Nuclear Magnetic Resonance Structure of an 8 × 8 Nucleotide RNA Internal Loop Flanked on Each Side by Three Watson–Crick Pairs and Comparison to Three-Dimensional Predictions

Abstract

The prediction of RNA 3D structure from sequence alone is a long-standing goal. High resolution, experimentally determined structures of simple noncanonical pairings and motifs are critical to the development of prediction programs. Here, we present the NMR structure of the duplex, (5′CCAGAAACGGAUGGA)2, which contains an 8x8 nucleotide internal loop flanked by three Watson-Crick pairs on each side. The loop is comprised of a central 5′AC/3′CA nearest neighbor flanked by two 3RRs motifs, a known stable motif consisting of three consecutive sheared GA pairs. Hydrogen bonding patterns between base pairs in the loop, all-atom RMSD for the loop, and deformation index were used to compare the structure to automated predictions by MC-sym, RNA FARFAR, and RNAComposer.

Nuclear Magnetic Resonance Reveals That GU Base Pairs Flanking Internal Loops Can Adopt Diverse Structures

“…Dihedral angles were restrained to typical A-form RNA values where the data indicated canonical pairs, and for angles with direct scalar-coupling evidence. 22 Tables S6–8 contain restraints used in simulated annealing.…”

“…However, spectral overlap prevented measurement of many such peaks, so a majority of NOE volumes were binned into four categories: strong (1.5–2.7 Å), medium (2.0–3.5 Å), weak (2–4.7 Å), and very weak (typically used for spin diffusion peaks, 2.5–6 Å). Dihedral angles were restrained to typical A-form RNA values where the data indicated canonical pairs and for angles with direct scalar-coupling evidence Tables S6–S8 contain restraints used in simulated annealing.…”

Differential Effects of Strand Asymmetry on the Energetics and Structural Flexibility of DNA Internal Loops

“…The presence of single-stranded regions within one or both strands of continuous helical domains alters the overall conformation and flexibility. Ensemble and single-molecule fluorescence studies show that the presence of single and tandem mismatches can enhance the flexibility and induce sharp bends. − Larger, single-stranded domains confined to one strand (i.e., bulges) produce a bent conformation that acts as a flexible hinge in which the degree of bending and flexibility is sensitive to the size and sequence of the unpaired nucleotides in the bulge. − , In contrast, symmetric loops (same number of unpaired nucleotides on both strands) produce an elongated form relative to a fully paired duplex. , Additional structural studies of RNA ,, and DNA symmetric internal loops show that the overall helical structure within these domains is maintained by significant loop stabilization through noncanonical pairings and base stacking interactions, yet they have larger end-to-end distances and greater flexibility relative to those of fully paired duplexes because of partial destabilization of local helical structure and base stacking . These conformational differences for bulge and symmetric loops highlight the structural implications of the contribution of strand asymmetry on loop entropy and enthalpy.…”

“…[2][3][4][5]43 In contrast, symmetric loops (same number of unpaired nucleotides on both strands) produce an elongated form relative to a fully paired duplex. 44,45 Additional structural studies of RNA 36,46,47 and DNA 48 symmetric internal loops show that the overall helical structure within these domains is maintained by significant loop stabilization through noncanonical pairings and base stacking interactions, yet they have larger end-to-end distances and greater flexibility relative to those of fully paired duplexes because of partial destabilization of local helical structure and base stacking. 44 These conformational differences for bulge and symmetric loops highlight the structural implications of the contribution of strand asymmetry on loop entropy and enthalpy.…”

Secondary structure prediction for RNA sequences including N6-methyladenosine

“…NOESY spectra with excitation optimized for imino protons were acquired with mixing times of 50, 150, and 250 ms, and with excitation optimized for aromatic protons and greater indirect resolution with mixing times of 75 and 400 ms. TOCSY spectra (30 ms mixing time) were used to identify pyrimidine H5-H6 cross-peaks and dynamic sugar residues. Spectra from 2D NMR were processed and assigned with NMRpipe 88 and NMRFAM-SPARKY 89 as described 90 .…”