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Design and Analysis of Hammerhead Ribozyme Activity Against an Artificial Gene Target

  • James R. Carter
  • Pruksa Nawtaisong
  • Velmurugan Balaraman
  • Malcolm J. FraserJr.
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1103)

Abstract

In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like dengue virus RNA. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays.

Key words

Dengue Flavivirus Ribozyme Catalysis Electrophoresis RNA In vitro transcription 

Notes

Acknowledgement

This work was supported by NIH grant AI097554 to MJF.

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • James R. Carter
    • 1
  • Pruksa Nawtaisong
    • 1
  • Velmurugan Balaraman
    • 1
  • Malcolm J. FraserJr.
    • 1
  1. 1.Department of Biology, Eck Institute for Global HealthUniversity of Notre DameNotre DameUSA

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