光谱法与共焦荧光成像法研究罗丹明B与ct-DNA的相互作用

采用吸收光谱、荧光光谱以及共焦荧光成像技术研究生理条件下罗丹明B(Rhodamine B,RB)与小牛胸腺DNA(ct-DNA)的相互作用以及相互作用模式。光谱研究结果显示,在450~650 nm的吸收光谱范围内,ct-DNA溶液对罗丹明B有减色作用;在560~660 nm荧光发射光谱范围内,ct-DNA对罗丹明B有荧光猝灭作用。同时,随着ct-DNA的加入,罗丹明B溶液的荧光偏振值发生变化,说明罗丹明B与ct-DNA能发生相互作用。在竞争性实验中,罗丹明B未能将亚甲基蓝(MB)从MB-ct-DNA复合体系中置换出来,说明罗丹明B与ct-DNA通过沟槽结合方式发生相互作用。共焦荧光成像结果显示,ct-DNA加入后,罗丹明B的荧光猝灭十分明显。利用罗丹明B和4',6-二脒基-2-苯基吲哚(DAPI)对He La细胞染色后进行共焦荧光成像,结果进一步证实罗丹明B与ct-DNA是通过沟槽结合作用将细胞核染成红色。

Study on Interaction between Rhodamine B and Calf Thymus DNA by Spectroscopy and Confocal Imaging

The interaction between Rhodamine B and calf thymus DNA (ct-DNA) at physiological conditions were studied by the techniques of absorption spectroscopy,fluorescence spectroscopy and confocal imaging.The results of spectroscopy experiments suggested that the hypochromicity of Rhodamine B could appear in the presence of ct-DNA from 450 nm to 650 nm in the absorption spectroscopy,and the fluorescence of Rhodamine B was quenched in the presence of ct-DNA from 560 nm to 660 nm in the fluorescence emission spectroscopy.At the same time,the fluorescence polarization of Rhodamine B varied with the addition of ct-DNA.It was indicated that Rhodamine B could interact with ct-DNA.In the competition experiments,Rhodamine B was unable to replace methylene blue (MB) from MB-ct-DNA comnplex.It was indicated that the interaction between Rhodamine B and ct-DNA took place by the mode of groove combination.The results of confocal imaging experiments showed that the fluorescence of Rhodanmine B was quenched quite obviously after the addition of ctDNA.HeLa cells were dyed with Rhodamine B and 4',6-diamidino-2-phenylindole (DAPI),then used for confocal imaging.It was further verified that the cell nucleus could be dyed red through the groove combination interaction between Rhodamine B and ct-DNA.