2016
DOI: 10.1002/0471142700.nc0136s64
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Synthesis of S‐Adenosyl‐L‐Methionine Analogs with Extended Transferable Groups for Methyltransferase‐Directed Labeling of DNA and RNA

Abstract: S-Adenosyl-L-methionine (AdoMet) is a ubiquitous methyl donor for a variety of biological methylation reactions catalyzed by methyltransferases (MTases). AdoMet analogs with extended propargylic chains replacing the sulfonium-bound methyl group can serve as surrogate cofactors for many DNA and RNA MTases enabling covalent deposition of these linear chains to their cognate targets sites in DNA or RNA. Here we describe synthetic procedures for the preparation of two representative examples of AdoMet analogs with… Show more

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Cited by 14 publications
(11 citation statements)
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“…The templates for in vitro transcription were prepared as detailed in Supplementary Methods . Cofactor AdoMet 1 analogues Ado-6-amine 2 , Ado-6-azide 3 and Ado-13-biotin 4 were prepared as outlined elsewhere ( 10 , 25 ). The synthesis of Ado-14-Cy3 5 is described in Supplementary Methods .…”
Section: Methodsmentioning
confidence: 99%
“…The templates for in vitro transcription were prepared as detailed in Supplementary Methods . Cofactor AdoMet 1 analogues Ado-6-amine 2 , Ado-6-azide 3 and Ado-13-biotin 4 were prepared as outlined elsewhere ( 10 , 25 ). The synthesis of Ado-14-Cy3 5 is described in Supplementary Methods .…”
Section: Methodsmentioning
confidence: 99%
“…In uTOP-seq, 4–10 ng of cfDNA (or 100 ng of CV tissue DNA, sheared to 200 bp by Covaris sonicator) were labeled with 0.11 μM eM.SssI [ 23 ] in 10 mM Tris–HCl (pH 7.4), 50 mM NaCl, 0.5 mM EDTA buffer supplemented with 200 μM Ado-6-azide cofactor [ 43 ] for 1 h at 30 °C followed by thermal inactivation at 65 °C for 20 min and Proteinase K treatment (0.2 mg/ml) for 30 min at 55 °C and finally column purified (GeneJET PCR purification kit, Thermo Fisher Scientific (TS)). In hmTOP-seq, 5hmC glycosylation was carried out with 5–10 ng of cfDNA (or 200 ng of CV tissue DNA, sheared to 200 bp by Covaris sonicator) supplemented with 50 µM UDP-6-azide-glucose (Jena Bioscience) and 2.5–5 U T4 β-glucosyltransferase (TS) for 1 h 37 °C followed by enzyme inactivation at 65 °C for 20 min and column purification (GeneJET PCR Purification kit (TS)).…”
Section: Methodsmentioning
confidence: 99%
“…For mTAG labeling, 50-500 ng of gDNA was incubated with eM.SssI (0.5-1 μM) in TNB buffer (10 mM Tris-HCl (pH7.4), 50 mM NaCl, 0.1 mg/ml BSA) supplemented with 200 μM Ado-6-azide cofactor (Kriukienė et al, 2013; Masevicius et al, 2016) for 1 hour at 30 °C followed by Proteinase K treatment (0.2 mg/ml) for 30 min at 55°C and column purification (GeneJet PCR Purification kit, Thermo Scientific (TS)).…”
Section: Methods Detailsmentioning
confidence: 99%