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

Nanomanipulation of single RNA molecules by optical tweezers.

Author information

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

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.

PMID:
25177917
PMCID:
PMC4540088
DOI:
10.3791/51542
[Indexed for MEDLINE]
Free PMC Article

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