Study of stochastic processes and RNA elasticity using optical tweezers

Abstract

Our main research project is to develop an experimental system to understand the ribosomes as molecular motors. For this purpose, we designed and developed the single molecule ribosome motility assay. In this thesis, we discuss the related steps which we have taken to develop this assay: the instrumentation and four experiments. In the introduction, we illustrate the current experimental scheme and show the preliminary data of the single molecule ribosome translation. In the first two chapters, we discuss two interesting experiments dealing with stochastic problems. In the first chapter, we introduce the idea that the noise can be reduced in certain conditions. We show that the noise can be really reduced but only if added fluctuations have to drive the input into states with reduced intrinsic noise. In the second chapter, we investigate two-state processes experimentally using a simple physical system in which a microscopic bead is trapped in a double-well potential. We discuss how we can control the characteristics of the escape process by adding a periodic force and illustrate the characteristics in terms of the residence time distribution and the escape phase. As last two chapters, we discuss the elastic and structural properties of homopolymeric RNA, polyadenylic acids (poly(A)), polycytidylic acids (poly(C)), and polyuridylic acids (poly(U)). First, we investigate the elastic property of poly(U) as a random coil. We find that the force-extension data is well predicted by a classic worm-like chain model at high Na⁺ concentrations, whereas at low such concentrations the introduction of a scale-dependent persistence length is required. As single-stranded helices, poly(A) and poly(C) are studied. We stretch the molecules to induce the conformational transition from folded states to unfolded states and use the elastically coupled two-state model to acquire their structural information (about 9 bases per turn and 8 bases per turn, for poly(A) and poly(C) respectively, at neutral pH and 500 mM [Na⁺]) and the free energy differences between two states (20 pN·nm for poly(A) and 17.5 pN·nm for poly(C)).