Nuclear magnetic resonance study of the impact of ribose 2'-O-methylation on the aqueous solution conformation of cytidylyl-(3' leads to 5')-cytidine.

Abstract

AbstractIn order to obtain information about the conformational characteristics at the nearestneighbor level in the 2′‐O‐methylated region of t‐RNA, as well as in the bizarre 5′‐terminus of eucaryotic mRNA, a detailed nuclear magnetic resonance study of 2′‐O‐methyl‐cytidylyl‐(3′ → 5′)‐cytidine (CmpC) was conducted. Proton spectra were recorded at 270 MHz in the Fourier mode in D2O solutions, 0.01M, pD 7.3 in the temperature range 5–80°C. Complete accurate sets of nmr parameters were derived for each of the nucleotidyl units by a combination of homo‐nuclear decouplings and simulation iteration methods. The data were translated into conformational parameters using procedures developed in earlier studies from these laboratories.It is shown that the ribofuranose ring exists at a 2E ⇄ 3E equilibrium with clear preference [(75–80)%] for the 3E mode. The C(4′)‐C(5′) and C(5′)‐O(5′) bonds form a stable conformational network with outspoken preference for conformers in which Ψ1, Ψ2 ≃ 60° and ϕ2 ≃ 180°. The orientation of the 3′‐phosphate and 2′‐O‐methyl groups is such that ϕ1′ ≃ 210° and ϕ″ ≃ 60°. The phosphodiester bonds are flexible and shift trends for base, H(1′), and H(5″) suggest the existence of a conformational blend of right‐handed stack (g−g−), left‐handed stack (g+g+), and unstacked arrays (tg− and tg+).Elevation of temperature perturbs the 2E ⇄ 3E equilibrium accompanied with modest depopulation of ψ1, ψ2 ≃ 60° and ϕ2 ≃ 180° conformers. The major effect of elevation of temperature is in the increase of unstacked arrays at the expense of g−g− and g+g+ conformers. The shift trend of Cmp‐H(3′) with temperature shows that torsional variation about O(3′)‐P is facilitated by increase in temperature and the preferred rotamer about O(3′)‐P in the unstacked form is t (ω1′ = 180°).A detailed comparison of the aqueous solution conformations of CpC and CmpC reveals that 2′‐O‐methylation causes: (i) a reduction in the magnitude of χ1; (ii) an increase in the population of 3E pucker at the 3′‐nucleotidyl unit; and (iii) modest perturbations in the O(3′)‐P and P‐O(5′) bond conformations. Comparison of the aqueous solution conformations of AmpA and CmpC makes clear that the conformational properties of pyrimidine‐pyrimidine and purine‐purine dimers which carry a 2′‐O‐methylated 3′‐nucleotidyl unit are significantly different.