- 作者: Hsiang-Ping Tsai; Li-Wei Lin; Zhi-Yang Lai; Jui-Yu Wu; Chiao-En Chen; Jaulang Hwang; Chien-Shu Chen; Chun-Mao Lin
- 作者服務機構: Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan, R.O.C.
- 中文摘要: --
- 英文摘要:
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 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.
Conclusions : 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. - 中文關鍵字: --
- 英文關鍵字: --