Origin of 4′,6-diamidino-2-phenylindole (DAPI) fluorescence dynamics in solution and DNA minor groove binding: Unveiled by femtosecond broadband fluorescence, transient absorption, and theoretical calculations

4′,6-Diamidino-2-phenylindole (DAPI) is a widely utilized DNA sensor, renowned for its strong affinity for the AT minor groove. Despite its essential role as a fluorescent probe in molecular biology and biomedical research, the origins of DAPI’s intrinsic fluorescence and the mechanisms behind its fluorescence enhancement upon DNA sensing remain unresolved. This study provides a comprehensive investigation into the fluorescence dynamics of DAPI, both in solution and when bound to detect the AT minor groove of a self-complementary dodecamer duplex DNA. We employed an integrated approach combining femtosecond broadband time-resolved fluorescence, transient absorption, and supported by theoretical calculations. The results reveal an unprecedented ultrafast inter-solute–solvent three-state excited-state proton transfer pathway, which clarifies the root cause of low fluorescence yield and fluorescence dynamics of free form DAPI in aqueous solution. Irrespective of the structural heterogeneity and rotamer conformation of the ground state of DAPI, this involves deprotonation of the excited state at a rate of ∼2.4 ps, followed by proton reuptake by the resulting weakly emissive deprotonated state, leading to direct reformation of DAPI’s ground state at 139 ps. Binding to the DNA minor groove completely inhibits this proton transfer, accounting for the significant fluorescence enhancement observed in the DAPI-DNA complex. Furthermore, the broadband capacity of our time-resolved fluorescence approach enables, for the first time, direct tracking of the fluorescence dynamic Stokes shift of minor groove-bound DAPI, revealing significant dispersive collective solvation dynamics specific to the sensing site. These findings provide valuable insights into how microenvironments dictate DAPI fluorescence dynamics and may assist in the strategic design of light-up sensors for recognizing interior hydration dynamics and local residue motions of DNA.