Structural probing of the HIV-1 polypurine tract RNA:DNA hybrid using classic nucleic acid ligands

The interactions of archetypical nucleic acid ligands with the HIV-1 polypurine tract (PPT) RNA:DNA hybrid, as well as analogous DNA:DNA, RNA:RNA and swapped hybrid substrates, were used to probe structural features of the PPT that contribute to its specific recognition and processing by reverse transcriptase (RT). Results from intercalative and groove-binding ligands indicate that the wild-type PPT hybrid does not contain any strikingly unique groove geometries and/or stacking arrangements that might contribute to the specificity of its interaction with RT. In contrast, neomycin bound preferentially and selectively to the PPT near the 5′(rA)4:(dT)4 tract and the 3′ PPT-U3 junction. Nuclear magnetic resonance data from a complex between HIV-1 RT and the PPT indicate RT contacts within the same regions highlighted on the PPT by neomycin. These observations, together with the fact that the sites are correctly spaced to allow interaction with residues in the ribonuclease H (RNase H) active site and thumb subdomain of the p66 RT subunit, suggest that despite the long cleft employed by RT to make contact with nucleic acids substrates, these sites provide discrete binding units working in concert to determine not only specific PPT recognition, but also its orientation on the hybrid structure

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 (Mr = 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