Dual 5′ Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions

Holstein, Josephin Marie; Muttach, Fabian; Schiefelbein, Stephan H. H.; Rentmeister, Andrea

doi:10.1002/chem.201604816

Cited by 44 publications

(36 citation statements)

References 68 publications

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“…184 By using the above-mentioned methyltransferases and exploring their regioselectivity, dual 5 0 Cap labelling of mRNAs was also achieved. 185 4.3.3 Enzyme-mediated modification of DNA. DNA IEDDA labelling has also been achieved using DNA polymerase, reverse transcriptase via primer extension (PEX) or reverse transcription.…”

Section: Alternative Enzymatic Methods or Enzyme Tags For Protein Modmentioning

confidence: 99%

Inverse electron demand Diels–Alder reactions in chemical biology

2017

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The emerging inverse electron demand Diels-Alder (IEDDA) reaction stands out from other bioorthogonal reactions by virtue of its unmatchable kinetics, excellent orthogonality and biocompatibility. With the recent discovery of novel dienophiles and optimal tetrazine coupling partners, attention has now been turned to the use of IEDDA approaches in basic biology, imaging and therapeutics. Here we review this bioorthogonal reaction and its promising applications for live cell and animal studies. We first discuss the key factors that contribute to the fast IEDDA kinetics and describe the most recent advances in the synthesis of tetrazine and dienophile coupling partners. Both coupling partners have been incorporated into proteins for tracking and imaging by use of fluorogenic tetrazines that become strongly fluorescent upon reaction. Selected notable examples of such applications are presented. The exceptional fast kinetics of this catalyst-free reaction, even using low concentrations of coupling partners, make it amenable for in vivo radiolabelling using pretargeting methodologies, which are also discussed. Finally, IEDDA reactions have recently found use in bioorthogonal decaging to activate proteins or drugs in gain-of-function strategies. We conclude by showing applications of the IEDDA reaction in the construction of biomaterials that are used for drug delivery and multimodal imaging, among others. The use and utility of the IEDDA reaction is interdisciplinary and promises to revolutionize chemical biology, radiochemistry and materials science.

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Section: Alternative Enzymatic Methods or Enzyme Tags For Protein Modmentioning

confidence: 99%

Inverse electron demand Diels–Alder reactions in chemical biology

2017

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“…At the same time, we found that hTgs1 did not accept any AdoMet analogues in previous studies . To this end, CMTR1 160–549 was incubated with the AdoMet analogues SeAdoYn or Hey‐SAM, synthetic RNA 8 equipped with a cap0, and MTAN (Figure S10) . Reaction progress was monitored by means of HPLC.…”

Section: Resultsmentioning

confidence: 83%

Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5′ Caps

et al. 2019

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Eukaryotic RNAs are heavily processed, including co‐ and post‐transcriptional formation of various 5′ caps. In small nuclear RNAs (snRNAs) or small nucleolar RNAs (snoRNAs), the canonical 7mG cap is hypermethylated at the N2‐position, whereas in higher eukaryotes and viruses 2′‐O‐methylation of the first transcribed nucleotide yields the cap1 structure. The function and potential dynamics of several RNA cap modifications have not been fully elucidated, which necessitates preparative access to these caps. However, the introduction of these modifications during chemical solid‐phase synthesis is challenging and enzymatic production of defined short and uniform RNAs also faces difficulties. In this work, the chemical synthesis of RNA is combined with site‐specific enzymatic methylation by using the methyltransferases human trimethylguanosine synthase 1 (hTgs1), trimethylguanosine synthase from Giardia lamblia (GlaTgs2), and cap methyltransferase 1 (CMTR1). It is shown that RNAs with di‐and trimethylated caps, as well as RNAs with caps methylated at the 2′‐O‐position of the first transcribed nucleotide, can be conveniently prepared. These highly modified RNAs, with a defined and uniform sequence, are hard to access by in vitro transcription or chemical synthesis alone.

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“…This is consistent with the in vitro results, showing that recombinant METTL3–METTL14 complex efficiently transfers the propargyl group to the N 6 -position in adenosine. Many other cellular MTases, including the ones from the eukaryotic 5′-capping machinery, also show a high level of promiscuity in vitro regarding the SAM analogues [ 47 ]. After the total RNA isolation, the transferred propargyl groups can be functionalized in a click-reaction with biotin azide, which enables enrichment of the RNA regions containing methylation sites on streptavidin beads.…”

Section: Approaches Involving Modification Steps In Vivo mentioning

confidence: 99%

Emerging approaches for detection of methylation sites in RNA

Ovcharenko¹,

Rentmeister²

2018

Open Biol.

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RNA methylations play a significant regulatory role in diverse biological processes. Although the transcriptome-wide discovery of unknown RNA methylation sites is essential to elucidate their function, the development of a bigger variety of detection approaches is desirable for multiple reasons. Many established detection methods for RNA modifications heavily rely on the specificity of the respective antibodies. Thus, the development of antibody-independent transcriptome-wide methods is beneficial. Even the antibody-independent high-throughput sequencing-based methods are liable to produce false-positive or false-negative results. The development of an independent method for each modification could help validate the detected modification sites. Apart from the transcriptome-wide methods for methylation detection de novo, methods for monitoring the presence of a single methylation at a determined site are also needed. In contrast to the transcriptome-wide detection methods, the techniques used for monitoring purposes need to be cheap, fast and easy to perform. This review considers modern approaches for site-specific detection of methylated nucleotides in RNA. We also discuss the potential of third-generation sequencing methods for direct detection of RNA methylations.

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Dual 5′ Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions

Cited by 44 publications

References 68 publications

Inverse electron demand Diels–Alder reactions in chemical biology

Inverse electron demand Diels–Alder reactions in chemical biology

Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5′ Caps

Emerging approaches for detection of methylation sites in RNA

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