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DNA‐ligand Binding Mode Discrimination by Characterizing Fluorescence Resonance Energy Transfer Through Lifetime Measurements with Picosecond Resolution
Author(s) -
Nardo Luca,
Bondani Maria,
Andreoni Alessandra
Publication year - 2007
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.2007.00204.x
Subject(s) - förster resonance energy transfer , picosecond , resolution (logic) , fluorescence , energy transfer , resonance (particle physics) , ligand (biochemistry) , dna , chemistry , biophysics , materials science , nuclear magnetic resonance , atomic physics , molecular physics , optics , physics , biology , computer science , receptor , biochemistry , laser , artificial intelligence
We describe a method for distinguishing between minor groove binders and base intercalators that is based on measurements of the fluorescence lifetime of a donor (D) in the presence of an acceptor (A). The D–A pair is separated by a short double helix DNA with which the ligands interact. By plotting the D fluorescence lifetime as a function of the ligand‐to‐base pair concentration ratio we find a clear signature that distinguishes between the two binding mechanisms: minor groove binding induces an asymptotic decrease of the D fluorescence lifetime, while intercalation gives a monotonically increasing lifetime and the appearance of an additional short lifetime. We assayed Quinacrine, Hoechst and 4′–6′diamidine‐2‐phenyl indole, which in control experiments performed on oligodeoxyribonucleotides (oligos) lacking the A are demonstrated not to interfere with the D fluorescence. The changes in fluorescence lifetimes measured in the case of dual‐labeled oligos are thus caused by structural changes in the DNA that modify the D–A distance. The appearance of the short‐lived transient in the fluorescence decay of Ds attached to dual‐labeled oligos upon binding of an intercalator can be interpreted as denaturation.