Folding and unfolding single RNA molecules under tension

Abstract

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.

Figure 1

Representative SMFS measurements. (a) An RNA molecule is attached to two “handles” bound to beads and manipulated using two optical traps. Unfolded RNA is stretched by the force applied between the traps. As RNA unfolds or refolds, the resulting extension change is monitored. (b) FECs for the HIV TAR hairpin. After unfolding in a single step (black), the hairpin refolds through multiple intermediate states (red), producing hysteresis. Occasionally, an unfolded hairpin refolds partially to a metastable misfolded state (blue), as revealed by the return to the fully-folded state in a subsequent unfolding curve (green). Adapted from [12•]. (c) Extension of a model DNA hairpin at equilibrium under constant force, using a passive force clamp. The G:C content of the stem sequence was engineered to produce a landscape containing a metastable, partially-folded intermediate.

Figure 2

Determining equilibrium free energies from non-equilibrium measurements using the Crooks fluctuation theorem. The point of intersection of the probability distributions for the irreversible work done when unfolding (red) and refolding (black) a 3-helix junction supplies the equilibrium free energy for the folding reaction. Adapted from [9].

Figure 3

SMFS of folding in an adenine riboswitch aptamer. (a) Binding of the adenine ligand alters the shape of the FECs. (b) Constant-force trajectories obtained at two different forces indicate five distinct states in the folding, corresponding to the formation of specific structural elements of the aptamer, as indicated. (c) Energy landscape for the complete folding reaction, both with (red) and without (black) ligand. Adapted from [26••].

Figure 4

Effect of co-transcriptional folding/unfolding of the histidine (his) terminator hairpin upon transcription. Adapted from [55••]. (a) Force is applied to an RNA transcript during transcription, biasing the structure (folded/unfolded) of the terminator hairpin as the motion of polymerase is continuously measured. (b) Efficiency of transcription is found to depend on force through two mechanisms that work in conjunction: (i) shearing of the RNA/DNA hybrid at a U-rich tract (yellow), most evident at high force, and (ii) folding/unfolding of the intrinsic terminator hairpin (black), most evident at low force. (c) Model of the energy landscape for termination, where the terminator hairpin lowers the barrier to termination. Force applied to the RNA can raise this barrier in the presence of the hairpin by causing the hairpin to unfold (dashed black line), but can lower the barrier in the absence of the hairpin by shearing the RNA/DNA hybrid at the U-tract (dashed yellow line).