The stability and unfolding of DNA: RNA hybrid G-quadruplexes

DNA:RNA hybrid G-quadruplexes (hG4s) are intermolecular G4 structures composed of guanine (G)-rich DNA and RNA strands. These structures play crucial regulatory roles in transcription and telomere stability. However, the absence of methods to produce uniform hG4s has limited systematic studies on their stability and unwinding. In this study, using human telomeric sequences as a model, we developed a PC (photocleavable)-linker-induced cleavage approach to generate hG4s with varying compositions and assessed their relative stability, revealing that hG4 stability increases with higher RNA content. We also showed that various G4-binding proteins recognize and bind to hG4 structures, with G4P exhibiting the highest affinity and effectively stabilizing hG4s. Additionally, the helicases Pif1 and RHAU were confirmed to efficiently unwind hG4 structures. Finally, we validated the roles of stabilizing and unwinding proteins in a transcription system. This study establishes a framework for investigating the stability and unwinding of hG4s and provides insights into modulating hG4 structures to regulate cellular processes.