Molecular dynamics reveals the stabilizing role of loop closing residues in kissing interactions: comparison between TAR-TAR* and TAR-aptamer

Abstract

A RNA aptamer (R06) raised against the trans- activation responsive (TAR) element of HIV-1 was previously shown to generate a loop-loop complex whose stability is strongly dependent on the selected G and A residues closing the aptamer loop. The rationally designed TAR* RNA hairpin with a loop sequence fully complementary to the TAR element, closed by U,A residues, also engages in a loop-loop association with TAR, but with a lower stability compared with the TAR-R06 complex. UV absorption monitored thermal denaturation showed that TAR-TAR*(GA), in which the U,A kissing residues were exchanged for G,A, is as stable as the selected TAR-R06 complex. Consequently, we used the TAR-TAR* structure deduced from NMR studies to model the TAR-R06 complex with either GA, CA or UA loop closing residues. The results of the molecular dynamics trajectories correlate well with the thermal denaturation experiments and show that the increased stability of the GA variant results from an optimized stacking of the bases at the stem-loop junction and from stable interbackbone hydrogen bonds.

Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex

“…In a control experiment, no effect was detected on the stability of the kissing complex when a similar modification to the sugar of A*12 was introduced. These results strongly support the notion that this intermolecular hydrogen bond involving H 2Ј of G*5 plays a substantial role in the stabilization of the kissing complex, as suggested earlier by molecular dynamics simulations (17). Similar analysis of the liquid-crystal NMR structure ensemble indicates that the H 2Ј of C5 may form a supplementary weak intermolecular hydrogen bond with U*6-O 2P .…”

“…A drop in the melting temperature is observed between the TAR* GA -TAR (27.2 Ϯ 0.5°C) and TAR* UA -TAR complexes (11.9 Ϯ 0.5°C). These results are consistent with the melting temperatures measured for complexes between TAR and RNA aptamers with longer stems (16,17). This validates the choice of shorter RNAs to investigate the role of the GA loop-closing bases of the aptamer.…”

“…Interestingly, all aptamers with the strongest binding properties also possess a GA combination at the end of the stem (loop-closing position). The role of loop-closing G and A nucleotides was investigated by systematic permutation (16) and by molecular dynamics studies (17). It was shown that the nature of the residues closing the loop of the aptamer was crucial for kissing complex stability, and that the selected G-A combination leads to the most stable complex.…”

Regulating eukaryotic gene expression with aptamers

“…In contrast an almost straight coaxial stacking of the helices was observed in the crystal structure of the 23S RNA kissing complex [17] and in the DIS-DIS one [18], a kissing complex that constitutes the initial step of the dimerization process of the human immunodeficiency virus type 1 (HIV-1) genome. Interestingly, molecular dynamics (MD) performed on the TAR-TAR* solution structure reconciled these observations [19]. The initial curvature of the structure disappeared during the first nanosecond of the simulation run and TAR-TAR* adopted an almost straight coaxial structure, indicating that this latter conformation was likely the most stable one.…”

Modulating viral gene expression by aptamers to RNA structures

“…In particular, C,A or C,U combinations led to very poor TAR ligands. Dynamic molecular modelling of TAR-aptamer kissing complexes demonstrated an increased stability of the association for the G,A variant (Beaurain et al, 2003). The large interglycosidic distance observed in this case compared to unfavourable combinations, optimised stacking interactions and the formation of interbackbone hydrogen bonds.…”