Solution structure of lariat RNA by 500 MHz NMR spectroscopy and molecular dynamics studies in water

A 500 MHz NMR study of the lariat RNA tetramer 1 and pentamer 2 mimicking the naturally occurring lariat RNA is reported. The conformational properties of 1 and 2 were compared with those of a linear branched RNA tetramer 3, which show that the conformational features of the two lariat RNAs, 1 and 2, are quite constrained and significantly different from those observed for the linear branched RNA tetramer 3. The conformation of all sugar residues forming the lariat ring in 1 and 2 are locked in a rigid South-type conformation. All residues in both lariat RNAs have a high population of gamma+ (67-85%) and beta t (95-100%) rotamers except guanosine where the gamma+ population is low. The conformation around the glycosidic bond is anti for all residues except for guanosine where NOE data indicates an equilibrium of syn<-->anti. In both lariat RNAs, 1 and 2, the temperature dependent 1H and 31P chemical shifts as well as the oligomerization shifts, with respect to adenosine 2',3',5'-triethyl-phosphate (Sund et al., 1992, Tetrahedron 48, 695) suggests that the 3'-->5' linked U4 or C4 residue is stacked on guanosine. Subsequently, 1H-1H, 1H-31P and 13C-31P coupling constants derived torsional constraints were used for molecular dynamics study in water with counter sodium ions for a total of 226 ps. The MD simulations were first carried out with harmonic torsional constraints which were derived from J couplings (0-86 ps) and then completely without constraints (96-226 ps). The lack of any major changes in the conformation of the two lariat-RNA structures upon releasing the NMR constraints indicate that the conformers generated in the MD simulation in water agree well with the structural features suggested by experimental observables. This means that the ensemble of conformers generated during the MD trajectory of 226 ps are not artificially held in these conformations due to the NMR constraints, suggesting that these conformers can be considered to be good representatives of the actual NMR observed solution structures.