Folding and unfolding single RNA molecules under tension

Single-molecule force spectroscopy constitutes a powerful method for probing RNA folding: It allows the kinetic, energetic, and structural properties of intermediate and transition states to be determined quantitatively, yielding new insights into folding pathways and energy landscapes. Recent advances in experimental and theoretical methods, including fluctuation theorems, kinetic theories, novel force clamps, and ultrastable instruments, have opened new avenues for study. These tools have been used to probe folding in simple model systems, for example, RNA and DNA hairpins. Knowledge gained from such systems is helping to build our understanding of more complex RNA structures composed of multiple elements, as well as how nucleic acids interact with proteins involved in key cellular activities, such as transcription and translation.

Introduction

RNA plays diverse roles in the cell, ranging from the transmission of genetic information to the regulation of genes, ligand binding, and catalysis. Driven by the desire to understand the myriad functions of RNA, there has been an intense effort to unravel details of structure formation and dynamics in RNA molecules. RNA folding is simplified by the independent stability of the (generally predictable) secondary structures, leading to a picture of ‘hierarchical’ folding [1] wherein secondary structure forms before tertiary structure. Folding nevertheless remains a formidable problem, characterized by multiple conformations arrayed across rugged energy landscapes, important chain entropy effects, and a sensitive dependence on electrostatic interactions between RNA and various metal ions [2, 3].

One of the newest techniques for studying RNA folding is single-molecule force spectroscopy (SMFS), where the extension of an individual molecule is measured under an applied tension [4]. By varying the load, a single RNA molecule can be unfolded and refolded repeatedly. Force thus functions as a mechanical denaturant acting selectively on a given molecule, in contrast to traditional denaturants, such as temperature or urea. This property allows individual folding trajectories to be observed, subpopulations and rare/transient states (including partially folded intermediates) to be distinguished, and the behavior of molecules with widely different stabilities to be compared under identical buffer conditions. Because unfolded states are fully stretched under load, the unfolded state is simplified from an ensemble of energetically similar, high-entropy configurations to a single low-entropy configuration: Both the initial and final states of the folding reaction are thus well defined. The vectorial nature of force also imposes a preferential direction upon the folding reaction, biasing particular pathways that can be isolated for study. The molecular extension measured in SMFS supplies a natural coordinate for describing the course of the reaction and can be interpreted in terms of specific structural elements. All these features make SMFS particularly well suited for probing folding reactions. Spatial resolution reaching the ångström level [5] permits subnucleotide extension changes to be measured. The broad temporal range currently achievable, ∼10⁻⁴–10³ s [6^•], is well matched to the time scales of RNA folding.

This review will focus on work with RNA using optical traps, as this has been the most commonly employed SMFS technique. We organize the discussion around four themes characterizing recent advances: (1) work on model systems for RNA folding, (2) advances in experimental and theoretical methods, (3) folding of complex functional RNAs, and (4) interactions between RNA folding and nucleic acid enzymes.