Extreme Kinetic Stability and RNase Resistance of Human Telomerase RNA G‐Quadruplexes Overcome by DHX36 Helicase

ABSTRACT RNA G‐quadruplexes (G4s) formed at the 5′‐end of the RNA component of human telomerase (hTR) are known to directly affect telomerase activity. However, the unfolding kinetics of hTR _1–18 G4s at physiological K ⁺ conditions have not been analyzed due to their extremely high thermal stability ( T _m > 80°C). Here, we overcome this challenge by measuring the unfolding rates of hTR _1–18 RNA G4s using single‐molecule magnetic tweezers and bulk RNase digestion assays. We found that hTR _1–18 RNA G4s exhibited exceptionally high mechanical stability and slow unfolding rates (10 ⁻⁷ s ⁻¹ ) in physiologically relevant 100 m m KCl buffer. Furthermore, we directly determined the digestion rate ( k _dig = 1.2 × 10 ⁻⁵ s ⁻¹ ) of hTR _1–18 RNA G4s in the presence of a high concentration of 1 U/µL RNase T1, which suggests that the RNase T1 can digest folded RNA G4 structure. Importantly, G4‐specific helicase DHX36 efficiently overcame this kinetic barrier, significantly reduced the fraction of folded hTR _1–18 G4s from 99% to 16%, and rendered them susceptible to RNase degradation. These results illuminate the remarkable kinetic stability of RNA G4s and highlight the crucial role of helicase‐dependent unfolding in controlling the persistence of RNA G4s in cells.