title: Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS.
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.
authors.affiliation: Novartis Institutes of Biomedical Research, 700 Main Street, Cambridge, MA, 02139, USA. michael.beverly@novartis.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Beverly
authors.affiliation: Novartis Institutes of Biomedical Research, 700 Main Street, Cambridge, MA, 02139, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Dell
authors.affiliation: Novartis Institutes of Biomedical Research, 700 Main Street, Cambridge, MA, 02139, USA.
authors.forename: Parul
authors.initials: P
authors.lastname: Parmar
authors.affiliation: Novartis Institutes of Biomedical Research, 700 Main Street, Cambridge, MA, 02139, USA.
authors.forename: Leslie
authors.initials: L
authors.lastname: Houghton
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry / Q000235:genetics; D012315:RNA Caps / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D015203:Reproducibility of Results; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D015335:Molecular Probes; D012315:RNA Caps; D012333:RNA, Messenger; D016914:Ribonuclease H
keywords: 5′ cap; LC-MS; N7-Methylguanosine; RNA; RNase H; mRNA