A novel form of RNA double helix based on G·U and C·A+ wobble base pairing

Abstract

Wobble base pairs are critical in various physiological functions and have been linked to local structural perturbations in double-helical structures of nucleic acids. We report a 1.38-Å resolution crystal structure of an antiparallel octadecamer RNA double helix in overall A conformation, which includes a unique, central stretch of six consecutive wobble base pairs (W helix) with two G·U and four rare C·A⁺ wobble pairs. Four adenines within the W helix are N1-protonated and wobble-base-paired with the opposing cytosine through two regular hydrogen bonds. Combined with the two G·U pairs, the C·A⁺ base pairs facilitate formation of a half turn of W-helical RNA flanked by six regular Watson-Crick base pairs in standard A conformation on either side. RNA melting experiments monitored by differential scanning calorimetry, UV and circular dichroism spectroscopy demonstrate that the RNA octadecamer undergoes a pH-induced structural transition which is consistent with the presence of a duplex with C·A⁺ base pairs at acidic pH. Our crystal structure provides a first glimpse of an RNA double helix based entirely on wobble base pairs with possible applications in RNA or DNA nanotechnology and pH biosensors.

Keywords: RNA double helix; adenine N1 protonation; pH-dependent structural variation; wobble base pairing; wobble helix.

FIGURE 1.

X-ray crystal structure of antiparallel RNA double helix with 2DF_o-mF_c difference density contoured at 1.0 σ with 1.6 Å radius of atoms. (A) The crystallographic asymmetric unit consists of a single 18-nt RNA strand. (B) Crystallographic dyad symmetry generates an RNA double helix (fl helix) that is divided into three segments of 6 bp each, abbreviated as WC, W, and WC helix. In the WC-helix segments, bases are paired in Watson–Crick geometry, while the WB helix contains six bases paired in wobble geometry.

FIGURE 2.

2DF_o-mF_c electron density contoured at 1.0 σ for the three unique wobble base pairs in the WB helix. Two characteristic hydrogen bonds are formed between bases, and a third hydrogen bond is mediated by a water molecule located in the major groove. (A,C) Two unique C·A⁺ pairs and (B) the unique G·U pair shown with their hydration networks in both helical grooves. (D) The C·A base pair after refinement with relaxed geometric restraints. Characteristic bond-angle changes in the adenine ring and positive difference density (green) at the position of adenine H1 both indicate adenine N1 protonation. The F_o–F_c difference density is contoured at the 2.5 σ level. Hydrogen-bond distances are given in Ångstrom units with hydrogen-acceptor distances shown in black and donor-acceptor distances (N…O or O…O) in cyan.

FIGURE 3.

Base pair parameters of the symmetric RNA double helix. Prominent variations in (A) Shear, (B) Opening, (C) Propeller, and (D) Twist steps are observed in the central wobble-base-paired W-helix segment as compared to the terminal Watson–Crick-paired A-RNA.

FIGURE 4.

pH-dependent variation of physical and thermodynamic properties of RNA. (A) UV thermal melting curves at pH 3.5, 6.5, and 8.0 (orange, green, and blue) indicate a pH-depen­dent structural transition in RNA. (B) RNA thermal melting curves recorded with differential scanning calorimetry (DSC) at pH 3.5, 6.5, and 8.0.

FIGURE 5.

Structural asymmetries of RNA element at different pH are probed by CD melting spectra. (A) An overlay of CD spectra at 20°C (solid line) and 90°C (dotted line) for RNA elements is represented at pH 3.5 (black) and 7.5 (blue). (B) The physiological structure of the RNA element is most likely an A-RNA stem–loop with either tri- or hexa-loop and not the double-helical structure observed at acidic pH.