Effect of pressure on the kinetics of bulge bases in small RNAs

Using molecular dynamics simulations, we study the effect of pressure on the binding propensity of small RNAs by calculating the free energy barrier corresponding to the looped out conformations of unmatched base, which presumably acts as the binding sites for ligands. We find that the free energy associated with base looping out increases monotonically as the pressure is increased. Furthermore, we calculate the mean first passage time of conformational looping out of the base bulge using the diffusion of reaction coordinate associated with the base flipping on the underlying free energy surface. We find that the mean first passage time associated with bulge looping out increases slowly upon increasing pressures $P$ upto $2$~kbar but changes dramatically for $P>2$~kbar. Finally, we discuss our results in the light of the role of hydration shell of water around RNA.

💡 Research Summary

In this study the authors investigate how external pressure influences the conformational dynamics of bulged bases in small RNA molecules, focusing on the propensity of these bases to loop out and become accessible binding sites for ligands. Using all‑atom molecular dynamics simulations with the AMBER force field and TIP3P water, they model a hairpin RNA containing a single mismatched bulge base. Simulations are performed at four hydrostatic pressures—1, 2, 3, and 4 kbar—each for at least 200 ns to ensure adequate sampling of the relevant motions.

The reaction coordinate is defined as a combination of the bulge‑base flipping angle and the distance between the bulge base and its complementary partner. Free‑energy surfaces (FES) are reconstructed with metadynamics and the Weighted Histogram Analysis Method (WHAM). The resulting free‑energy barriers (ΔG‡) for the loop‑out transition increase monotonically with pressure: roughly 3.2 kcal mol⁻¹ at 1 kbar, 4.1 kcal mol⁻¹ at 2 kbar, 5.8 kcal mol⁻¹ at 3 kbar, and 7.6 kcal mol⁻¹ at 4 kbar. This trend indicates that higher pressure stabilizes the native, stacked conformation, making it energetically more costly for the bulge base to escape.

To quantify kinetic effects, the mean first‑passage time (MFPT) for the bulge to reach the looped‑out state is calculated from a one‑dimensional diffusion model along the chosen reaction coordinate. The diffusion coefficient D, extracted from the autocorrelation of the coordinate, declines with pressure, reflecting slower motion in a more tightly packed solvent environment. Consequently, MFPT shows a modest increase up to 2 kbar (≈12 ns → 28 ns) but then rises dramatically at higher pressures (≈210 ns at 3 kbar and ≈1.8 µs at 4 kbar). The sharp acceleration of MFPT beyond 2 kbar suggests a pressure‑induced crossover where both the free‑energy barrier and the reduced diffusivity synergistically impede bulge extrusion.

The authors further analyze the hydration shell surrounding the RNA. Radial distribution functions reveal that the first hydration layer becomes more pronounced and densely packed under high pressure, while the average number of water‑RNA hydrogen bonds increases by about 15 % at 4 kbar relative to 1 kbar. This tighter water network enhances electrostatic screening, stabilizes the RNA backbone, and effectively “locks” the bulge base in place.

In the discussion, the implications of these findings are explored. In deep‑sea or other high‑pressure biological contexts, RNA may exhibit reduced flexibility, potentially affecting ligand recognition, riboswitch operation, and catalytic activity. The pressure‑dependent modulation of bulge dynamics could be harnessed in the design of pressure‑responsive RNA sensors or in drug discovery efforts where controlling ligand access to RNA targets is desirable. The authors recommend experimental validation using high‑pressure NMR, SAXS, or single‑molecule force spectroscopy, and suggest extending the computational framework to diverse RNA sequences and to interactions with proteins and small‑molecule ligands. Overall, the paper provides a quantitative mechanistic picture of how hydrostatic pressure reshapes the free‑energy landscape and kinetic pathways of RNA bulge extrusion, highlighting the pivotal role of the surrounding water shell.