The 1.19 A X-ray structure of 2'-O-Me(CGCGCG)(2) duplex shows dehydrated RNA with 2-methyl-2,4-pentanediol in the minor groove

Abstract

The crystal and molecular structure of 2'-O-Me(CGCGCG)(2) has been determined at 1.19 A resolution, at 100 K, using synchrotron radiation. The structure in space group P3(2)12 is a half-turn right-handed helix that includes two 2-methyl-2,4-pentanediol (MPD) molecules bound in the minor groove. The structure deviates from A-form RNA. The duplex is overwound with an average value of 9.7 bp per turn, characterised as having a C3'-endo sugar pucker, very low base pair rise and high helical twist and inclination angles. The structure includes 65 ordered water molecules. Only a single row of water molecules is observed in the minor groove due to the presence of hydrophobic 2'-O-methyl groups. As many as five magnesium ions are located in the structure. Two are in the major groove and interact with O(6) and N(7) of guanosine and N(4) of cytidine residues through their hydration spheres. This work provides the first example of molecular interactions of nucleic acids with MPD, which was used as a precipitant, cryo-solvent and resolution enhancing agent. The two MPD molecules intrude into the hydration network in the minor groove, each forming hydrogen bonds between their secondary hydroxyl group and exo-amino functions of guanosine residues. Comparison of the 2'-O-Me(CGCGCG)(2) structure in the P3(2)12 and P6(1)22 crystals delineates stability of the water network within the minor groove to dehydration by MPD and is of interest for evaluating factors governing small molecule binding to RNA. Intrusion of MPD into the minor groove of 2'-O-Me(CGCGCG)(2) is discussed with respect to RNA dehydration, a prerequisite of Z-RNA formation.

Figure 1

Crystal packing of 2′-O-Me(CGCGCG)₂ in the P3₂12 form. The crystal lattice consists of pseudo-infinite columns of duplexes base stacked head-to-tail, arranged in layers one molecule thick, perpendicular to the 3-fold screw axis. The neighbouring layers are at 120° to each other.

Figure 2

Two orthogonal stereo views of the molecular structure of the 2′-O-Me(CGCGCG)₂ duplex in its P3₂12 form showing 65 water molecules (red spheres), five magnesium ions (violet spheres) and two MPD molecules (thick sticks) bound to the minor groove. The relationship between views (A) and (B) is a 90° rotation about a horizontal axis in the plane of the paper.

Figure 3

The pattern of inter-strand stacking within CG (A and B) and GC steps (C and D) of the 2′-O-Me(CGCGCG)₂ duplex in the P3₂12 form. In CG steps inter-strand guanines are parallel while cytosines are not parallel; in GC steps this motif is reversed.

Figure 4

Hydration of the 2′-O-Me(CGCGCG)₂ duplex in the P3₂12 form within the major groove. Atoms lying in the further perspective are omitted for clarity (A). The pattern of fused 5- and 6-membered rings seen in the P6₁22 crystal (33) is observed. The (3F_o – 2F_c) electron density is shown for magnesium ions MG4 (B) and MG5 (of a second strand) (B), together with their three coordinating ordered water molecules; two of them are hydrogen bonded to N⁷ and O⁶ of guanine and the third to N⁴ of cytosine. The hydration pattern is disrupted near the 5′-ends by electron density [blue contours, (3F_o – 2F_c) map at 1 r.m.s. level], probably corresponding to partially disordered MPD.

Figure 5

Hydration and MPD binding within the minor groove of 2′-O-Me(CGCGCG)₂ in its P3₂12 form. A single row of seven water molecules spans both duplex strands: each water bridges, by donating a pair of hydrogen bonds, either to two self-parallel cytosines or two self-parallel guanines. Two MPD molecules intrude into the minor groove. For each MPD molecule the oxygen of its secondary hydroxyl group is the hydrogen bond acceptor from the exo-NH₂ group of the G2A (MPD1) or G2B (MPD2) guanine residue (A). ‘Omit’ (F_o – F_c) maps, contoured at the 3 r.m.s. level, are shown for the two MPD molecules (B and C). The maps were calculated after omitting MPD from the model and 10 cycles of least squares minimisation.