NDLI: Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS

Content Provider	Springer Nature Link
Author	Beverly, Michael Dell, Amy Parmar, Parul Houghton, Leslie
Copyright Year	2016
Abstract	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. Graphical abstract mRNA 5′ end analysis with RNAse H cleavage and capture probe
Starting Page	5021
Ending Page	5030
Page Count	10
File Format	PDF
ISSN	16182642
Journal	Analytical and Bioanalytical Chemistry
Volume Number	408
Issue Number	18
e-ISSN	16182650
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2016-05-18
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	mRNA RNase H 5′ cap LC-MS N7-Methylguanosine RNA Analytical Chemistry Biochemistry Laboratory Medicine Characterization and Evaluation of Materials Food Science Environmental Monitoring/Analysis
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry Biochemistry

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