Abstract

RNA aptamers are oligonucleotides that bind with high specificity and affinity to target ligands. In the absence of bound ligand, secondary structures of RNA aptamers are generally stable, but single-stranded and loop regions, including ligand binding sites, lack defined structures and exist as ensembles of conformations. For example, the well-characterized theophylline-binding aptamer forms a highly stable binding site when bound to theophylline, but the binding site is unstable and disordered when theophylline is absent. Experimental methods have not revealed at atomic resolution the conformations that the theophylline aptamer explores in its unbound state. Consequently, in the present study we applied 21 microseconds of molecular dynamics simulations to structurally characterize the ensemble of conformations that the aptamer adopts in the absence of theophylline. Moreover, we apply Markov state modeling to predict the kinetics of transitions between unbound conformational states. Our simulation results agree with experimental observations that the theophylline binding site is found in many distinct binding-incompetent states and show that these states lack a binding pocket that can accommodate theophylline. The binding-incompetent states interconvert with binding-competent states through structural rearrangement of the binding site on the nanosecond to microsecond timescale. Moreover, we have simulated the complete theophylline binding pathway. Our binding simulations supplement prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline form an initial complex, most notably, a major rearrangement of the C27 base from a buried to solvent-exposed orientation. Theophylline appears to bind by a combination of conformational selection and induced fit mechanisms. Finally, our modeling indicates that when Mg2+ ions are present the population of binding-competent aptamer states increases more than twofold. This population change, rather than direct interactions between Mg2+ and theophylline, accounts for altered theophylline binding kinetics.

Highlights

  • Many RNAs exist as large ensembles of conformations in solution when they are unbound[1,2,3,4,5,6]

  • Several ribozymes have been shown to exist as a distribution of conformational states, and only a minor fraction of the equilibrium population is in the catalytically active state; a conformational change is necessary for the active state to become populated[13,14,15,16]

  • We report essential features of the modeled aptamer landscape, namely the secondary and tertiary structures and equilibrium populations of kinetically connected metastable conformational states, along with the timescales and free energy barriers of conformational transitions between these metastable states

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Summary

Introduction

Many RNAs exist as large ensembles of conformations in solution when they are unbound[1,2,3,4,5,6]. Whereas in most proteins individual α-helices and β-sheets are unstable individually[7], individual helical secondary structural elements in RNA are often highly stable[8] This stability enables RNAs to form partially folded states containing defined secondary structure but limited or no tertiary structure[4,8,9,10]. These partially folded states are often structurally heterogeneous, forming a complex structural landscape, and RNA molecules transition frequently between different conformational states in the landscape[11,12]. Several ribozymes have been shown to exist as a distribution of conformational states, and only a minor fraction of the equilibrium population is in the catalytically active state; a conformational change is necessary for the active state to become populated[13,14,15,16]

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