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Structural Probing Techniques on Natural Aptamers

  • Catherine A. Wakeman
  • Wade C. Winkler
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 535)

Abstract

RNA sequences fold in a hierarchical manner to form complex structures. This folding pathway proceeds first with formation of secondary structure elements followed by the compilation of tertiary contacts. Although bioinformatics-based tools are commonly used to predict secondary structure models, it is notoriously difficult to achieve a high degree of accuracy via these approaches alone. Therefore, a diverse assortment of biochemical and biophysical techniques are regularly used to investigate the structural arrangements of biological RNAs. Among these different experimental techniques are structural probing methods, which are often times employed to determine which nucleotides for a given RNA polymer are paired or unpaired. Yet other probing methods assess whether certain RNA structures undergo dynamical structure changes. In this chapter we outline a general protocol for in-line probing, a method for analyzing secondary structure (and backbone flexibility) and describe a basic experimental protocol for hydroxyl radical footprinting as a method of investigating RNA folding.

Key words

In-line probing riboswitch hydroxyl radical footprinting RNA folding RNA secondary and tertiary structure 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Catherine A. Wakeman
    • 1
  • Wade C. Winkler
    • 1
  1. 1.Department of BiochemistryThe University of Texas Southwestern Medical CenterDallasUSA

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