Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides.

Area of Science:

• Biophysical Chemistry
• Molecular Biophysics
• Fluorescence Spectroscopy

Background:

• 4',6-diamidino-2-phenylindole (DAPI) is a fluorescent dye used for DNA staining.
• Understanding DAPI's photophysical properties is crucial for accurate biological imaging.
• DAPI exhibits complex fluorescence decay dynamics influenced by its environment.

Purpose of the Study:

• To investigate the fluorescence decay kinetics and spectral properties of DAPI.
• To explore how DAPI interacts with natural and synthetic polydeoxynucleotides.
• To elucidate the structural basis of DAPI's fluorescence heterogeneity.

Main Methods:

• Multifrequency phase-modulation fluorometry was employed to study DAPI.
• Fluorescence decay measurements were performed on DAPI in solution and complexed with DNA.
• Phase- and modulation-resolved emission spectra were collected across a range of wavelengths.

Main Results:

• DAPI in solution exhibits a double exponential fluorescence decay with lifetimes of 2.8 ns and 0.2 ns.
• Complexation with natural polydeoxynucleotides altered DAPI's decay, increasing the long lifetime component to ~4 ns.
• AT polymers/DAPI complexes showed single exponential decay, while poly(GC)/DAPI complex resembled free DAPI, indicating sequence-dependent binding.

Conclusions:

• DAPI's fluorescence heterogeneity arises from ground-state conformers affected by pH.
• DNA binding influences DAPI's solvation and stabilizes longer-lived excited states.
• Fluorescence decay analysis reliably distinguishes different DNA-binding modes of DAPI.