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US 6,183,956 B2Granted
High throughput in vitro screening assays for transcription modulators
AssigneeTularik Inc.
InventorsMohanram Sivaraja, Berta Strulovici, Osvaldo A. Flores
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Abstract
High-throughput in vitro assays are provided for identifying modulators of transcription activity and RNA expression. New assays, related compositions, apparatus and integrated systems are provided.
Claims38
A method of measuring the expression of a selected RNA in the presence of a potential nucleic acid transcriptional activity modulator in a sample, the method comprising the steps of:
- (i) providing a sample comprising a potential transcriptional activity modulator and an expression cassette comprising a promoter operably linked to a selected nucleic acid encoding the selected RNA;
- (ii) incubating the sample with a DNA oligonucleotide, wherein a region of the oligonucleotide is complementary to a region of the selected RNA, forming an RNA:DNA heteroduplex;
- (iii) cleaving single-stranded RNA in the sample with an RNase enzyme;
- (iv) binding the RNA:DNA heteroduplex to a first recognition reagent comprising an antibody that binds to RNA:DNA heteroduplexes, the antibody immobilized on a solid substrate, thereby immobilizing the RNA:DNA heteroduplex on the solid substrate;
- and
- (v) detecting the RNA:DNA heteroduplex bound to the solid substrate.
3 more elements
A method of measuring expression of a selected RNA in the presence of a potential nucleic acid transcriptional activity modulator in a sample, the method comprising the steps of:
- (i) incubating a sample comprising a potential transcriptional activity modulator and an expression cassette comprising a promoter operably linked to a selected nucleic acid encoding a selected G-less or a selected A-less RNA;
- (ii) cleaving single-stranded RNA in the sample with an RNase enzyme that cleaves at guanine or adenine positions;
- (iii) binding the selected RNA to a first recognition reagent directly or indirectly immobilized on a solid substrate, thereby immobilizing the selected RNA;
- and
- (iv) detecting the level of expression of the selected RNA in the sample.
2 more elements
Dependencies are inferred from the claim text.