荧光探针法研究阳离子多聚物基因载体Polybrene与DNA的相互作用

Studies on the interaction between polybrene and DNA with fluorescence probe

目的:用荧光探针法研究海美溴铵(PB)和DNA之间的相互作用。方法:将一定量的PB溶液逐渐加入到DNA/溴化乙锭(EB)复合物的水溶液中,在535nm处激发,测定595nm的荧光强度,观察体系荧光强度随PB浓度变化而发生的改变。用同样的方法观察PB对Hoe-DNA和4,6-二脒基-2-苯基吲哚(DAPI)-DNA体系荧光强度的影响,Hoe的激发和发射波长分别设置为350nm和460nm,DAPI为340nm和450nm。通过荧光Scatchard分析法获得PB对DNA的结合常数。结果:随着PB浓度的增加,EB与DNA的结合减弱。通过对PB存在下EB-DNA体系的荧光分析,获得了PB与DNA之间的结合常数为1.8×105/mol。PB的加入大大提高了Hoe-DNA体系的荧光强度,同时Hoe的最大发射波长由最初的490nm蓝移至460nm。Hoe-DNA体系和DAPI-DNA体系的荧光强度均随着聚合物单体的浓度与DNA磷酸基团的浓度比值(N∶P比值)的增加而增加,并且在N∶P比值等于或接近1.0时达到最大。结论:DNA与PB之间复合物的形成减弱了DNA与嵌入剂的结合,但却能增强DNA和沟区结合探针之间的相互作用。提示PB与DNA的结合可能特异性地影响了DNA的二级结构。

Objective：To study the interaction of polybrene （PB） with hsDNA by fluorescence probes. Methods.. The effect of PB on the DNA/ethidium bromide （EB） complex was investigated by adding a certain volume of PB solution into the DNA/EB complex solution. The fluorescence spectra and intensity of the solutions were measured using a fluorescence spectrophotometer. The fluorescence intensity was measured at 595 nm with excitation wave-length at 535 nm. In the similar methods, the influences of PB on the fluorescence intensity of Hoe DNA and 4,6- diamidino-2-phenylindole （DAPI）-DNA systems were also examined. The fluorescence intensity was measured at 460 nm with excitation wavelength at 350 nm for Hoechst 33258（Hoe） while at 450 nm and 340 nm for DAPI, and binding of PB to hsDNA was studied by fluorescence Scatchard analysis. Results：Both the binding constant of EB with DNA and the number of binding sites per nucleotide were decreased with the increase in concentration of PB. From the Scatchard analysis, the association constant of PB to DNA（1.8 × 10^5/mol）were obtained. The addition of PB greatly enhanced the fluorescence intensity of Hoe - DNA system. Meanwhile, the maximum emission wavelength of Hoe blue-shifted from 490 nm to 460 nm. The fluorescence intensities of Hoe - DNA systems were enhanced greatly with the increase in concentration of PB, reaching the maximum when the N/P ratios were equal or close to 1.0. Conclusion：The formation of the complex between DNA and PB is not in favor of the interaction between DNA and intercalator, but can strengthen the interaction between DNA and groove-binding probes. These data suggest that the binding of PB on DNA may have specific influence on the secondary structure of DNA.