title: PCR-free telomerase assay using chronocoulometry coupled with hexaammineruthenium(III) chloride.
abstract: An electrochemical method based on chronocoulometry coupled with hexaammineruthenium chloride (RuHex) is proposed for simple and rapid assay of telomerase without relying on PCR and gel electrophoresis. Thus, DNA extended by telomerase in extracts of as small as 5-1,000 HeLa cells on the TS primer-immobilized electrodes was quantified successfully. This method is suitable for quick screening of drug candidates which inhibit telomerase. When 10 compounds were tested, the multiplicity of extension (x in (TTAGGG)(x)) varied from 11 to 0, suggesting that there is more than one mechanism of inhibition. IC(50) values of telomerase inhibitors TMPyP4 and PIPER were determined as 5.5 and 15 μM, respectively, though their mechanisms of inhibition are different. This method is capable of discriminating two possible mechanisms of telomerase inhibition: direct binding of inhibitors to telomerase and indirect inhibition through their binding to the quadruplex generated by telomerase. As this method is easy and quick to run, it will be useful for high-throughput screening of drug candidates which inhibit telomerase.
authors.affiliation: Department of Applied Chemistry, Kyushu Institute of Technology, Sensui-cho 1-1, Kitakyushu-shi, Fukuoka 804-8550, Japan.
authors.forename: Shinobu
authors.initials: S
authors.lastname: Sato
authors.forename: Shigeori
authors.initials: S
authors.lastname: Takenaka
mesh_terms: D055664:Electrochemical Techniques; D004566:Electrodes; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D010569:Perylene / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011166:Porphyrins / Q000737:chemistry / Q000494:pharmacology; D017975:Ruthenium Compounds / Q000737:chemistry*; D019098:Telomerase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; C116108:N,N'-bis(2-1(piperidino)-ethyl)-3,4,9,10-perylene-tetracarboxylic diimide; D010880:Piperidines; D011166:Porphyrins; D017975:Ruthenium Compounds; C031584:hexammineruthenium; C021096:tetra(4-N-methylpyridyl)porphine; D010569:Perylene; D019098:Telomerase