Solution structure of RNA duplexes containing alternating CG base pairs: NMR study of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 under low salt conditions

Abstract

Structures of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 have been determined by NMR spectroscopy under low salt conditions. All protons and phosphorus nuclei resonances have been assigned. Signals of H5'/5" have been assigned stereospecifically. All 3JH,H and 3JP,H coupling constants have been measured. The structures were determined and refined using an iterative relaxation matrix procedure (IRMA) and the restrained MD simulation. Both duplexes form half-turn, right-handed helices with several conformational features which deviate significantly from a canonical A-RNA structure. Duplexes are characterised as having C3'-endo sugar pucker, very low base-pair rise and high helical twist and inclination angles. Helices are overwound with <10 bp per turn. There is limited inter-strand guanine stacking for CG steps. Within CG steps of both duplexes, the planes of the inter-strand cytosines are not parallel while guanines are almost parallel. For the GC steps this pattern is reversed. The 2'-O-methyl groups are spatially close to the 5'-hydrogens of neighbouring residues from the 3'-side and are directed towards the minor groove of 2'-O-Me(CGCGCG)2 forming a hydrophobic layer. Solution structures of both duplexes are similar; the effect of 2'-O-methylation on the parent RNA structure is small. This suggests that intrinsic properties imposed by alternating CG base pairs govern the overall conformation of both duplexes.

Thermodynamics and kinetics for base-pair opening in the P1 duplex of the Tetrahymena group I ribozyme

“…This modification also causes a small 2-fold destabilization of the U-1·G22 two base pairs away, but had no measurable effect on the directly neighboring C-2·G23 and C-4·G25 base pairs. Previous studies of unmodified and fully modified, r(CGCGCG) 2 , showed that 2′-O-methylation stabilized the duplex to thermal denaturation but had little effect on the NMR solution structure (43). The X-ray crystal and NMR solution structural studies of this duplex showed that the 2′-OMe group points towards the minor group (43–45) and is very close to the H5′ of the 3′-neighboring residue (43).…”

“…Previous studies of unmodified and fully modified, r(CGCGCG) 2 , showed that 2′-O-methylation stabilized the duplex to thermal denaturation but had little effect on the NMR solution structure (43). The X-ray crystal and NMR solution structural studies of this duplex showed that the 2′-OMe group points towards the minor group (43–45) and is very close to the H5′ of the 3′-neighboring residue (43). The X-ray structure of the fully modified, r(CGCGCG) 2 duplex shows that the 2′-OMe group affects the minor groove hydration pattern (44,45), thus stabilization of the U-3·A24 base pair may arise from protection of the 2′-OMe group from water.…”

Consequences of P‐Chirality in Chimeric 2′‐O‐Methyloligoribonucleotides with Stereoregular Methylphosphonothioate Linkages

“…The small coupling constant (J 1Ј,2Ј = 2.9 Hz) for cytidine indicates that this sugar moiety prefers the N conformation, whereas a value of J 1Ј,2Ј = 5.85 Hz for the uridine moiety is indicative of the greater conformational flexibility of this moiety in solution and a significant content of the 2Ј-endo puckered conformer in dynamic equilibrium between the 2Ј-endo and 3Ј-endo conformations. [43] The conformational analysis based on individual coupling constants J 1Ј2Ј , J 2Ј3Ј , and J 3Ј4Ј was performed using the PSEUROT 6.2 program and confirmed that a relatively high proportion of the uridine moiety adopts the S conformer (62 %). These results indicate that the (S P )-methylphosphonothioate modification of the internucleotide bond in 2Ј-O-methylribonucleotides has significantly increased the population of the S conformers relative to the unmodified internucleotide phosphodiester bonds in 2Ј-O-methylribonucleotides.…”

Hydrolysis of Phosphodiester Bonds within RNA Hairpin Loops in Buffer Solutions: the Effect of Secondary Structure on the Inherent Reactivity of RNA Phosphodiester Bonds

“…± The loop structures studied are shown in the Figure. To study the cleavage of one particular phosphodiester bond at a time, all but one of the ribonucleoside units were converted to 2'-O-methylribonucleotides. 2'-O-Methylnucleosides were chosen, because the ring conformation of a 2'-O-methylribose closely resembles that of the parent ribose [12]. Compound 1 is a linear molecule that serves as a reference material.…”

“…The rate constants for cleavage of 5 ± 7, which appear to be more reactive than a linear oligonucleotide, were also determined in the presence of the Zn 2 [12]aneN 3 chelate. This catalyst was employed in our previous study on metal-ion-promoted cleavage of hairpin structures [9].…”