Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening
1999; Elsevier BV; Volume: 4; Issue: 5 Linguagem: Inglês
10.1177/108705719900400505
ISSN2472-5560
AutoresDavid L. Earnshaw, Keith J. M. Moore, Catherine J. Greenwood, Hakim Djaballah, Anthony J. Jurewicz, Kenneth Murray, Andrew J. Pope,
Tópico(s)Polyomavirus and related diseases
ResumoDNA helicases are responsible for the unwinding of double-stranded DNA, facilitated by the binding and hydrolysis of 5'-nucleoside triphosphates. These enzymes represent an important class of targets for the development of novel anti-infective agents particularly because opportunity exists for synergy with existing therapies targeted at other enzymes involved in DNA replication. Unwinding reactions are conventionally monitored by low throughput, gel-based radiochemical assays; to overcome the limitations of low throughput to achieve comprehensive characterization of adenosine triphosphate (ATP)-dependent unwinding by viral and bacterial helicases and the screening for unwinding inhibitors, we have developed and validated homogeneous time-resolved fluorescence energy transfer (TRET) assays. Rapid characterization and screening of DNA helicase has been performed in 96- and 384-well plate densities, and the ability to assay in 1536-well format also demonstrated. We have successfully validated and are running full high throughput runs using 384-well TRET helicase assays, culminating in the identification of a range of chemically diverse inhibitors of viral and bacterial helicases. For screening in mixtures, we used a combination of quench correction routines and confirmatory scintillation proximity (SP) assays to eliminate false-positives due to the relatively high levels of compound quenching (unlike other Ln(3+)-based assays). This strategy was successful yet emphasised the need for further improvements in helicase assays.
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