Abstract
Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.
Highlights
The search for specific inhibitors of helicases strongly relies on the development of easy, cheap, fast, reproducible biochemical assays, suitable for high-throughput (HT) screening
In most RecQ helicases the catalytic core is followed by a RecQ-C-terminal (RQC) domain, that has been proposed to have a crucial role in the helicase activity, by providing an aromatic residue acting as essential “pin” that physically disrupts the dsDNA base-pairing[25,26]; a bioinformatic analysis recently suggested the presence of a non-canonical RQC domain in RecQ427
Surface plasmon is the collective oscillations of the free electrons on the surface of nanoparticles which is in resonance with the frequency of the visible light interacting with them
Summary
The search for specific inhibitors of helicases strongly relies on the development of easy, cheap, fast, reproducible biochemical assays, suitable for high-throughput (HT) screening. The ones that can more be adapted for HT screens are those based on fluorescent resonance energy transfer (FRET), but are subjected to some drawbacks, including high costs, poor stability of the substrate and compound interference[2,9,10,11]. RecQ helicases are ubiquitous nucleic acid unwinding enzymes, playing a vital role in maintaining genomic stability by acting at the interface of replication, repair and recombination. They are involved in DNA repair, homologous recombination, telomere maintenance, mitochondrial genome maintenance and DNA replication[15,16,17]. A number of site-directed mutants have been analysed, confirming the importance of key residues in the putative RQC domain for the helicase activity
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