SHAMS: combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids.

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TitleSHAMS: combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids.
Publication TypeJournal Article
Year of Publication2009
AuthorsTurner, KB, Yi-Brunozzi, HYoung, Brinson, RG, Marino, JP, Fabris, D, Le Grice, SFJ
JournalRNA
Volume15
Issue8
Pagination1605-13
Date Published2009 Aug
ISSN1469-9001
KeywordsAcylation, Base Sequence, DNA, DNA, Fungal, DNA, Viral, HIV-1, Nuclear Magnetic Resonance, Biomolecular, Nucleic Acid Heteroduplexes, Nucleic Acid Hybridization, RNA, RNA, Fungal, RNA, Viral, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Tandem Mass Spectrometry
Abstract

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2'-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)-containing RNA/DNA hybrids, their size (20-25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (M(r) = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2'-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed.

DOI10.1261/rna.1615409
Alternate JournalRNA
PubMed ID19535461
PubMed Central IDPMC2714758
Grant ListGM59107 / GM / NIGMS NIH HHS / United States
GM643208 / GM / NIGMS NIH HHS / United States