title: Immobilizing topoisomerase I on a surface plasmon resonance biosensor chip to screen for inhibitors.
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 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.
authors.affiliation: Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Hsiang-Ping
authors.initials: HP
authors.lastname: Tsai
authors.forename: Li-Wei
authors.initials: LW
authors.lastname: Lin
authors.forename: Zhi-Yang
authors.initials: ZY
authors.lastname: Lai
authors.forename: Jui-Yu
authors.initials: JY
authors.lastname: Wu
authors.forename: Chiao-En
authors.initials: CE
authors.lastname: Chen
authors.forename: Jaulang
authors.initials: J
authors.lastname: Hwang
authors.forename: Chien-Shu
authors.initials: CS
authors.lastname: Chen
authors.forename: Chun-Mao
authors.initials: CM
authors.lastname: Lin
mesh_terms: D002166:Camptothecin / Q000378:metabolism / Q000494:pharmacology; D020552:Comet Assay; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004800:Enzymes, Immobilized; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D010936:Plant Extracts / Q000378:metabolism / Q000494:pharmacology; D011799:Quinazolines / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*; D059004:Topoisomerase I Inhibitors
chemical_list: D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D010936:Plant Extracts; D011799:Quinazolines; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; D004247:DNA; C049639:evodiamine; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin