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Label-free analysis of mRNA capping efficiency using RNAse H probes and LC-MS

Beverly, Michael and Dell, Amy and Parmar, Parul and Houghton, Leslie (2016) Label-free analysis of mRNA capping efficiency using RNAse H probes and LC-MS. Analytical and Bioanalytical Chemistry, 408 (18). pp. 5021-5030. ISSN 1618-2650

Abstract

A label free method for determining the 5’ end cap identity and orientation on 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 good sensitivity for detecting capping reaction impurities and uncapped triphosphate mRNA spiked into 100picomoles 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.

Item Type: Article
Keywords: mRNA, RNA capping, ARCA
Date Deposited: 12 Oct 2016 00:45
Last Modified: 12 Oct 2016 00:45
URI: https://oak.novartis.com/id/eprint/27051

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