Urea Destabilizes RNA by Forming Stacking Interactions and Multiple Hydrogen Bonds with Nucleic Acid Bases

Abstract

Urea has long been used to probe the stability and folding kinetics of proteins. 1 In contrast only recently it was shown that the RNA molecules that have a high propensity to misfold can be resolved using moderate amounts of urea. 2 Urea titrations can also be used to probe the interactions that stabilize the folded states of RNA. 2c Although the mechanism by which urea denatures proteins is now fairly well understood 3 the nature of interactions by which urea destabilizes RNA is not known. In order to provide a microscopic basis for the action of urea on RNA we have carried out extensive all atom molecular dynamics (MD) simulations on two RNA constructs using two urea force fields. Destabilization of RNA is due to disruption of base-pair interactions by direct hydrogen bonding of urea with the bases. The simulations also reveal a novel mechanism in which urea molecules engage in stacking interactions with the purine bases. 4 Analyses of 20 ns trajectories generated using MD simulations with a urea force field that was created as a part of the present work (see SI for simulation details, SI Figs. 1 and 2 and Tables 1-3 for urea parameter development, and for assessing the validity of the force field) of the 22-nucleotide RNA hairpin P5GA 5 (Fig. 1A) in various urea concentrations ([C]s) reveal that at high [C] the solvent-exposed stem regions lead to disruption of base pairing. The fraction of intact hydrogen bonds associated with the bases in the stem decreases from about 0.71 in the absence of urea to 0.46 in 8M urea. The loss of the Watson-Crick (WC) hydrogen bonds is accompanied by opening of the base pairs, which is reflected in the distribution of the hydrogen bond donor-acceptor distances (R HB ) in the hairpin stem (Fig. 1B). The base-paired state is indicated by a sharp peak at R HB = 3Å, whose height decreases as [C] increases to 6M. The probability of sampling R HB distances that are greater than 10Å (Fig. 1B) increases greatly in high [C], which results in a rotation of the bases of the helix leading to N1-N3 distances of about 16Å. 6 Examination of opening at the individual base pair level reveals considerable heterogeneity 7 with the largest fluctuations occurring at the GA and GU mismatches. We also show that urea-induced disruption of the base opening due to the loss of WC hydrogen bonds is nonspecific in the sense that urea does not preferentially interact with a specific base pair. These finding suggests that denaturation of RNA is due to favorable non-specific interactions with amide-like surfaces of the nucleic acids. The average base-base interaction energies (GC, thirum@umd.edu. amackere@rx.umaryland Table 4). When averaged over all base pair interactions in the stem the interactions become less favorable by about 2.7 kcal/mol at 6M relative to [C] = 0 (SI Table 4). The average interaction energies for certain base pairs (for example A6G17 and U8A15) are substantially less at high [C] relative to their values in water (see SI Table 4).In contrast, the backbone conformational proper...

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Balancing target flexibility and target denaturation in computational fragment‐based inhibitor discovery

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