用光波导光模光谱技术研究DNA-DNA结合蛋白相互作用

Application of Optical Waveguide Lightmode Spectroscopy in Studying the Interaction of DNA to DNA-Binding Protein

探讨了应用光波导光模光谱(Optical waveguide lightmode spectroscopy,OWLS)技术研究DNA-DNA结合蛋白相互作用的可行性和灵敏性。以固定在传感器芯片表面的DNA探针为捕捉分子,溶液中同时含有探针结合序列和NF—κB结合位点序列的寡核苷酸与NF-κB亚单位p50同源二聚体形成的DNA-蛋白质复合物为检测分子,用光波导光模光谱检测技术建立非标记DNA-DNA结合蛋白相互作用检测研究体系。利用这一体系对不同样品中NF-κB p50浓度和具不同NF-κB结合位点序列的寡核苷酸与NF-κB p50亲合和力进行检测。样品中低至0．33 nmol/1的NF-κB p50被光波导光模光谱检测出,不同的NF-κB结合序列与NF-κB p50亲合力有显著的差异。研究发现,光波导光模光谱技术可以用于DNA-DNA结合蛋白相互作用研究,所建立的非标记检测研究体系可以进行样品中结合蛋白含量高灵敏检测和核酸序列与结合蛋白的亲合力的检测研究。

The feasibility and sensitivity of optical waveguide lightmode spectroscopy （OWLS） in studying DNA- protein interaction are dissused. In the label-free DNA and DNA-protein interaction detection assay based on optical waveguide lightmode spectroscopy （OWLS）, the probe was located online onto the amino silanized sensor chip of optical waveguide lightmode spectroscopy by glutaraldehyde covalent coupling; the DNA-protein complex, combination of NF-κB p50 homodimer and oligonucleotide with binding site to NF-κB p50 homodimer and binding sequence to probe, was used as the detect molecule. The different concentrations of p50 in detection samples and affinity of oligonucleotides with different binding site sequence of NF-κB to p50 were tested in this system. With this detection system, as low as 0. 33 nmol/l of NF-κB p50 in the samples was detected, and the affinity of oligonucleotides with different binding site sequence to NF-κB is quite different. The label-free studying system of DNA-protein interaction based on the optical waveguide lightmode spectroscopy technique is feasible and high sensitivity, and this system can be used in the research targeted at the interaction of DNA and DNA-binding proteins and detection of the affinity of oligonucleotides to binding protein.