Investigating temperature-induced unfolding pathways of DNA G-quadruplexes via 2D UV resonant Raman spectroscopy

G-quadruplexes are highly polymorphic noncanonical secondary structures of nucleic acids that play important biological roles. Understanding their unfolding mechanisms is essential for elucidating their stability and interactions. In this study, we investigated the thermal unfolding pathways of four biologically relevant G-quadruplex-forming DNA sequences using ultraviolet resonance Raman (UVRR) spectroscopy combined with advanced spectral analysis techniques. Singular value decomposition (SVD) analysis identified distinct conformational states, while perturbation-correlation moving-window 2D (PCMW2D) and generalized 2D correlation spectroscopy (2D-COS) provided insights into the sequential molecular events during unfolding. Our results reveal distinct pathways for temperature-induced hydrogen bond breaking and structural rearrangements that are unique to each G-quadruplex. This study demonstrates the power of UVRR spectroscopy in resolving structural transitions at the molecular level, providing self-consistent models for the unfolding of G-quadruplexes and contributing to a deeper understanding of their stability and structural features.