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Biochemical Characterization of G4 Quadruplex Telomerase RNA Unwinding by the RNA Helicase RHAU

  • Evan P. Booy
  • Ewan K. S. McRae
  • Sean A. McKennaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

G4 quadruplexes are stable secondary structures prevalent in DNA and RNA that exhibit diverse regulatory functions. Herein, we describe an in vitro technique using the purified RNA helicase RHAU to unwind a G4 quadruplex identified near the 5′ end of the human telomerase RNA (hTR). A synthetic RNA corresponding to the quadruplex forming region of hTR (hTR10–43), as well as a predicted complementary strand (25P1), are combined in a reaction containing the purified helicase and ATP. Reaction products and appropriate controls are resolved by native gel electrophoresis. Gels can be stained using a combination of total RNA and quadruplex-specific dyes to observe the expected quadruplex to duplex conversion. This straightforward method can be extended to study structural changes in other inter- or intramolecular quadruplex containing DNA/RNA molecules with the RHAU helicase or other RNA/DNA remodeling enzymes.

Key words

RHAU Quadruplex Helicase Telomerase RNA DHX36 G4R1 P1-helix Unwinding Secondary structure 

Notes

Acknowledgements

Evan Booy is supported by the Manitoba Health Research Council postdoctoral fellowship. This work is supported by the Canadian Institutes of Health Research (CIHR)/Manitoba Health Research Council (MHRC) regional partnership program and a Cancer Research Society Operating Grant.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Evan P. Booy
    • 1
  • Ewan K. S. McRae
    • 1
  • Sean A. McKenna
    • 1
    • 2
    Email author
  1. 1.Department of ChemistryUniversity of ManitobaWinnipegCanada
  2. 2.Department of Biochemistry & Medical GeneticsUniversity of ManitobaWinnipegCanada

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