Biochemical characterization of G4 Quadruplex Telomerase RNA unwinding by the RNA helicase RHAU

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 purifi ed RNA helicase RHAU to unwind a G4 quadruplex identifi ed 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 purifi ed 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-specifi c 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.