Nanomanipulation of Single RNA Molecules by Optical Tweezers

William Stephenson; Gorby Wan; Scott A. Tenenbaum; Pan T. X. Li

doi:10.3791/51542

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Method Article

Nanomanipulation of Single RNA Molecules by Optical Tweezers

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DOI:

10.3791/51542

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August 20th, 2014

William Stephenson¹ , Gorby Wan² , Scott A. Tenenbaum³^,⁴ , Pan T. X. Li⁴^,⁵

¹Nanoscale Engineering Graduate Program, College of Nanoscale Science and Engineering, University at Albany, State University of New York, ²Nanoscale Science Undergraduate Program, College of Nanoscale Science and Engineering, University at Albany, State University of New York, ³Nanobioscience Constellation, College of Nanoscale Science and Engineering, University at Albany, State University of New York, ⁴The RNA Institute, University at Albany, State University of New York, ⁵Department of Biological Sciences, University at Albany, State University of New York

Corresponding Authors: Pan T. X. Li <pli@albany.edu>

Summary

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5’ and 3’ ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

Abstract

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Introduction

The development of the optical tweezers technique has long been accompanied with its application in biological research. When the optical trapping effect was first discovered, Arthur Ashkin observed that bacteria in contaminated water could be trapped at the laser focus¹. Since then, bacteria trapping has become an unintentional, fun experiment for generations of biophysics students as well as a serious research tool to study microbial physiology^2,3. The optical trapping technique uses focused laser beam to immobilize a microscopic object¹. Practically, a laser trap functions as an optical spring, which also measures force (F)....