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High Resolution Optical Tweezers as a Tool to Study RNA Folding Pathways

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Abstract

Optical tweezers utilizes light energy to trap small neutral particles due to the transfer of momentum from the trapping beam to the particle. The method of trapping has been successfully used to trap a single cell, or an organelle that has opened numerous possibilities to study biological systems. In more advanced systems, the technique is used to study sub-cellular motions such as molecular motors or folding of biomolecules e.g., DNA, RNA and proteins. Typically, the linear fluctuations in these systems are in the nanometer scale that can be manipulated by applying microscopic external force in the order of pico-Newtons. Recently, we have built a high resolution dual beam optical-tweezers that can measure forces at the resolution of ± 0.15 pN, distances at 1 nm and a temporal resolution of sub-milliseconds (< 0.25 msec). Using our custom built instrument we investigated the folding of a model RNA hairpin P5ab, which has been earlier studied as a two-state folder. P5ab forms the structural domain of group I ribozymes that is involved in intronic splicing. Our results show that P5ab folds with atleast three intermediate states that has been missed in the earlier studies. By holding the molecule at a constant force, we allowed the RNA to fluctuate through various intermediates before it folded into its native conformation. The kinetics reveals a complex mechanism of folding that is taken by P5ab. By performing multiple rounds of equilibrium experiments, we located the position and the height of the activation barrier in the folding scheme that must be crossed by the RNA molecule to achieve the native conformation. The findings are important as it highlights how a single base change can alter the folding pathways and the barrier heights that often trap biomolecules in misfolded states, which is the hallmark of diseased condition such as cancer and prion.

© 2014 Optical Society of America

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