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Enzymatic Ligation Strategies for the Preparation of Purine Riboswitches with Site-Specific Chemical Modifications

  • Renate Rieder
  • Claudia Höbartner
  • Ronald MicuraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 540)

Summary

One of the most versatile riboswitch classes refers to purine nucleoside metabolism. In the cell, purine riboswitches of the respective mRNAs either act at the transcriptional or translational level and off- or on-regulate genes upon binding to their dedicated ligands. Biophysical studies on ligand-induced folding of these RNA domains in vitro contribute to understanding their regulation mechanisms in vivo. For such studies, in particular, for approaches using fluorescence spectroscopy, the preparation of large RNAs with site-specific chemical modifications is required. Here, we describe a strategy for the preparation of riboswitch aptamers and aptamers adjoined to their expression platforms by chemical synthesis and enzymatic ligation. The modular design enables fast access to a large number of purine riboswitch derivatives with the modification of interest at any strand position. We exemplarily provide a detailed protocol for the preparation of adenosine deaminase (add) A-riboswitch variants with 2-aminopurine (AP) modifications at the 40-nmol scale.

Key words

Ligation Riboswitch Aminopurine RNA modification 

Notes

Acknowledgments

We thank the Austrian Science Fund FWF (P17864) and the BMWF (Gen-AU program; projects “Non-coding RNAs” No. P7260-012-011 and No. P7260-012-012) for funding.

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Copyright information

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

Authors and Affiliations

  • Renate Rieder
    • 1
  • Claudia Höbartner
    • 1
  • Ronald Micura
    • 1
    Email author
  1. 1.Institute of Organic ChemistryLeopold Franzens UniversityAustria

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