title: In vitro high-throughput screening assay for modulators of transcription.
abstract: We developed a 96-well microtiter-plate high-throughput screening (HTS) assay for the detection of modulators of transcription. This HTS assay consists of three steps: (1) the in vitro transcription reaction; (2) modification and hybridization of RNA products; and (3) washing and quantification. During the first step, a DNA template containing the promoter of interest upstream of a cassette lacking guanosine residues in one of its strands (G-less cassette) is incubated with nuclear extract and the necessary cofactors/activators and substrates. During the second step, the in vitro synthesized transcripts are digested with RNase T1 and hybridized to two DNA oligonucleotides. One oligonucleotide is biotinylated for trapping of the RNA products to a streptavidin-coated plate, and the other is europium-labeled for detection by time-resolved fluorescence. We show that this assay is highly reproducible and robust, yielding results comparable to those obtained by standard methodologies employing radioactive nucleotide incorporation and gel electrophoresis while offering a very significant advantage in terms of throughput (>2000 assay points per operator per day). We demonstrate the usefulness of the assay for the discovery of small molecule inhibitors of transcription, and applications of this approach for the high-throughput discovery of transcriptional modulators are discussed.
authors.affiliation: Signal Research Division of Celgene, 5555 Oberlin Drive, San Diego, California 92121, USA.
authors.forename: León F
authors.initials: LF
authors.lastname: García-Martínez
authors.forename: Graham K
authors.initials: GK
authors.lastname: Bilter
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.forename: Joe
authors.initials: J
authors.lastname: O'Neill
authors.forename: Miguel S
authors.initials: MS
authors.lastname: Barbosa
authors.forename: Robert
authors.initials: R
authors.lastname: Kovelman
mesh_terms: D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D015536:Down-Regulation; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D008322:Mammals; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D000546:Amanitins; D012313:RNA; D012319:RNA Polymerase II; D012259:Ribonuclease, Pancreatic