Nucleic Acids Research, 2003, Vol. 31, No. 8 2178-2186
© 2003 Oxford University Press
Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA
1 Physikalisch-Chemisches Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany and 2 Department of Chemistry, Rajdhani College, University of Delhi, Raja Garden, New Delhi 110015, India
Correspondence may also be addressed to Markus Sauer. Tel: +49 6221 548460; Fax: +49 6221 544255; Email: sauer{at}urz.uni-heidelberg.de
Ensemble and single-molecule fluorescence measurements of 2'-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)– calf thymus (CT) DNA complexes at various [H-258]/[DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a [H-258]/[DNA bp] ratio of 0.05 the relative fluorescence quantum yield, 
f, of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of 
3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low [H-258]/[DNA bp] ratios. With increasing [H-258]/[DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher [H-258]/[DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5–2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher [H-258]/[DNA bp] ratios reflected by a lifetime component of 1.5–2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher [H-258]/[DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of 500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.
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