Kinase assays using polycations

• US 6,472,141 B2
• Filed: 05/11/2000
• Issued: 10/29/2002
• Est. Priority Date: 05/21/1999
• Status: Expired due to Term

• Current Assignee: Caliper Technologies Corp (Revvity, Inc.)
• Original Assignee: Caliper Technologies Corp (Revvity, Inc.)
• Inventors: Nikiforov, Theo T.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/569,193
• Publication Number:
  US 20020076697A1
• Time in Patent Office: 901 Days
• Field of Search: 435/4, 435/6, 435/7.1, 435/91.1, 435/91.2, 435/194, 435/183, 435/283.1, 435/285.1, 435/285.2, 435/287.2, 435/287.1, 436/164, 436/171, 436/172, 536/22.1, 536/23.1, 536/24-- 2, 204/155
• US Class Current: 435/4
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• 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 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

Methods, systems, kits for carrying out a wide variety of different assays that comprise providing a first reagent mixture which comprises a first reagent having a fluorescent label. A second reagent is introduced into the first reagent mixture to produce a second reagent mixture, where the second reagent reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent. A polyion is introduced into at least one of the first and second reagent mixtures, and the fluorescent polarization in the second reagent mixture relative to the first reagent mixture is determined, this fluorescent polarization being indicative of the rate or extent of the reaction.

Claims

1. A method of monitoring the activity of a kinase enzyme, comprising:

providing a first mixture comprising a phosphorylatable compound that includes a fluorescent label, a kinase enzyme and a phosphate donor group, wherein the kinase enzyme is capable of phosphorylating the phosphorylatable compound to produce a phosphorylated product;

contacting the phosphorylated product with a polycationic component; and

monitoring a level of phosphorylated product produced by the activity of the kinase enzyme by monitoring binding of the polycationic component to the phosphorylated product.

2. The method of claim 1, wherein the phosphorylatable compound comprises a phosphorylatable peptide.

3. The method of claim 1, wherein the monitoring step comprises monitoring a rate of rotation of the phosphorylated product, a decrease in the rate of rotation of the phosphorylated product relative to a rate of rotation of the phosphorylatable compound being indicative of binding of the polycationic component to the phosphorylated product.

4. The method of claim 1, wherein the monitoring step is carried out using fluorescence polarization detection, triplet state absorbance anisotropy, fluorescent correlation spectroscopy or detection of fluorescence recovery after photobleaching.

5. The method of claim 1, wherein binding of the polycationic component to the phosphorylated product is monitored by fluorescence polarization detection.

6. The method of claim 1, further comprising introducing at least a first test compound into the first mixture and monitoring whether there is a change in an amount of phosphorylated product produced in a presence of the test compound as compared to an amount of phosphorylated product produced in an absence of test compound.

7. The method of claim 6, comprising repeating the providing, introducing and monitoring steps with a plurality of different test compounds.

8. The method of claim 1, wherein the polycationic component is sufficiently large to induce a shift in an amount of polarized fluorescence emitted from the phosphorylated product when the polycationic component binds to the phosphorylated product.

9. The method of claim 1, wherein the polycationic component is between 5 kD and 1000 kD.

10. The method of claim 8, wherein the polycationic component comprises multivalent metal ions.

11. The method of claim 8, wherein the multivalent metal ions are selected from the group consisting of Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺.

12. The method of claim 8, wherein the polycationic component comprises a polymeric material having multivalent metal cations associated therewith.

13. The method of claim 12, wherein the multivalent metal cations are selected from the group consisting of Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺.

14. The method of claim 12, wherein the multivalent metal cations are chelated to the polymeric material.

15. The method of claim 1, wherein the polycationic component is contained in a homogeneous mixture.

16. The method of claim 1, wherein the polycationic component comprises a polyamino acid.

17. The method of claim 1, wherein the polycationic component is selected from the group consisting of polylysine, polyarginine, polyhistidine, a copolymer of polylysine, a copolymer of polyarginine and a copolymer of polyhistidine.

18. A method of monitoring a kinase reaction, comprising:

contacting a fluorescently labeled kinase substrate with a kinase enzyme and a phosphate donor in a first mixture, the kinase enzyme reacting with the kinase substrate and the phosphate donor to produce a fluorescently labeled phosphorylated product;

introducing a binding component into the first mixture to produce a second mixture, the binding component comprising multivalent metal ions associated therewith, and being sufficiently large to induce a shift in a level of polarized fluorescence emitted from the second mixture when the binding component binds the fluorescently labeled phosphorylated product; and

monitoring a level of polarized fluorescence emitted from the second mixture, the level of polarized fluorescence emitted from the second mixture being indicative of an amount of fluorescently labeled phosphorylated product produced by the kinase reaction.

19. The method of claim 18, wherein the binding component comprises a polymeric material.

20. The method of claim 19, wherein the polymeric material comprises protein.

21. The method of claim 18, wherein the multivalent metal ions are chelated to the binding component.

22. The method of claim 18, wherein the multivalent metal ions are selected from the group consisting of Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺.

23. The method of claim 18, further comprising comparing a level of polarized fluorescence emitted from the second mixture produced the first mixture in the presence of a first test compound to a level of polarized fluorescence emitted from the second mixture produced from the first mixture in the absence of the first test compound.

24. The method of claim 18, wherein the binding component is between 5 kD and 1000 kD.

25. The method of claim 18, wherein the binding component comprises a polyamino acid.

26. The method of claim 18, wherein the binding component is selected from the group consisting of polylysine, polyarginine, polyhistidine, a copolymer of polylysine, a copolymer of polyarginine and a copolymer of polyhistidine.

27. A method of screening for effectors of a kinase mediated reaction, comprising:

providing a first mixture comprising a phosphorylatable compound that includes a fluorescent label, a kinase enzyme and a phosphate donor group;

adding at least a first test compound to the first mixture;

introducing a polycationic component to the first mixture; and

determining whether the test compound is an effector of the kinase enzyme mediated reaction by comparing binding of the polycationic component to a product of the kinase mediated reaction carried out in the presence of the test compound to a product of the kinase mediated reaction carried out in the absence of the test compound.

28. The method of claim 27, wherein the polycationic component is sufficiently large to induce a shift in an amount of polarized fluorescence emitted from the phosphorylated product when the polycationic component binds to the phosphorylated product.

29. The method of claim 27, wherein the polycationic component is between 5 kD and 1000 kD.

30. The method of claim 27, wherein the polycationic component comprises multivalent metal cations.

31. The method of claim 30, wherein the multivalent metal ions are selected from the group consisting of Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺.

32. The method of claim 27, wherein the polycationic component comprises a polymeric material.

33. The method of claim 32, wherein the polyeationic component comprises a polyamino acid.

34. The method of claim 32, wherein the polycationic component is selected from the group consisting of polylysine, polyarginine, polyhistidine, a copolymer of polylysine, a copolymer of polyarginine and a copolymer of polyhistidine.

35. The method of claim 27, wherein the first mixture is provided in a well of a multiwell plate.

36. The method of claim 27, wherein the first mixture is provided in a plurality of different wells in a multiwell plate, and a different test compound is separately added to the first mixture in a plurality of different wells, and the determining step comprises comparing binding of the polycationic component to the product of the kinase enzyme and the phosphorylatable compound in the first mixture in a plurality of wells with binding of the polycationic component to the product of the kinase enzyme and the phosphorylatable compound in the first mixture in another well of the multiwell plate that does not include a test compound.