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| 국가/구분 | United States(US) Patent 등록 |
|---|---|
| 국제특허분류(IPC7판) |
|
| 출원번호 | UP-0241872 (2005-09-30) |
| 등록번호 | US-7632651 (2009-12-24) |
| 발명자 / 주소 |
|
| 출원인 / 주소 |
|
| 대리인 / 주소 |
|
| 인용정보 | 피인용 횟수 : 5 인용 특허 : 341 |
Systems for detecting molecular modifications and the presence and/or activity of enzymes and/or other agents involved in facilitating or otherwise regulating such modifications.
We claim: 1. A method of detecting the activity of an enzyme that catalyzes formation of a product from a substrate, the method comprising: contacting the substrate with the enzyme and with a binding partner to form a sample mixture, the binding partner binding selectively either to the substrate r
We claim: 1. A method of detecting the activity of an enzyme that catalyzes formation of a product from a substrate, the method comprising: contacting the substrate with the enzyme and with a binding partner to form a sample mixture, the binding partner binding selectively either to the substrate relative to the product or to the product relative to the substrate, the binding partner including at least one metal that is required for selective binding of the binding partner to the substrate or to the product via at least one phosphate moiety of the substrate or product, where the substrate and/or product includes an energy transfer donor and the binding partner includes an energy transfer acceptor, or the substrate and/or product includes an energy transfer acceptor and the binding partner includes an energy transfer donor, such that luminescence energy transfer can occur between the donor and the acceptor when the binding partner binds to the substrate or the product, where the enzyme is selected from the group consisting of kinases, phosphatases, nucleotide cyclases, and nucleotide phosphodiesterases, where the binding partner includes a lanthanide chelate and the metal, where the lanthanide chelate includes an organic chelator and a lanthanide, and where the organic chelator includes at least one metal-binding functional group that associates the lanthanide chelate with the metal; exposing the sample mixture to light capable of inducing luminescence from the energy transfer donor; measuring a detectable luminescence energy transfer response from the sample mixture, without separating the bound substrate or product from the unbound substrate or product, where the detectable luminescence energy transfer response is indicative of the extent of binding between the substrate or product and the binding partner; and correlating the response with the activity of the enzyme. 2. The method of claim 1, where the metal is selected from the group consisting of aluminum, iron, gallium, europium, and terbium. 3. The method of claim 2, where the metal is Ga(III). 4. The method of claim 2, where the metal is Fe(III). 5. The method of claim 1, where measuring the detectable luminescence energy transfer response includes measuring luminescence intensity. 6. The method of claim 1, where the energy transfer acceptor is luminescent. 7. The method of claim 1, where the energy transfer acceptor is a quencher. 8. The method of claim 1, where the binding partner includes the energy transfer donor. 9. The method of claim 1, the energy transfer donor having a luminescence lifetime, where the luminescence lifetime is at least about 400 nanoseconds. 10. The method of claim 1, where the energy transfer donor comprises the lanthanide chelate. 11. The method of claim 1, where the metal-binding functional group is a phosphate, sulfonic acid, or carboxylic acid functional group. 12. The method of claim 11, where the metal-binding functional group is a phosphate functional group. 13. The method of claim 1, where the organic chelator includes a sensitizer moiety. 14. The method of claim 13, where the organic chelator has the formula where the R4, R5, and R6 substituents are independently hydroxy, alkyl groups having 1-6 carbons, alkoxy groups having 1-6 carbons, or amine groups, each of which is optionally further substituted by additional aliphatic groups, aromatic groups, amide groups, and heteroatom-substituted aliphatic groups; where Z is a sensitizer moiety that is a polycyclic heteroaromatic ring system that is bound via a covalent linkage; where the organic chelator is substituted by the metal-binding functional group; and where the organic chelator is optionally substituted by one or more reactive functional groups or conjugated substances. 15. The method of claim 14, where Z is a sensitizer moiety having the formula where K is the covalent linkage; where X, Y, V, and J are carbon or a heteroatom that is nitrogen, oxygen, sulfur, or selenium; and where the sensitizer is optionally further substituted at one or more positions by alkyl, alkoxy, halogen, or a metal-binding functional group. 16. The method of claim 10, where the energy transfer donor includes a complexed lanthanide ion that is selected from terbium, europium, dysprosium, or samarium. 17. The method of claim 1, where the substrate is a polypeptide, and where the substrate and product are related by phosphorylation or dephosphorylation of the polypeptide. 18. The method of claim 1, further comprising: contacting the substrate and enzyme with a candidate compound; and determining the ability of the candidate compound to enhance or inhibit enzyme activity by its effects on the response. 19. The method of claim 1, further comprising: providing a sample holder having a plurality of sample sites supporting a corresponding plurality of sample mixtures; and repeating the steps of contacting, exposing, measuring, and correlating for the plurality of sample mixtures.
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