Dual resonance energy transfer nucleic acid probes
First Claim
1. A composition comprising:
- (a) a stem-loop donor molecular beacon; and
(b) a stem-loop acceptor molecular beacon, wherein resonance energy transfer occurs between the donor molecular beacon and the acceptor molecular beacon when the donor and acceptor molecular beacons hybridize adjacent to each other on a target nucleic acid and are exposed to an exciting amount of electromagnetic radiation, and(c) means for delivering the stem-loop donor molecular beacon and the stem-loop donor molecular beacons into a cell.
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Accused Products
Abstract
Dual nucleic acid probes with resonance energy transfer moieties are provided. In particular, fluorescent or luminescent resonance energy transfer moieties are provided on hairpin stem-loop molecular beacon probes that hybridize sufficiently near each other on a subject nucleic acid, e.g. mRNA, to generate an observable interaction. The invention also provides lanthanide chelate luminescent resonance energy transfer moieties on linear and stem-loop probes that hybridize sufficiently near each other on a subject nucleic acid to generate an observable interaction. The invention thereby provides detectable signals for rapid, specific and sensitive hybridization determination in vivo. The probes are used in methods of detection of nucleic acid target hybridization for the identification and quantification of tissue and cell-specific gene expression levels, including response to external stimuli, such as drug candidates, and genetic variations associated with disease, such as cancer.
20 Citations
39 Claims
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1. A composition comprising:
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(a) a stem-loop donor molecular beacon; and (b) a stem-loop acceptor molecular beacon, wherein resonance energy transfer occurs between the donor molecular beacon and the acceptor molecular beacon when the donor and acceptor molecular beacons hybridize adjacent to each other on a target nucleic acid and are exposed to an exciting amount of electromagnetic radiation, and (c) means for delivering the stem-loop donor molecular beacon and the stem-loop donor molecular beacons into a cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A composition comprising:
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(a) a shared-stem acceptor molecular beacon; and (b) a shared-stem donor molecular beacon, wherein fluorescence resonance energy transfer occurs between the acceptor molecular beacon and the donor molecular beacon when the acceptor and donor molecular beacons hybridize adjacent to each other on a target nucleic acid and are exposed to an exciting amount of electromagnetic radiation. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for detecting expression of a target nucleic acid in a cell comprising:
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(a) delivering a stem-loop acceptor molecular beacon and a stem-loop donor molecular beacon to an interior of a cell; (b) exposing the molecular beacons with an exciting amount of electromagnetic energy so that fluorescence resonance energy transfer occurs between the acceptor molecular beacon and the donor molecular beacon when the acceptor and donor molecular beacons hybridize adjacent to each other on the target nucleic acid; and (c) detecting a signal resulting from fluorescence resonance energy transfer between the stem-loop donor molecular beacon and the stem-loop acceptor molecular beacon in response to the exciting amount of electromagnetic energy wherein a detectable signal indicates the presence of the target nucleic acid. - View Dependent Claims (22, 23)
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24. A method for detecting cancer comprising:
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(a) delivering a stem-loop acceptor molecular beacon and a stem-loop donor molecular beacon to an interior of a cell suspected of being cancerous, wherein the molecular beacons specifically hybridize to a subject nucleic acid that is differentially expressed in cancer cells compared to non-cancer cells; (b) exposing the molecular beacons with an exciting amount of radiation so that fluorescence resonance energy transfer occurs between the acceptor molecular beacon and the donor molecular beacon when the acceptor and donor molecular beacons hybridize adjacent to each other on the subject nucleic acid; and (c) detecting emissions from the acceptor molecular beacon resulting from resonance energy transfer, wherein a detectable signal is indicative of cancer. - View Dependent Claims (25, 26, 27, 28, 29, 30)
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31. A method for identifying compounds that increase or decrease expression of a subject nucleic acid comprising:
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(a) delivering stem-loop acceptor molecular beacons and a stem-loop donor molecular beacons to an interior of a cell suspected of being cancerous; (b) exposing the molecular beacons to an exciting amount of electromagnetic radiation so that fluorescence resonance energy transfer occurs between the acceptor molecular beacons and the donor molecular beacons when the acceptor and donor molecular beacons hybridize adjacent to each other on the subject nucleic acid; (c) detecting a first signal from the acceptor molecular beacons resulting from resonance energy transfer; (d) contacting the cell comprising the molecular beacons with a test compound; (e) detecting a second signal from the acceptor molecular beacons resulting from resonance energy transfer, wherein in an increased second signal compared to the first signal indicates that the test compound increases expression of the target nucleic acid and a decreased second signal compared to the first signal indicates that the test compound decreases expression of the subject nucleic acid. - View Dependent Claims (32, 33, 34)
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35. A system for detecting a target nucleic acid comprising:
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(a) a stem-loop donor molecular beacon; (b) a stem-loop acceptor molecular beacon, wherein resonance energy transfer occurs between the donor molecular beacon and the acceptor molecular beacon when the donor and acceptor molecular beacons hybridize adjacent to each other on a target nucleic acid and are exposed to an exciting amount of electromagnetic radiation; (c) means for exciting the molecular beacons; (d) means for detecting emissions from the molecular beacons; and (e) means for delivering the beacons inside the cell. - View Dependent Claims (36)
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37. A kit comprising:
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a) a stem-loop acceptor molecular beacon that specifically hybridizes to a target nucleic acid; b) a stem-loop donor molecular beacon that specifically hybridizes to the target nucleic acid, wherein fluorescence resonance energy transfer occurs between the acceptor molecular beacon and the donor molecular beacon when the acceptor and donor molecular beacons hybridize adjacent to each other on a target nucleic acid and are exposed to an exciting amount of electromagnetic radiation; and c) instructions for detecting the target nucleic acid using the molecular beacons. - View Dependent Claims (38, 39)
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Specification