An interference-free fluorescent assay of telomerase for the high-throughput analysis of inhibitors.
We have developed a telomerase assay that can quickly and accurately rank the ability of molecules to inhibit telomerase activity. It is based on the method of Orlando and co-workers which utilizes PicoGreen to detect dsDNA formed during the polymerase chain reaction (PCR) amplification of telomerase products. PCR cycles were optimized to give as linear a signal as possible relative to telomerase products; 96-well streptavidin-coated PCR plates were used to isolate the preamplification telomerase products and to wash inhibitors away before the amplification step. The inhibitor removal step is critical to prevent false positives potentially caused by inhibition of Taq polymerase during amplification. Use of the streptavidin-coated PCR plate allows this step to be done much more rapidly than use of the liquid/liquid extraction adopted by others. We have demonstrated that this assay can correctly order the ability of four inhibitors to inhibit telomerase and reproduce within a factor of two the absolute IC(50) values determined by the more time-consuming direct assay. We have shown that the difference in IC(50) values determined in this assay versus the direct assay can be corrected for by using the standard curve appropriately. Using this method 96 compounds can be assessed in 3-5h.
Francis
Rawle
R
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri, 5005 Rockhill Road, Room 108, Kansas City, MO 64110-2499, USA.
Friedman
Simon H
SH
D002457:Cell Extracts; D004247:DNA / Q000032:analysis / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D005470:Fluorometry; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D019098:Telomerase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013997:Time Factors
D002457:Cell Extracts; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA; D019098:Telomerase