Biochemical characterization of G4 quadruplex telomerase RNA unwinding by the RNA helicase RHAU.

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.