Real-time control of the energy landscape by force directs the folding of RNA molecules

Abstract

The rugged folding-energy landscapes of RNAs often display many competing minima. How do RNAs discriminate among competing conformations in their search for the native state? By using optical tweezers, we show that the folding-energy landscape can be manipulated to control the fate of an RNA: individual RNA molecules can be induced into either native or misfolding pathways by modulating the relaxation rate of applied force and even be redirected during the folding process to switch from misfolding to native folding pathways. Controlling folding pathways at the single-molecule level provides a way to survey the manifold of folding trajectories and intermediates, a capability that previously was available only to theoretical studies. mechanical force ͉ misfolding ͉ optical tweezers ͉ RNA folding ͉ single molecule T he folding of a macromolecule can be thought of as a biased diffusion over an energy surface that describes the thermodynamic and kinetic constraints of possible intramolecular interactions. RNAs differ from proteins in the nature, strength, specificity, and degeneracy of the interactions that stabilize their native structures. Whereas proteins are made of 20 amino acids, it takes only 4 nucleotides to build RNAs. This simpler composition, endowed with robust base-pairing rules, greatly increases the promiscuity of interactions between any given nucleotide and the rest of the RNA structure. These RNA-protein differences are reflected in the topography of the energy surface over which the molecules diffuse in their search for the native structure: the energy surfaces of RNAs are significantly more rugged than those of proteins, containing many competing local minima (1-4). Even relatively small RNAs, such as tRNA (4-6), tend to fold into stable alternate secondary structures with energies only a few kilocalories (1 kcal ϭ 4.18 kJ on first use) per mole apart (4). Once trapped kinetically in a secondary structure with suboptimal folding energy, it is difficult for an RNA molecule to reach its native structure (1).The promiscuous nature of secondary interactions in RNA begs answers for three questions: How do RNAs find their native structure? How do they avoid the kinetic traps on their rugged folding-energy landscape? And, how can RNAs switch between alternate structures in response to changes in the cellular environment, as happens, for example, during transcription attenuation in bacteria (7)? To address these questions, we need to follow the folding trajectories of RNA molecules as they diffuse over their energy landscape in search of stable conformations. Because of the structural polymorphism and manifold pathways of RNA, singlemolecule approaches (8-12) are particularly useful for answering these questions by identifying folding intermediates and distinguishing native pathways from those that misfold.Here we use optical tweezers to unfold and control the refolding of single RNA molecules by force (11, 13). Two micrometer-sized beads are attached to the ends of an RNA molecule and are manipulated by ...

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

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Scaling of hysteresis loop of interacting polymers under a periodic force

“…Though, hysteresis has been observed in single molecule experiments [24][25][26][27][28][29][30][31], however, many aspects of these phenomena are yet to be explored. In a recent work [32], Kapri showed that using the work theorem [33], it is possible to extract the equilibrium force-extension curve from the hysteresis loops.…”

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