Fig. 1.

NOESY spectra indicate an intrahelical adenosine (A8) at the expected branch site and a partially syn conformer for the preceding guanosine (G7) for the pseudouridine (Ψ)-modified U2 snRNA – BPS duplex. (a) Top panel: One-dimensional spectrum of the imino protons at 0 °C. The RNA sequences are inset (expected branch site A8, red; preceding G7, blue; other consensus sequences, black; terminal nucleotides to facilitate NMR analysis, grey). Four lower panels: Two-dimensional NOESY spectra of the aromatic/amino region of the pseudouridine-containing duplex in 100% D₂O at 15 °C with 400 ms mixing time (panels spanning 4.5–7.1 ppm) and in 95/5% H₂O/D₂O at 0 °C with 50 ms mixing time (bottom panel). Gray lines in the D₂O NOESY trace the H8/6-H1′ backbone walk with intra-residue peaks labeled. Cross-peaks between G-imino and C-amino protons and between U-imino and A-H2 protons indicating Watson-Crick base-pairs are connected by light gray lines in the H₂O NOESY. Key peaks for defining the branch site conformation are circled and labeled in bold font. Contours in the boxed region of the H₂O spectrum are drawn five-fold lower than in the rest of the spectrum to show the Ψ35H3-A8H2 cross-peak. (b) Diagram of major branch site interactions: Double-headed arrows, canonical Watson-Crick base-pairs; blue lines, NOESY interactions used to determine the relative position of G7; red lines, NOESY interactions used to identify the relative position of A8. Dashed lines represent weak NOEs.