Oligonucleotide‐Addressed Covalent 3′‐Terminal Derivatization of Small RNA Strands for Enrichment and Visualization

Osipenko, Aleksandr; Plotnikova, Alexandra; Nainytė, Milda; Masevičius, Viktoras; Klimašauskas, Saulius; Vilkaitis, Giedrius

doi:10.1002/ange.201701448

Cited by 7 publications

(12 citation statements)

References 23 publications

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“…In combination with non‐natural substrates and click chemistry, this approach is attractive to isolate, enrich and identify subclasses of RNAs or targets of these RNA‐modifying enzymes. Impressive examples are NAD‐captureSeq and different Hen1‐MTases for enrichment for RNA‐species (Cahova et al, ; Mickutė et al, ; Osipenko et al, ).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to the isolation via substrate‐bound, complementary oligonucleotides, the 3′ installed handle remains on the RNA and can be used again for purification after subsequent reactions. Additionally, the covalent 3′‐modification allows for more stringent washes than the hybridized alternative (Osipenko et al, ).…”

Section: Enrichment and Detection Of Rna Modifications In Isolated Tomentioning

confidence: 99%

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Chemo‐enzymatic treatment of RNA to facilitate analyses

2019

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Labeling RNA is a recurring problem to make RNA compatible with state-of-theart methodology and comes in many flavors. Considering only cellular applications, the spectrum still ranges from site-specific labeling of individual transcripts, for example, for live-cell imaging of mRNA trafficking, to metabolic labeling in combination with next generation sequencing to capture dynamic aspects of RNA metabolism on a transcriptome-wide scale. Combining the specificity of RNAmodifying enzymes with non-natural substrates has emerged as a valuable strategy to modify RNA site-or sequence-specifically with functional groups suitable for subsequent bioorthogonal reactions and thus label RNA with reporter moieties such as affinity or fluorescent tags. In this review article, we will cover chemoenzymatic approaches (a) for in vitro labeling of RNA for application in cells, (b) for treatment of total RNA, and (c) for metabolic labeling of RNA.

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Section: Discussionmentioning

confidence: 99%

Section: Enrichment and Detection Of Rna Modifications In Isolated Tomentioning

confidence: 99%

Chemo‐enzymatic treatment of RNA to facilitate analyses

2019

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“…In addition, groups carrying an alkene [15][16][17][18], a norbornene moiety (for bioorthogonal tetrazine ligation [13]), a photocrosslinker [19] and a photoremovable cage [20] have recently become available. An increasingly popular choice is onestep mTAG labeling with fluorophores or biotin [21][22][23][24][25].…”

Section: Engineering Adomet-dependent Transmethylation Reactionsmentioning

confidence: 99%

“…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).…”

Section: Engineering Adomet-dependent Transmethylation Reactionsmentioning

confidence: 99%

“…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][21][22][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).…”

Section: Engineering Adomet-dependent Transmethylation Reactionsmentioning

confidence: 99%

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

Tomkuvienė

Mickutė

Vilkaitis

et al. 2019

Current Opinion in Biotechnology

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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.

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Tag‐Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases

2020

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The mRNA modification N6‐methyladenosine (m6A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3‐METTL14 and METTL16 act as “writers” for different target transcripts and sequence motifs. The modification is perceived by dedicated “reader” and “eraser” proteins, but not by polymerases. We report that METTL3‐14 shows remarkable cosubstrate promiscuity, enabling sequence‐specific internal labeling of RNA without additional guide RNAs. The transfer of ortho‐nitrobenzyl and 6‐nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase‐catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo‐enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo‐enzymatic modification of different sites in a single RNA.

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Oligonucleotide‐Addressed Covalent 3′‐Terminal Derivatization of Small RNA Strands for Enrichment and Visualization

Cited by 7 publications

References 23 publications

Chemo‐enzymatic treatment of RNA to facilitate analyses

Chemo‐enzymatic treatment of RNA to facilitate analyses

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

Tag‐Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases

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