High salt solution structure of a left-handed RNA double helix

Abstract

Right-handed RNA duplexes of (CG)n sequence undergo salt-induced helicity reversal, forming left-handed RNA double helices (Z-RNA). In contrast to the thoroughly studied Z-DNA, no Z-RNA structure of natural origin is known. Here we report the NMR structure of a half-turn, left-handed RNA helix (CGCGCG)2 determined in 6 M NaClO4. This is the first nucleic acid motif determined at such high salt. Sequential assignments of non-exchangeable proton resonances of the Z-form were based on the hitherto unreported NOE connectivity path [H6(n)-H5'/H5''(n)-H8(n+1)-H1'(n+1)-H6(n+2)] found for left-handed helices. Z-RNA structure shows several conformational features significantly different from Z-DNA. Intra-strand but no inter-strand base stacking was observed for both CpG and GpC steps. Helical twist angles for CpG steps have small positive values (4-7 degrees), whereas GpC steps have large negative values (-61 degrees). In the full-turn model of Z-RNA (12.4 bp per turn), base pairs are much closer to the helix axis than in Z-DNA, thus both the very deep, narrow minor groove with buried cytidine 2'-OH groups, and the major groove are well defined. The 2'-OH group of cytidines plays a crucial role in the Z-RNA structure and its formation; 2'-O-methylation of cytidine, but not of guanosine residues prohibits A to Z helicity reversal.

Figure 1

Contour plot of the 2D-NOESY spectrum (250 ms) of (CGCGCG)₂ in the Z-RNA form showing wide-ranging connectivity path [H6_(n)-H5′/H5″_(n)-H8_(n+1)-H1′_(n+1)-H6_(n+2)] for Z-helices. Intra-residue cross peaks of cytidine H6–H5′ and guanosine H8-H1′ are marked.

Figure 2

First column: superposition of 12 conformers representing the refined structure of (CGCGCG)₂ in the Z-RNA form as viewed from the minor groove and along the helix z-axis. Second and third column: stacking pattern within the CpG and GpC steps in Z-RNA as viewed from the minor groove and along the helix z-axis. For both steps only intra- but no inter-strand stacking is observed.

Figure 3

Full-turn model of the Z-RNA helix (12.4 bp per turn) (left) compared to the 12 bp per turn models of Z_I- (middle) and Z_II-DNA (right) (41). Models are visualized in two different conventions in order to show their base pair location and groove architecture. Atoms are coloured by type (carbon in black, hydrogen in white, nitrogen in blue, phosphorus in yellow, oxygen in red; cytidine 2′ oxygens are given in green).

Figure 4

Monitoring of the A to Z RNA helicity reversal. From bottom to top: ³¹P NMR spectra of the (CGCGCG)₂, its residue-specific 2′-O-methylated analogues [C(2′-OMe)G]₃, [(2′-OMe)CG]₃ and of 2′-O-Me(CGCGCG)₂ at 0 and 6 M NaClO₄. ³¹P chemical shifts of A- and Z-forms are coded as follows: circles, GpC (A); black circles, GpC (Z); rectangles, CpG (A); black rectangles, CpG (Z).