photo-electrochemical biosensor for hmC quantitation [52]. In epigenomic research, hmC, the product of enzymatic oxidation of m5C, is now extensively recognized both as a part of demethylation pathway and as an independent epigenetic mark (reviewed in [3,53,54]).

Other DNA modifying enzymes for epigenomic analysis

Besides MTases, only a few other enzymatic reactions have undergone engineering for biotechnological labeling of DNA. The most broadly used is the hmC-specific beta-glucosyltransferase (BGT) from bacteriophage T4. Engineered BGT reactions rely on the uridine 5’-diphosphate glucose cofactor in which the 6-position of the naturally transferable D-glucose moiety is functionalized with an amine, ketone or azide group [55–57] (Figure 1B). The latter click-compatible derivative (UDP-6-N3-Glc) proved the most popular – it is already available commercially and can also be prepared in situ from inexpensive precursor compounds [58].

Recent analytical applications, with hmC as a biomarker (Figure 4B), include a biotin-based enrichment and next generation sequencing of cell-free DNA for cancer diagnostics and monitoring [59,60] (see also accompanying review [61]). Besides the elaborate genomic analyses, nanopore sensing permitted quantitation of the biotin-derivatized hmC in the murine genome [62], whereas optical quantitation of fluorescently labeled genomic hmC in minute blood samples proved valuable for diagnostics of colorectal and blood cancer [63] (see accompanying review by Heck et al.). In addition, a one-pot combination of TET oxygenase and BGT activities enabled detection of proximal m5C and hmC modifications using single-molecule FRET [64]. Recently, single-molecule dual optical mapping of genetic and epigenetic features in large fragments of human DNA has been demonstrated using commercial nanochannel arrays [65].

RNA modifying enzymes for transcriptome and epitranscriptome analysis

Every living cell contains multiple types of RNA molecules. A big variety of chemical groups as well as different positions in an RNA molecule can be modified by methyltransferases resulting in over 70 distinct modifications [66]. To date, a handful of these enzymes have been adapted for targeted covalent tagging of RNA (Figures 3B and 4A).

mRNA cap methyltransferases

Covalent labeling of the 5’-end of messenger RNA could provide tools for studies of trafficking and localization of genetic transcripts in eukaryotic cell. A substantial progress in the mRNA cap modification has been made by exploiting two enzymes: the GlaTgs2 MTase, which methylates the N2 position of the m7G 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 m7G capped RNAs, respectively, [13,15,17,19,32,37,67] as well as dinucleotide cap variants m7GpppA 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 [13,15–17,19,32,37,67,68]. Using the Ecm1-directed labeling, it was demonstrated that photo-reactive arylazide or diazirine groups can be used to detect specific RNA-protein interaction in vitro [19]. Combining the above tagging reactions in a one-pot cascade format, dual labeling of the 5’-cap with different reporters has been demonstrated [68].

3’ terminal RNA methyltransferases

Another type of RNA methyltransferases, HEN1 enzymes, modify the 2’-OH position of 3’-terminal nucleosides regardless of their identity. In cells, HEN1 transferases are guided by additional proteins to methylate distinct classes of regulatory RNAs such as miRNA and siRNA duplexes in plants [69] or single stranded piRNA and siRNA molecules in animals [25]. As mentioned above, the broad cofactor tolerance by both types of HEN1 MTases permits robust targeted deposition of a wide spectrum of functional or reporter groups including biotin or Cy3 fluorophore [24,25,36]. In particular, the AtHEN1 MTase was shown to be instrumental for DNA-addressed tagging of miRNA and siRNA strands. This reaction proved tolerant to the presence of additional chemical moieties (such as aptamers or fluorophores) on the probing DNA strand [24] permitting further development of two-reporter analytical systems for sequence-specific miRNA analysis in biological samples. Meanwhile a truncated variant of DmHen1 showed efficient derivatization of a wide range of ssRNA substrates regardless of their length (20–80 nt) or the nature of the 3’-terminal base, even in the presence of endogenous AdoMet [25] suggesting its potential applicability for in vivo RNA profiling experiments [70].

Internal RNA methyltransferases

The majority of biological RNA modifications occur at internal nucleosides [66]. It was shown that the substrates of tRNA:m22G26-methyltransferase Trm1 and tRNA:m2G10-methyltransferase Trm11 can be specifically modified with terminal alkynyl group and further visualized after Cu+-catalyzed AAC or even applied for single-molecule fluorescence experiment [71]. Synthetically tunable sequence-specific 2’-O-tagging and click labeling was demonstrated using an archaeal box C/D ribonucleoprotein complex [40]. Besides the described general labeling tools, profiling of internal m6A modifications in mRNA has recently gained increased attention as this modification has been associated with regulation of mRNA metabolism and cell differentiation [72]. However, the N6-methyl groups are “invisible” to general transcriptomic techniques and dedicated approaches for the identification of residues targeted by the heterodimeric METTL3-METTL14 MTase have recently been proposed. In one instance, it was shown that the action of the human MTase in the presence of an engineered cofactor can produce N6-allyladenosine residues; subsequent treatment with aqueous iodine leads to a N1,N6-cyclic derivative, which is revealed by the appearance of misincorporated bases in RNA sequencing data [18]. Independently, METTL3-METTL14 was applied to modify its target sites with propargyl groups, which were then used for click-biotinylation and immobilization of the RNA to streptavidin beads. The created steric barrier prompted high frequency termination of reverse transcription at the modified nucleosides enabling their mapping [33].

Other tRNA modifying enzymes for post-transcriptional RNA labeling

Two other tRNA modifying enzymes have been successfully applied for sequence-specific RNA labeling using modified co-substrate analogs (Figures 1C, D and 4C). In both cases, enzyme-directed covalent modification of a target sequence (prokaryotic tRNA or its fragment) genetically fused to an RNA of interest was exploited. The incorporated functional handles or direct reporters were used to detect the presence and location of the RNA in cellular extracts or fixed cells [73,74]. A tRNAIle2-agmatidine synthetase (Tias), which catalyses the attachment of the agmatine (4-aminobutylguanidine) moiety to C34 of A. fulgidus tRNAIle2, was used to modify RNA substrates with agmatine analogs bearing azide or alkyne functional groups [73]. E. coli tRNA guanine transglycosylase (TGT) was shown to use a shorter 17-nt fragment of tRNATyr for derivatization of RNA. TGT functions by replacing G34 with the preQ1 nucleobase, which can be appended with a fluorophore or biotin beforehand [74,75] or in a subsequent step [76]. Notably, the latter system can even be configured for spatiotemporal regulation of translation in transfected HeLa cells: modification of the inserted target sequences in the 5’ UTR with bulky light-sensitive groups leads to stalled translation, which is readily reversed by light–induced uncaging of mRNA [77].

Conclusions

Repurposing enzymatic reactions for covalent derivatization of selected targets in DNA and RNA (Figure 1) is coming of age, counting presently over 20 examples. By far the most widely exploited are MTases, followed by BGT (Figure 4). The mTAG technique [4,9] offers a very broad selection of tagging chemistries (Figure 2) as well as molecular features on DNA and RNA (Figure 3) that can be targeted in a highly specific manner in vitro. In vivo tagging of biological methylation sites has been attempted in model systems and currently appears to suffer from technical limitations: the size restrictions of accepted methionine analogs, competition by endogenous methionine and AdoMet and the MTase selectivity; bypassing these hurdles will permit deciphering deeper layers of the cellular mechanisms underlying transcriptional and translational regulation in mammals. Both types of reactions (DNA MTases and BGT) have been implemented in validated sequencing-based protocols for epigenome analysis ready for routine laboratory and, potentially, clinical applications [39,45,59,60] (see also accompanying reviews [61] and Heck et al.). However, the utility of mTAG labeling is further increased with recent technological advances in physical detection of individual tagged reporter molecules on DNA and RNA. Optical (super-resolution microscopy) and/or electrochemical (nanopore sensing) positioning of covalently tagged reporters along the contour of DNA (currently at 50–200 bp resolution) offers single-molecule multiplexed readout permitting high throughput automated mapping of genetic and epigenetic features in minute biological samples. The techniques are at different stages of technical maturity, awaiting further refinement and clinical validation. Two other RNA modifying enzymes (Figures 1C and D) have been successfully adapted for in vivo labeling of RNA at short genetically fused affinity tags (Figure 4C). Altogether, a handful of developed powerful techniques have collected enough of proofs of principle and are currently on the brink of real benchtop applications in both fundamental research and next generation diagnostics.

Highlights.

  • Enzymatic transfer of chemically extended groups enables targeted covalent functionalization of DNA and RNA

  • Methyltransferase-directed labeling of DNA and RNA provides novel tools for (epi)genomic and (epi)transcriptomic studies

  • Detection of individual covalently-bound reporters on DNA permits single-molecule analysis of (epi)genomes

Acknowledgements

The authors wish to acknowledge support from the European Research Council [grant ERC-2016-ADG 742654 to S.K.], the Lithuanian Research Council [grant SEN-17/2015 to G.V.] and the H2020 COST consortium CM1406 for facilitating constructive discussion platforms.

Footnotes

Declarations of interest

M.M, G.V and S.K are inventors on related patents and patent applications

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