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

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 mm 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.