Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei, Taiwan.
J Biomed Sci. 2010 Jun 17;17(1):49. doi: 10.1186/1423-0127-17-49.
The topoisomerase I (TopI) reaction intermediate consists of an enzyme covalently linked to a nicked DNA molecule, known as a TopI-DNA complex, that can be trapped by inhibitors and results in failure of re-ligation. Attempts at new derivative designs for TopI inhibition are enthusiastically being pursued, and TopI inhibitors were developed for a variety of applications. Surface plasmon resonance (SPR) was recently used in TopI-inhibition studies. However, most such immobilized small molecules or short-sequence nucleotides are used as ligands onto sensor chips, and TopI was used as the analyte that flowed through the sensor chip.
We established a sensor chip on which the TopI protein is immobilized to evaluate TopI inhibition by SPR. Camptothecin (CPT) targeting the DNA-TopI complex was used as a representative inhibitor to validate this label-free method.
Purified recombinant human TopI was covalently coupled to the sensor chip for the SPR assay. The binding of anti-human (h)TopI antibodies and plasmid pUC19, respectively, to the immobilized hTopI was observed with dose-dependent increases in resonance units (RU) suggesting that the immobilized hTopI retains its DNA-binding activity. Neither CPT nor evodiamine alone in the analyte flowing through the sensor chip showed a significant increase in RU. The combination of pUC19 and TopI inhibitors as the analyte flowing through the sensor chip caused increases in RU. This confirms its reliability for binding kinetic studies of DNA-TopI binders for interaction and for primary screening of TopI inhibitors.
TopI immobilized on the chip retained its bioactivities of DNA binding and catalysis of intermediates of the DNA-TopI complex. This provides DNA-TopI binders for interaction and primary screening with a label-free method. In addition, this biochip can also ensure the reliability of binding kinetic studies of TopI.
拓扑异构酶 I（TopI）反应中间体由酶与切口 DNA 分子共价连接组成，称为 TopI-DNA 复合物，可被抑制剂捕获，导致重新连接失败。人们正在积极尝试设计新的 TopI 抑制剂衍生物，并且已经开发出多种应用的 TopI 抑制剂。最近，表面等离子体共振（SPR）被用于 TopI 抑制研究。然而，大多数此类固定的小分子或短序列核苷酸被用作传感器芯片上的配体，而 TopI 则被用作流过传感器芯片的分析物。
我们建立了一种传感器芯片，将 TopI 蛋白固定在上面，以通过 SPR 评估 TopI 抑制作用。喜树碱（CPT）靶向 DNA-TopI 复合物被用作代表性抑制剂来验证这种无标记方法。
纯化的重组人 TopI 被共价偶联到传感器芯片上进行 SPR 分析。分别观察到抗人（h）TopI 抗体和质粒 pUC19 与固定的 hTopI 的结合，随着 RU 的剂量依赖性增加，表明固定的 hTopI 保留其 DNA 结合活性。分析物中单独的 CPT 或吴茱萸碱本身都不会导致 RU 显著增加。当 pUC19 和 TopI 抑制剂作为分析物流过传感器芯片时，会导致 RU 增加。这证实了它用于 DNA-TopI 结合物相互作用的结合动力学研究以及 TopI 抑制剂的初步筛选的可靠性。
固定在芯片上的 TopI 保留了其 DNA 结合和催化 DNA-TopI 复合物中间产物的活性。这为相互作用提供了 DNA-TopI 结合物，并提供了无标记方法的初步筛选。此外，这种生物芯片还可以确保 TopI 结合动力学研究的可靠性。
