Conformational analysis of single-base bulges in A-form DNA and RNA using a hierarchical approach and energetic evaluation with a continuum solvent model.

The analysis and prediction of non-canonical structural motifs in RNA is of great importance for an understanding of the function and design of RNA structures. A hierarchical method has been employed to generate a large variety of sterically possible conformations for a single-base adenine bulge structure in A -form DNA and RNA. A systematic conformational search was performed on the isolated bulge motif and neighboring nucleotides under the constraint to fit into a continuous helical structure. These substructures were recombined with double-stranded DNA or RNA. Energy minimization resulted in more than 300 distinct bulge conformations. Energetic evaluation using a solvation model based on the finite-difference Poisson-Boltzmann method identified three basic classes of low-energy structures. The three classes correspond to conformations with the bulge base stacked between flanking nucleotides (I), location of the bulge base in the minor groove (II) and conformations with a continuous stacking of the flanking helices and a looped out bulge base (III). For the looped out class, two subtypes (IIIa and IIIb) with different backbone geometries at the bulge site could be distinguished. The conformation of lowest calculated energy was a class I structure with backbone torsion angles close to those in standard A -form RNA. Conformations very close to the extra-helical looped out bulge structure determined by X-ray crystallography were also among the low-energy structures. In addition, topologies observed in other experimentally determined bulge structures have been found among low-energy conformers. The implicit solvent model was further tested by comparing an uridine and adenine bulge flanked by guanine:cytosine base-pairs, respectively. In agreement with the experimental observation, a looped out form was found as the energetically most favorable form for the uridine bulge and a stacked conformation in case of the adenine bulge. The inclusion of solvation effects especially electrostatic reaction field contributions turned out to be critically important in order to select realistic low-energy bulge structures from a large number of sterically possible conformations. The results indicate that the approach might be useful to model the three-dimensional structure of non-canonical motifs embedded in double-stranded RNA, in particular, to restrict the number of possible conformations to a manageable number of conformers with energies below a certain threshold.