2'- O-Methyl-8-methylguanosine as a Z-Form RNA Stabilizer for Structural and Functional Study of Z-RNA

Abstract

In contrast to Z-DNA that was stabilized and well-studied for its structure by chemical approaches, the stabilization and structural study of Z-RNA remains a challenge. In this study, we developed a Z-form RNA stabilizer m⁸Gm, and demonstrated that incorporation of m⁸Gm into RNA can markedly stabilize the Z-RNA at low salt conditions. Using the m⁸Gm-contained Z-RNA, we determined the structure of Z-RNA and investigated the interaction of protein and Z-RNA.

Keywords: NMR; Z-RNA structure; circular dichroism; synthesis of oligonucleotide.

Figure 1

CD spectra of native RNA r(CGCGCG)₂ (a) and m⁸Gm-contained r(CGC[m⁸Gm]CG)₂ (b) (0.15 mM base concentration) at 10 °C with various NaClO₄ concentrations in 5 mM sodium phosphate buffer (pH 7.0).

Figure 2

(a) CD melting curves for native RNA r(CGCGCG)₂ (back line), r(CGC[m⁸Gm]CG)₂ containing one m⁸Gm (blue line), and r(C[m⁸Gm]C[m⁸Gm]CG)₂ containing two m⁸Gms (red line). (b) CD melting curves for RNA sequences containing an AU base pair, native RNA r(CGCGUGCG)/r(CGCACGCG) (back line), r(CGCGUGCG)/r(C[m⁸Gm]CAC[m⁸Gm]CG) having two m⁸Gms in one strand (blue line), r(C[m⁸Gm]CGU[m⁸Gm]CG)r(C[m⁸Gm]CAC[m⁸Gm]CG) having four m⁸Gms in two strands (red line). In 5 mM sodium phosphate buffer (pH 7.0), 7 M NaClO₄ concentrations (0.15 mM base concentration).

Figure 3

(a–c) H6/H5′′ of C and H8/H1′ of G (8CH₃/H1′ of m⁸Gm) proton region of NOESY spectra of r(CGC[m⁸Gm]CG)₂ in NaClO₄ solution. The NOE connectivity pathway is shown as red line. Intraresidue NOE cross-peaks are labeled with residue numbers. The Z-RNA structure specific cross-peaks were observed between C₅ amino protons and C₁H5 protons from the opposite strand (indicated as red words), as well as between C₁ amino protons and C₅H5 protons from the opposite strand (c). (d) The cross peaks of imino proton of G₂ and amino proton of C₅, as well as imino proton of G₆ and amino proton of C₁, indicated Watson–Crick-type base pairs of Z-RNA. Intraresidue NOE cross-peaks of imino and amino proton of G₂ and G₆ are shown.

Figure 4

Monitoring of the A to Z-RNA transition by ³¹P NMR. ³¹P NMR spectra of the RNAs at 5 mM-7 M NaClO₄. Green and red dots indicate the ³¹P peaks resulting from A-RNA and Z-RNA, respectively.

Figure 5

The model for r(CGC[m⁸Gm]CG)₂ Z-RNA structure. (a) m⁸Gm is expanded in green with the C8-methyl group, hydrogen and carbon of methyl group are coloured in yellow and purple. (b) Hydrogen and carbon of methyl group are coloured in green and white. C8-methyl groups were located outside of Z-RNA.

Figure 6

Visualization of Z-RNA and Zα-EGFP protein. Lane 1: Z-RNA only, Lane 2: RNA + Zα-EGFP, Lane 3: Zα-EGFP only. The different modes are indicated in upper. [RNA] = 1 μM, [Zα-EGFP] = 10 μM, [NaCl] = 100 mM, [Tris-HCl (pH7.0)] = 10 mM, [DTT] = 5 mM, 10% glycerol, 10 μg/mL BSA. Z-RNA: r(CGCGUGCG)-Cy3/r(C[m⁸Gm]CAC[m⁸Gm]CG).