MOLECULAR MODIFICATION ASSAYS

• US 20100304404A1
• Filed: 12/14/2009
• Published: 12/02/2010
• Est. Priority Date: 09/15/1997
• Status: Abandoned Application

• Current Assignee: Molecular Devices
• Original Assignee: Molecular Devices
• Inventors: YI, GEORGE G., GAUDET, ELIZABETH, BOGE, ANNEGRET, SPORTSMAN, J. RICHARD
• Application Number:
  US12/637,598
• Publication Number:
  US 20100304404A1
• Time in Patent Office: Days
• US Class Current: 435/7.4
• CPC Class Codes: B01L 3/50853 with covers or lids
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/25 involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/0405 manipulating closing or opening means, e.g. stoppers, screw caps, lids or covers
• CPC Class Codes: G01N 2035/0425 Stacks, magazines or elevators for plates
• CPC Class Codes: G01N 2035/0462 Buffers [FIFO] or stacks [LIFO] for holding carriers between operations
• CPC Class Codes: G01N 21/6445 Measuring fluorescence polarisation
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/1011 : Control of the position or alignment of the transfer device
• CPC Class Codes: G01N 35/1074 : arranged in a two-dimensional array

Abstract

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.

Claims

1. A method of detecting the activity of an enzyme that operates on an enzyme substrate to form an enzyme product in a sample, comprising:

contacting the substrate or product with a binding partner that specifically binds to the substrate or to the product but not to both, where the binding partner includes a metal that is involved in binding between the binding partner and the substrate or product;

contacting the substrate with the enzyme;

exposing the sample to light capable of inducing luminescence from the sample; and

measuring a detectable luminescence energy transfer response, 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 luminescence energy transfer 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.

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 at least one of the substrate and/or product, and the binding partner includes an energy transfer donor, and at least the other of the substrate and/or product, and the binding partner includes an energy transfer acceptor, such that luminescence energy transfer can occur between the donor and the acceptor.

9. The method of claim 6, where the binding partner includes an energy transfer donor or acceptor, and that energy transfer donor or acceptor includes the metal that is involved in binding between the binding partner and the substrate or product.

12. The method of claim 9, where the energy transfer donor comprises a lanthanide.

16. The method of claim 12, where the energy transfer donor further comprises an organic chelator and a sensitizer moiety.

20. The method of claim 12, where the lanthanide chelate has the formula

21. The method of claim 1, where the substrate is a polypeptide, and where the substrate and product are related by a posttranslational modification.

22. The method of claim 21, where the posttranslational modification is phosphorylation or dephosphorylation of the polypeptide and the enzyme is selected from the group consisting of kinases and phosphatases.

25. The method of claim 1, where the substrate is a nucleotide, the substrate and product are related by a cyclization or decyclization of the nucleotide, and the enzyme is selected from the group consisting of cyclases and phosphodiesterases.

26. The method of claim 1, where the substrate is a lipid, lipid precursor or lipid fragment, and the enzyme is selected from the group of lipases and lipid ligases.

27. The method of claim 1, where the substrate includes a phosphorylated polypeptide or a nonphosphorylated polypeptide.

28. The method of claim 1, where the substrate includes a cyckzed nucleotide or a noncyclized nucleotide.

29. 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.

30. The method of claim 1, further comprising:

providing a sample holder having a plurality of sample sites supporting a corresponding plurality of samples; and

repeating the steps of contacting, detecting, and correlating for the plurality of samples.

31. A kit for performing the method of claim 1, comprising:

a phosphorylated or nonphosphorylated polypeptide; and

a binding partner that specifically binds to one of the phosphorylated and nonphosphorylated polypeptide but not both, wherein the binding partner includes a metal required for specific binding of the binding partner to the one form of the polypeptide.

32. A kit for performing the method of claim 1, comprising:

a cyclized or noncyclized form of a nucleotide; and

a binding partner that specifically binds to one of the cyclized form and the noncyclized form of the nucleotide, but not both, wherein the binding partner includes a metal required for specific binding of the binding partner to the one form of the nucleotide.

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33. A method of detecting phosphorylation or nonphosphorylation of a polypeptide in a sample, comprising:

contacting the polypeptide with a binding partner that specifically binds to the phosphorylated polypeptide or to the nonphosphorylated polypeptide but not to both, where the binding partner includes a metal that is required for binding between the binding partner and the phosphorylated polypeptide or nonphosphorylated polypeptide;

exposing the sample to light capable of inducing luminescence from the sample; and

measuring a detectable luminescence energy transfer, where the luminescence energy transfer is indicative of the extent of binding between the polypeptide and the binding partner without separating the bound polypeptide from the unbound polypeptide; and

correlating the luminescence energy transfer with the extent of phosphorylation or nonphosphorylation of the polypeptide, or with the activity of an enzyme that affects phosphorylation or nonphosphorylation of the polypeptide.

34. A method of detecting cyclization or noncyclization of a nucleotide in a sample, comprising:

contacting a nonradioactive nucleotide with a binding partner that specifically binds to a cyclized nucleotide or to a noncyclized nucleotide but not to both, substantially without regard to the nucleoside portion of the nucleotide, where the binding partner includes a metal that is required for binding between the binding partner and the cyclized nucleotide or noncyclized nucleotide;

exposing the sample to capable of inducing luminescence from the sample; and

measuring a detectable luminescence energy transfer, where the luminescence energy transfer is indicative of the extent of binding between the nucleotide and the binding partner; and

correlating the response with the extent of cyclization or noncyclization of the nucleotide, or with the activity of an enzyme that affects cyclization or noncyclization of the nucleotide.