Force field based conformational analysis of RNA structural motifs: GNRA tetraloops and their pyrimidine relatives

The protocol of conformational analysis applied here to ribonucleotide olig omers combines conformational search in the space of torsion angles and ene rgy minimization using the AMBER4.1 force field with a continuum treatment of electrostatic solute-solvent interactions. RNA fragments with 5'-CGGCCNN AGCCU-3' sequences commonly fold into hairpins with four-membered loops. Th e combinatorial search for acceptable conformations using the MC-SYM progra m was restricted to loop nucleotides and yielded roughly 1500 structures be ing compatible with a double-stranded stem. After energy minimization by th e JUMNA program (without applying any experimental constraints), these stru ctures converged into an ensemble of 74 different conformers including 26 s tructures which contained the sheared G-A base pair observed in experimenta l studies of GNRA tetraloops. Energetic analysis shouts that inclusion of s olvent electrostatic effects is critically important for the selection of c onformers that agree with experimentally determined structures. The continu um model accounts for solvent polarization by means of the electrostatic re action field. In the case of GNRA loop sequences, the contributions of the reaction field shift relative stabilities towards conformations showing mos t of the structural features derived from NMR studies. The agreement of com puted conformations with the experimental structures of GAAA, GCAA, and GAG A tetraloops suggests that the continuum treatment of the solvent represent s a definitive improvement over methods using simple damping models in elec trostatic energy calculations. Application of the procedure described here to the evaluation of the relative stabilities of conformers resulting from searching the conformational space of RNA structural motifs provides some p rogress in (non-homology based) RNA 3D-structure prediction.