A homogeneous cell-based bicistronic fluorescence assay for high-throughput identification of drugs that perturb viral gene recoding and read-through of nonsense stop codons.
Recoding mechanisms are programmed protein synthesis events used commonly by viruses but only very rarely in cells for cellular gene expression. For example, HIV-1 has an absolute reliance on frameshifting to produce the correct ratio of key proteins critical for infectivity. To exploit such recoding sites as therapeutic targets, a simple homogeneous assay capable of detecting small perturbations in these low-frequency (<5%) events is required. Current assays based on dual luciferase reporters use expensive substrates and are labor-intensive, both impediments for high-throughput screening. We have developed a cell-based bifluorophore assay able to measure accurately small recoding changes (<0.1%) with a high Z'-factor in 24- or 96-well formats that could be extended to 384 wells. In cases of nonsense mutations arising within coding regions of genes, the assay is suitable for assessing the potential of screened compounds to increase read-through at these nonprogrammed stop signals of variable termination efficiency.
Cardno
Tony S
TS
Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand.
Poole
Elizabeth S
ES
Mathew
Suneeth F
SF
Graves
Ryan
R
Tate
Warren P
WP
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D018389:Codon, Nonsense / Q000187:drug effects*; D005456:Fluorescent Dyes; D018965:Frameshifting, Ribosomal / Q000187:drug effects; D005796:Genes / Q000235:genetics; D017930:Genes, Reporter; D005814:Genes, Viral / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008826:Microbial Sensitivity Tests / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D013050:Spectrometry, Fluorescence; D000097573:Red Fluorescent Protein
D018389:Codon, Nonsense; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012367:RNA, Viral; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein