Nucleoside Digestion Mix Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro"
Article Title: RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro
Figure Legend Snippet: RNA-mediated restriction of methyltransferase like-3 (METTL3)-METTL14 activity. ( a ) Methyltransferase activity of METTL3-METTL14 in the presence of various DNA or RNA substrates measured by radiometric assay. CPM, counts per minute. The highest activity was measured with the d6T* oligo. ( b ) FP-based binding assay for DNA and RNA oligos showing highest affinity for rNEAT2 RNA (green) and lowest affinity for d6T* oligo (red). Equilibrium dissociation constants (Kd) for each oligo are shown on the right side of the isotherms. The data were fit into one site-specific binding model (Y = Bmax*X/(Kd+ X)). See Materials and methods section and source data for details. ( c ) Methyltransferase activity of METTL3-METTL14 on the respective RNA oligos (red), d6T* DNA alone (black) and equimolar mixture of the two (blue), measured by radiometric assay. ( d ) Predicted secondary structures of each oligonucleotide. Yellow, RNA strand; black, DNA strand. The values of the equilibrium dissociation constants (Kd) shown for each oligonucleotide indicate an inverse relationship between binding affinity and methyltransferase activity. ( e ) Dose-dependent inhibition of METTL3-METTL14 activity by RNA oligos rNEAT2 or rTCE23, as measured by radiometric assay in a reaction buffer containing 5.0 mM NaCl (upper panel) and 50.0 mM NaCl (lower panel). IC 50 , concentration of RNA required to achieve 50% inhibition of the METTL3-METTL14 activity. ( f ) Attenuation of the methyltransferase activity in presence of rNEAT2 or rTCE23, as measured by oligonucleotide intact mass analysis. Quantitation of modified dA (black circle) or rA (red square) is shown in absence or presence of equivalent amounts of rNEAT2 or rTCE23. ( g ) Nucleoside composition analysis of the METTL3-METTL14 reactions. UHPLC chromatograms showing the reaction in absence (blue trace) or in the presence of either rNEAT2 (red trace) or rTCE23 (green trace). The quantitation of the fraction of modified bases in the nucleoside pool was consistent with the results from the oligonucleotide intact mass analysis shown in ( f ). The insert shows the full chromatographic trace with all detected nucleosides. Results presented in panels ( a–c and e–f ) are the average of three independent experiments (n = 3) with one standard deviation (s.d.) for each oligonucleotide (shown as error bars). Source data are provided as a Source Data file. MTase activity on individual RNA/DNA substrates. Binding isotherms of METTL3-METTL14. Comparison of MTase activity on different RNA/DNA substrates. Dose-dependent inhibition of MTase activity by RNA. RNA-mediated attenuation of MTase activity by intact mass analysis.
Techniques Used: Activity Assay, FP-binding Assay, Binding Assay, Inhibition, Concentration Assay, Quantitation Assay, Modification, Standard Deviation
2) Product Images from "Strategies to Avoid Artifacts in Mass Spectrometry‐Based Epitranscriptome Analyses"
Article Title: Strategies to Avoid Artifacts in Mass Spectrometry‐Based Epitranscriptome Analyses
Journal: Angewandte Chemie (International Ed. in English)
Figure Legend Snippet: Impact of RNA hydrolysis conditions on detection of RNA modifications. (A) Abundance of peak with GpsG mass transition from synthetic GpsG containing RNA and native RNA from HEK cells digested with Nuclease P1 (NP1) and calf intestine phosphatase (CIP) at 37 °C for different incubation times. (B–D) Abundance of canonical nucleosides (rN) and various modified nucleosides from HEK total RNA digested with: 1, Benzonase/PDE1/CIP;  2, NP1/CIP;  or 3 a commercial RNA hydrolysis kit (NEB, Nucleoside Digestion Mix). (E) Abundance of ribose methylated nucleosides from HEK total RNA digested in the absence (−) and presence (+) of phosphodiesterase 1 (PDE1) using either: 1, Benzonase+CIP  or 2, NP1+CIP.  All data represent mean ± SD for 3 experimental replicates.
Techniques Used: Incubation, Modification, Methylation