Conformational flexibility in RNA: the role of dihydrouridine

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Abstract
In order to further understand the structural role of the modified nucleoside dihydrouridine in RNA the solution conformations of Dp and ApDpA were analyzed by one-and two-dimensional proton NMR spectroscopy and compared with those of the related uridine-containing compounds. The analyses indicate that dihydrouridine significantly destabilizes the C3′-endo sugar conformation associated with base stacked, ordered, A-type helical RNA. Equilibrium constants (Keq = [C2′-endo]/[C3′-endo]) for C2′-endo-C3′-endo interconversion at 25°C for Dp, the 5′-terminal A of ApDpA and D in ApDpA are 2.08, 1.35 and 10.8 respectively. Stabilization of the C2′-endo form was shown to be enhanced at low temperature, indicating that C2′-endo is the thermo-dynamically favored conformation for dihydrouridine. ΔH values show that for Dp the C2′-endo sugar conformation is stabilized by 1.5 kcal/mol compared with Up. This effect is amplified for D in the oligonucleotide ApDpA and propagated to the 5′-neighboring A, with stabilization of the C2′-endo form by 5.3 kcal/mol for D and 3.6 kcal/mol for the 5′-terminal A. Post-tran-scriptional formation of dihydrouridine therefore represents a biological strategy opposite in effect to ribose methylation, 2-thiolation or pseudouridylation, all of which enhance regional stability through stabilization of the C3′-end Conformer. Dihydrouridine effectively promotes the C2′-endo sugar conformation, allowing for greater conformational flexibility and dynamic motion in regions of RNA where tertiary interactions and loop formation must be simultaneously accomodated.