Abstract
To evaluate the potency of potential helicase modulators, we developed an assay of helicase enzyme activity. Using a DNA or RNA biotin labelled oligonucleotide and after the addition of a recombinant helicase, the nucleic acid unwinds, causing the emission of luminescence, which is quantified with a particular antibody. In our assay, one of the DNA oligos was biotinylated, while the other was labelled with digoxygenin (DIG), both in their 5’ termini. The biotin molecule immobilises the DNA duplex on a neutravidin-coated plate and the helicase activity is measured through the unwinding of DNA, due to ATP activation. The subsequent release of DIG-labelled oligos results in a luminescence signal measured with a chemiluminescence antibody. Our goal was to provide a high throughput screening method for potential helicase inhibitors. The method described in this paper has been demonstrated to be fast, easy and reproducible and doesn’t use radiochemicals.
Highlights
Helicase activity assays include analysis of ATPase activity, but it was shown that measuring the helicase unwinding activity is the best method for evaluating modulators of this class of enzyme (Borowski et al, 2002)
This type of assay depends on the ability of the enzyme to separate the release strand of DNA or RNA from the template strand
We propose a combination of the methods as mentioned above without radio-labelled molecules that can detect the residual release strand with a chemiluminescent antibody, giving a robust helicase assay and a stable readout, well suited to high-throughput screening
Summary
Helicase activity assays include analysis of ATPase activity, but it was shown that measuring the helicase unwinding activity is the best method for evaluating modulators of this class of enzyme (Borowski et al, 2002). Other methods measure the deposition of radio-labelled release strands after gel electrophoresis, thin-layered chromatography or scintillation counting (Borowski et al, 2001; Bartelma and Padmanabhan 2002; AlaouiIsmaili et al, 2000). These methods could be enhanced via high-throughput screening, the radioactive materials would be a problem. Article history Received: 21 December 2019 Accepted: 31 December 2019 Published: 13 February 2020
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