Banerjee Debapriya, Pal Samir Kumar
Unit for Nano Science & Technology, Department of Chemical, Biological & Macromolecular Sciences, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India.
J Phys Chem B. 2008 Jan 24;112(3):1016-21. doi: 10.1021/jp077090f. Epub 2008 Jan 3.
Two distinct modes of interaction of the fluorescent probe 4',6-diamidino-2-phenylindole (DAPI), depending on the sequence of DNA, have been reported in the literature. In the present study, the dynamics of solvation has been utilized to explore the binding interaction of DAPI to DNA oligomers of different sequences. Picosecond-resolved fluorescence and polarization-gated anisotropy have been used to characterize the binding of DAPI to the different oligomers. In the double-stranded dodecamer of sequence CGCGAATTCGCG (oligo1), the solvation relaxation dynamics of the probe reveals time constants of 0.130 ns (75%) and 2.35 ns (25%). Independent exploration of the minor-groove environment of oligo1 using another well-known minor-groove binder Hoechst 33258 (H258) shows similar timescales, further confirming minor-groove binding of DAPI to oligo1. In the double-stranded dodecamer (oligo2) having the sequence GCGCGCGCGCGC, where intercalation has been reported in the literature, no solvation is observed in our experimental window. DAPI bound to oligo2 shows quenching of fluorescence compared to that of DAPI in a buffer. The quenching of fluorescence of DAPI intercalated in DNA is also borne out by the appearance of a fast component of 30 ps in the fluorescence lifetime, revealing electron transfer to DAPI from GC base pairs, between which it intercalates. In addition to this, the excited-state lifetime of the probe in the DAPI-DNA complex also shows a time constant similar to that of the dye in a buffer, indicating that the excited-state photoprocesses associated with the free dye is also operative in this binding mode, consistent with the binding geometry of the DAPI in the DNA. The dynamics of DAPI in calf thymus DNA having a random sequence of base pairs is similar to that associated with the DNA minor groove. Our studies clearly explore the structure-dynamics correlation of the DAPI-DNA complex in the two distinct modes of interaction of DAPI with DNA.
文献报道了荧光探针4',6-二脒基-2-苯基吲哚（DAPI）根据DNA序列存在两种不同的相互作用模式。在本研究中，利用溶剂化动力学来探究DAPI与不同序列DNA寡聚物的结合相互作用。皮秒分辨荧光和偏振门控各向异性已被用于表征DAPI与不同寡聚物的结合。在序列为CGCGAATTCGCG的双链十二聚体（oligo1）中，探针的溶剂化弛豫动力学显示时间常数为0.130纳秒（75%）和2.35纳秒（25%）。使用另一种著名的小沟结合剂Hoechst 33258（H258）对oligo1的小沟环境进行独立探究，显示出相似的时间尺度，进一步证实了DAPI与oligo1的小沟结合。在文献报道存在嵌入作用的序列为GCGCGCGCGCGC的双链十二聚体（oligo2）中，在我们的实验窗口内未观察到溶剂化现象。与缓冲液中的DAPI相比，与oligo2结合的DAPI显示出荧光猝灭。嵌入DNA中的DAPI的荧光猝灭也通过荧光寿命中30皮秒的快速成分的出现得到证实，这表明从其嵌入的GC碱基对向DAPI发生了电子转移。除此之外，DAPI-DNA复合物中探针的激发态寿命也显示出与缓冲液中染料相似的时间常数，表明与游离染料相关的激发态光过程在这种结合模式中也起作用，这与DAPI在DNA中的结合几何结构一致。具有随机碱基对序列的小牛胸腺DNA中DAPI的动力学与与DNA小沟相关的动力学相似。我们的研究清楚地探究了DAPI与DNA相互作用的两种不同模式下DAPI-DNA复合物的结构-动力学相关性。
