A New Dual Luciferase Assay Using NanoLuc Enables a Second Generation Coincidence Reporter System to Reduce False Hits in HTS Poster
Part # PS200
Christopher Eggers1, Samuel Hasson2, Brock Binkowski1, Matt Robers1, James Unch3, Braeden Butler1, , Keith Wood1, James Inglese2 and Frank Fan1
1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711-5399; 2National Center for Advancing Translational Sciences, 9800 Medical Center Dr, Rockville, MD 20850; 3Promega Biosciences LLC, 277 Granada Dr, San Luis Obispo, CA 93401
Luciferase-based reporter-gene assays remain a cornerstone of high-throughput screening of compounds because of their high sensitivity and dynamic range. However, a substantial number of non-relevant hits can be generated due to direct interaction of compounds with the luciferase reporter. To help differentiate compounds modulating the biological pathway of interest from those affecting the stability or activity of the reporter enzyme, we have developed a second-generation coincidence reporter system in which transcriptional activation leads to stoichiometric expression of two orthologous reporters that have dissimilar profiles of compound interference. In this system, firefly luciferase (Fluc) and PEST-destabilized NanoLuc® luciferase (NlucP) are expressed off the same promoter using ribosome skipping mediated by the P2A peptide.
To sensitively measure both Fluc and NanoLuc (Nluc) in the same sample, we have developed the Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay System, a homogeneous lytic assay performed in an “add-read-add-read” format, in which the Fluc signal is quenched over a million-fold by addition of the Nluc reagent. The increased brightness of Nluc and improved Fluc inhibition means that Nluc can be detected at over 2-3 orders of magnitude lower molar concentration than Renilla luciferase in the existing homogenous firefly/Renilla dual-luciferase assay (Dual-Glo), allowing both luciferases to be dynamic reporters. Following single-copy integration of the Fluc-2A-NlucP biocircuit into a gene locus relevant to Parkinson’s disease, HTS using NanoDLR easily distinguished compounds affecting one of the reporters from those affecting transcription, yielding a >5-fold decrease in the number of hits.
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