Nanomanipulation of single RNA molecules by optical tweezers

J Vis Exp. 2014 Aug 20;(90):51542. doi: 10.3791/51542.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Video-Audio Media

MeSH terms

  • Calibration
  • Nanotechnology / instrumentation
  • Nanotechnology / methods
  • Nucleic Acid Conformation
  • Optical Tweezers*
  • RNA / chemical synthesis
  • RNA / chemistry*
  • RNA, Small Interfering / chemistry

Substances

  • RNA, Small Interfering
  • RNA