We are analyzing https://link.springer.com/article/10.1007/s00216-016-9605-x.

Title:
Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS | Analytical and Bioanalytical Chemistry
Description:
A label-free method for determining the 5′-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the 5′ end of target mRNA were used with RNase H to cleave the 5′ end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quantitative and qualitative information on the 5′ cap was determined from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5′ RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5′ cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA preparations was determined to be between 88 and 98 % depending on the modification type and length of the mRNA. mRNA of 2.2K and 9K nucleotides in length and containing the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping.
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Keywords {🔍}

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Topics {✒️}

parul parmar & leslie houghton mrna-based therapeutics—developing month download article/chapter liquid chromatography/mass spectrometry �anti-reverse” cap analogs anti-reverse cap analogs streptavidin-coated magnetic beads vaccinia-capped mrna preparations bioanalytical chemistry aims capture probe methods enzymol biotin-tagged probes privacy choices/manage cookies label-free method bmc mol biol phage rna polymerases label-free analysis full article pdf full-size enzyme synthetic modified mrna high translational efficiency rna cap synthesis vitro transcribed rna sequencing control nucleic acid vaccines mrna 5′ cap-analogues fully capped mrna cap-binding proteins study mrna capping european economic area enzymatically active xrn1 xenopus oocyte nuclei biochim biophys acta truncated cd40l protein superior translational properties sequence-dependent hydrolysis modified oligonucleotide splints strong anion-exchange highly efficient reprogramming increased translational capacity de la pena darpa grant hr-0011-13-3-0003 electronic supplementary material mrna decapping status chemically modified mrna sequence-engineered mrna mrna capping efficiency 5′-end cap identity lc-ms

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