Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA
Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA
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DOI：
10.1093/nar/gkg308
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发表时间：
2003-04-15
影响因子：
14.9
通讯作者：
Sauer, M
中科院分区：
文献类型：
--
作者：
Adhikary, A;Buschmann, V;Sauer, M
用包合法和单分子荧光法测定了不同[H-258]/[DNA bp]比的2 '-（4-羟基苯基）-5-[5-（4-甲基哌嗪-1-基）苯并咪唑-2-基]-苯并咪唑（H-258）-小牛胸腺（CT）DNA复合物，以阐明H-258与DNA的结合。在以0.05的[H-258]/[DNA bp]比率与双链CT DNA（CT ds DNA）结合时，H-258的相对荧光量子产率Phi（f）从0.02增加到0.58。荧光衰减几乎可以用单指数模型拟合，寿命约为3.6ns。这表明H-258在低[H-258]/[DNA bp]比下几乎定量地结合在DNA的小沟中。随着[H-258]/[DNA bp]比率的增加，例如0.15和0.20，H-258的荧光量子产率分别降低至0.28和0.19。对较高[H-258]/[DNA bp]比率测量的荧光衰减的拟合揭示了在0.5-2.0 ns范围内存在额外的较短荧光寿命组分。我们的研究结果表明，H-258部分嵌入G：C序列在较高的[H-258]/[DNA bp]的比例反映了1.5-2 ns的寿命组件。此外，H-258分子在DNA上的堆积或吸附发生在较高的[H-258]/[DNA bp]比率下。这些分子表现出类似于500 ps的短荧光寿命，并且更多地暴露于水性环境。单H-258 CT ds DNA的强度和寿命的荧光瞬变表明，弱（非特异性）结合的H-258分子表现出较短的荧光寿命和强烈降低的光稳定性。
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, Phi(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 similar to3.6 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 similar to500 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.