SHAMS: combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids.
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.
Turner
Kevin B
KB
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, USA.
Yi-Brunozzi
Hye Young
HY
Brinson
Robert G
RG
Marino
John P
JP
Fabris
Daniele
D
Le Grice
Stuart F J
SF
D000215:Acylation; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004271:DNA, Fungal; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; C034477:RNA primers; D012331:RNA, Fungal; D012367:RNA, Viral; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase; C496084:reverse transcriptase Ty3, S cerevisiae