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首页> 外文期刊>Langmuir: The ACS Journal of Surfaces and Colloids >Surface plasmon resonance study of DNA polymerases binding to template/primer DNA duplexes immobilized on supported lipid monolayers
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Surface plasmon resonance study of DNA polymerases binding to template/primer DNA duplexes immobilized on supported lipid monolayers

机译：DNA聚合酶与固定在支持的脂质单分子层上的模板/引物DNA双链体结合的表面等离子体共振研究

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摘要

Recent advances in biosensor technology have contributed significantly to our understanding of the mechanisms of molecular recognition processes. In this study, a surface plasmon resonance (SPR)-based optical sensor was employed to study the binding interactions between a series of template/primer double-stranded DNA (dsDNA) and DNA polymerase enzymes containing both polymerase and exonuclease activities. The DNA molecules were immobilized on the SPR gold surface by the controlled assembly of a biotinylated phospholipid monolayer, an avidin monolayer, and a layer of biotinylated DNA. By introducing a number of different mismatches atear the 3'-end of the primer, the effects of the mismatches on the overall binding affinities of the Klenow fragment (XF) of Escherichia coil DNA polymerase I and T4 and T7 bacteriophage polymerases were measured. While no obvious trend in the overall binding affinity of the Klenow fragment was observed with the introduction of mismatched base pair(s), its polymerase domain binds with the fully matched DNA 20 times more strongly than with the DNA containing three consecutive terminal mismatches. On the other hand, ids exonuclease domain binds with the DNA containing three terminal mismatches 12 times more strongly than with the fully matched DNA. For both T4 and T7 polymerases, the overall affinities of the enzymes toward DNA increased as the number of mismatches increased, consistent with previous reports that the enhanced melting ability of terminally mismatched DNA duplex leads to the preferential binding at the exonuclease site. The results have demonstrated the feasibility of using the SPR biosensor to study molecular recognition events such as single base discrimination involved in protein-DNA interactions. [References: 77]
机译：生物传感器技术的最新进展为我们对分子识别过程机理的理解做出了重要贡献。在这项研究中，基于表面等离子体共振（SPR）的光学传感器用于研究一系列模板/引物双链DNA（dsDNA）与同时具有聚合酶和核酸外切酶活性的DNA聚合酶之间的结合相互作用。通过生物素化磷脂单层，亲和素单层和生物素​​化DNA层的受控组装将DNA分子固定在SPR金表面上。通过在引物的3'末端/附近引入许多不同的错配，可以测量错配对大肠杆菌线圈DNA聚合酶I和T4和T7噬菌体聚合酶的Klenow片段（XF）整体结合亲和力的影响。 。通过引入错配的碱基对，没有观察到Klenow片段的整体结合亲和力的明显趋势，但是其聚合酶结构域与完全匹配的DNA结合的强度是含有三个连续末端错配的DNA的结合强度的20倍。另一方面，ids核酸外切酶结构域与包含三个末端错配的DNA的结合强度是与完全匹配的DNA结合的12倍。对于T4和T7聚合酶，随着错配数目的增加，酶对DNA的整体亲和力也随之增加，这与先前报道的末端错配DNA双链体熔解能力增强导致核酸外切酶位点优先结合的报道一致。结果证明了使用SPR生物传感器研究分子识别事件（例如参与蛋白质-DNA相互作用的单碱基识别）的可行性。 [参考：77]

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