New insights into the structure and dynamics of the epigenetic modifications on DNA.

DNA methylation is a key epigenetic modification involved in genomic imprinting, X-chromosome inactivation, and repression of repetitive element transcription and transposition. Despite its biological significance, the impact of epigenetic modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) on the structural and UV-induced dynamics of DNA remains poorly understood. Here, we employed the fluorescent nucleobase analogue 2-aminopurine (2Ap) in combination with steady-state and time-resolved spectroscopy, molecular dynamics, and quantum mechanical calculations to investigate these effects. Our findings reveal distinct differences in base stacking and helical stability between mC and hmC-modified DNA. mC-modified DNA predominantly adopts a stacked conformation, promoting efficient fluorescence quenching of 2Ap. In contrast, hmC-modified DNA displays both stacked and non-stacked conformations, leading to reduced base stacking and a more hydrophobic local environment, as indicated by blue-shifted emission spectra. Furthermore, although charge-transfer quenching occurs in all systems, hmC shows weaker charge-transfer character, resulting in higher fluorescence quantum yields and longer lifetimes. These results highlight the subtle but crucial role of hmC in modulating local DNA conformation and stability. Moreover, they demonstrate the effectiveness of 2Ap as a sensitive probe for detecting epigenetic modifications, offering deeper insights into the molecular mechanisms of DNA methylation and demethylation pathways.