Abstract
Turn-on fluorescent probes show enhanced emission upon DNA binding, advocating their importance in imaging cellular DNA. We have probed the DNA binding mode of thiazole-coumarin (TC) conjugate, a recently reported hemicyanine-based turn-on red fluorescent probe, using a number of biophysical techniques and a series of short oligonucleotides. TC exhibited increased fluorescence anisotropy and decreased absorbance (~50%) at low [DNA]/[TC] ratio. Although the observed hypochromicity and the saturating value of [DNA base pair]:[TC] ratio is consistent with a previous study that suggested intercalation to be the DNA binding mode of TC, a distinctly different and previously unreported binding mode was observed at higher ratios of [DNA]:[TC]. With further addition of DNA, only oligonucleotides containing AnTn or (AT)n stretches showed further change—decreased hypochromicity, red shifted absorption peaks and concomitant fluorescence enhancement, saturating at about 1:1 [DNA]: [TC]. 1H-NMR chemical shift perturbation patterns and H1’-H6/H8 NOE cross-peaks of the 1:1 complex indicated minor groove binding by TC. ITC showed the 1:1 DNA binding event to be endothermic (ΔH° ~ 2 kcal/mol) and entropy driven (ΔS° ~ 32 cal/mol/K). Taken together, the experimental data suggest a dual DNA binding mode by TC. At low [DNA]/[TC] ratio, the dominant mode is intercalation. This switches to minor groove binding at higher [DNA]/[TC], only for sequences containing AnTn or (AT)n stretches. Turn-on fluorescence results only in the previously unreported minor groove bound state. Our results allow a better understanding of DNA-ligand interaction for the newly reported turn-on probe TC.
Highlights
DNA is a ubiquitous biological macromolecule whose specific detection under various conditions forms the basis of probing molecular mechanisms behind various cellular processes [1]
We show that TC, in addition to intercalation, binds at the minor groove of AnTn or (AT)-rich DNA–a binding mode responsible for the fluorescence enhancement
Addition of DNA resulted in red shifts (Fig 1E): highest for A3T3 and A2T2 (~ 20 nm), intermediary for (AT)2, t-(AT)2 and AT (~ 10–15 nm) and no detectable shift for CG
Summary
DNA is a ubiquitous biological macromolecule whose specific detection under various conditions forms the basis of probing molecular mechanisms behind various cellular processes [1]. A typical double helical DNA molecule can bind appropriate molecular partners at the major or the minor groove. The binding partner can intercalate between stacked base pairs. The major groove is much wider than the minor groove and typically accommodates protein domains. Small molecules, both natural and synthetic, display either minor groove binding or intercalation [2].
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