Two-dimensional (P/T) studies of secondary/tertiary conformational dynamics in nucleic acids: pressure induced melting and Maxwell relations at the single molecule level

Abstract

A predictive understanding of conformational folding of nucleic acids depends crucially on the underlying competition between enthalpic and entropic contributions to overall free energy changes. In extreme environments (e.g. deep ocean vents), such free energy changes are in turn impacted by both pressure and temperature as strongly coupled intensive variables, emphasizing the importance of a detailed molecular level understanding of the underlying thermodynamics. In this work, single-molecule fluorescence energy resonance transfer (smFRET) microscopy methods are implemented for quantitative kinetic study of secondary structure (i.e., DNA hybridization) and tertiary structure (i.e., RNA Mn²⁺ riboswitch folding) equilibria as a function of both pressure (P = 1 to 1000 bar) and temperature (T = 21 to 27 °C). Temperature dependent studies at a series of fixed pressures reveal the single molecule DNA and RNA constructs to be stabilized and destabilized, respectively, with increasing T. Interestingly, results for the Mn²⁺ riboswitch indicate a positive entropy change (ΔS > 0) for achieving the native tertiary conformation at all external pressures. This is contrary to more common physical expectations of increased order and thus an entropically penalty for tertiary folding of the RNA. On the other hand, pressure dependent scans for a series of constant temperature conditions confirm that both the DNA hairpin and RNA riboswitch constructs destabilize (“melt”) under increasing pressure, which by van’t Hoff analysis implies a positive volume change (ΔV⁰ > 0) for both secondary and tertiary folding into the native state. Furthermore, slices through these two-dimensional free energy surfaces permit parameters for isobaric thermal expansion in both secondary and tertiary conformational folding coordinates to be extracted. Finally, experimental control of multiple variables allows determination of the folding free energies as a two-dimensional function of pressure and temperature (ΔG(P, T)). To the best of our knowledge, this facilitates a first experimental confirmation of the underlying Maxwell relation (∂ΔS/∂P)_T = −(∂ΔV/∂T)_P for the exact free energy differential dG(P, V, S, T) at the single molecule level.