tailieunhanh - Immobilizing topoisomerase I on a surface plasmon resonance biosensor chip to screen for inhibitors

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. | Tsai et al. Journal of Biomedical Science 2010 17 49 http content 17 1 49 a NSC Tha cost of publication In Journal of Blomodlcal Science Is boms by tlM National Science Council Taiwan JOURNAL OF BIOMEDICAL SCIENCE RESEARCH Open Access Immobilizing topoisomerase I on a surface plasmon resonance biosensor chip to screen for inhibitors Hsiang-Ping Tsait1 2 Li-Wei Lin 3 Zhi-Yang Lai43 Jui-Yu Wu2 Chiao-En Chen4 Jaulang Hwang4 Chien-Shu Chen5 and Chun-Mao Lin 2 Abstract Background 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. Methods 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. Results 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 dosedependent 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 .