Multiscale modelling of phosphate…π contacts in RNA U-turns reveals AMBER force-field deficiencies

Phosphate…π, also called anion…π, contacts occur between nucleobases and phosphate OP oxygens in r(GNRA) and r(UNNN) U-turn motifs (N = A,G,C,U; R = A,G). We investigated these contacts in detail by using state-of-the-art quantum chemical methods (QM) to characterize some of their physico-chemical properties and to evaluate the ability of the AMBER force field (AFF) to describe these contacts. We found that AFF interaction energies of phosphate…π contacts calculated for model dimethyl phosphate…nucleobase systems are less stabilizing in comparison with double-hybrid DFT methods and that the minimum contact distances are stretched for all nucleobase systems. This distance stretch is also observed in large-scale AFF computations on several r(gcGNRAgc) tetraloop hairpins when compared to QM/MM. Further, classical molecular dynamics (MD) simulations of these tetraloop hairpins confirm this distance stretch and reveal shifted OP2/nucleobase positions when compared to experimental data extracted from high-resolution X ray/cryo EM structures (≤ 2.5 Å) of r(GNRA) tetraloops using the WebFR3D bioinformatic tool. We propose that discrepancies between QM and AFF are caused by a combination of missing polarization, too large AFF Lennard-Jones (LJ) radii of nucleobase carbon atoms and exaggerated short-range repulsion due to an approximate r−12 LJ repulsive term. We put these results in regard with those obtained in earlier investigations on lone pair…π contacts occurring in CpG Z-steps. Charge-transfer calculations do not support any significant n->π* donation effects and hence this label is inappropriate. We also investigated thiophosphate…π contacts for which we calculated less stabilizing interaction energies than for the phosphate…π contacts. We thus challenge suggestions that the experimentally observed enhanced thermodynamic stability of phosphorothioated r(GNRA) tetraloops can be straightforwardly explained by larger London dispersion.