Molecular modification assays

• US 20040249586A1
• Filed: 12/23/2003
• Published: 12/09/2004
• Est. Priority Date: 09/15/1997
• Status: Active Grant

• Current Assignee: Molecular Devices
• Original Assignee: Molecular Devices
• Inventors: Huang, Wei, Boge, Annegret, Sportsman, J., Hoekstra, Merl, Lavis, Luke
• Granted Patent: US 7,745,142 B2
• Time in Patent Office: Days
• US Class Current: 702/57
• 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 1/42 involving phosphatase
• 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 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

Assays for detecting molecular modifications such as phosphate modifications and the presence and/or activity of enzymes and other agents involved in facilitating or otherwise regulating such modifications.

Claims

1. A method of detecting a cyclized or noncyclized form of a nucleotide in a sample, comprising:

contacting the nucleotide with a binding partner that specifically binds to one of a cyclized form and a noncyclized form of the nucleotide, but not both, the binding partner including a metal required for specific binding of the binding partner to the one form of the nucleotide; and

detecting a response indicative of the extent of binding between the one form of the nucleotide and the binding partner.

2. The method of claim 1, further comprising a step of contacting the nucleotide with at least one enzyme selected from cyclases and phosphodiesterases.

3. The method of claim 2, wherein the step of contacting the nucleotide with at least one enzyme is performed before the step of contacting the nucleotide with a binding partner.

4. The method of claim 2, further comprising (1) a step of contacting the at least one enzyme with a candidate compound;

and (2) a step of determining the ability of the candidate compound to enhance or inhibit enzyme activity by its effect on the response.

5. The method of claim 2, further comprising a step of correlating the response with a cyclization or decyclization activity of the at least enzyme on the nucleotide.

6. The method of claim 1, wherein the step of contacting includes (1) a step of placing a metal salt in a liquid, the metal salt including the metal, and (2) a step of contacting the nucleotide with the metal salt after the step of placing.

7. The method of claim 1, wherein the step of contacting includes a step of contacting the nucleotide with one or more metal ions selected from the group consisting of aluminum, gallium, and iron ions, and wherein the one or more metal ions are required for specific binding of the binding partner to the one form of the nucleotide.

8. The method of claim 1, wherein the step of contacting includes a step of contacting the nucleotide with one or more metal ions selected from the group consisting of europium, strontium, terbium, and zirconium ions, and wherein the one or more metal ions are required for specific binding of the binding partner to the one form of the nucleotide.

9. The method of claim 1, wherein the step of contacting includes a step of contacting with gallium, the gallium being required for specific binding of the binding partner to the one form of the nucleotide.

10. The method of claim 1, wherein the steps of contacting and detecting are performed a plurality of times for a plurality of samples disposed in different wells of a microplate.

11. The method of claim 1, further comprising a step of associating the metal with a distinct solid support that does not include the metal.

12. The method of claim 11 wherein the step of associating includes associating the metal with beads that do not include the metal.

13. The method of claim 1, wherein the step of contacting is performed with the metal not associated substantially with any distinct solid support that does not include the metal.

14. The method of claim 1, wherein the step of detecting is performed after the step of contacting without separation of bound and unbound species of the nucleotide.

15. The method of claim 1, wherein the step of detecting includes (1) a step of exposing the nucleotide to a condition capable of producing luminescence, and (2) a step of detecting a luminescence response produced by the step of exposing.

16. The method of claim 15, wherein the nucleotide is luminescent.

17. The method of claim 15, wherein the step of detecting a luminescence response includes a step of detecting at least one of luminescence polarization, luminescence intensity, luminescence lifetime, and luminescence resonance energy transfer.

18. 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.

19. The kit of claim 18, further comprising an enzyme capable of converting the cyclized form of the nucleotide into the noncyclized form, or vice versa.

20. The kit of claim 18, wherein the kit includes a luminescently labeled, cyclized form of the nucleotide and a binding partner that specifically binds to the noncyclized form of the nucleotide.

21. The kit of claim 20, further comprising a phosphodiesterase enzyme.

22. The kit of claim 18, wherein the kit includes a luminescently labeled, noncyclized form of the nucleotide and a binding partner that specifically binds to the noncyclized form of the nucleotide.

23. The kit of claim 22, further comprising a cyclase enzyme.

24. A method of detecting a cyclized or noncyclized form of a nucleotide in a sample, comprising:

contacting the nucleotide with a binding partner that specifically binds to one of a cyclized form and a noncyclized form of the nucleotide, but not both, substantially without regard to the base portion the nucleotide; and

detecting a response indicative of the extent of binding between the one form of the nucleotide and the binding partner.

25. The method of claim 24, further comprising a step of contacting the nucleotide with at least one enzyme selected from cyclases and phosphodiesterases.

26. The method of claim 25, further comprising a step of correlating the response with a cyclization or decylization activity of the at least enzyme on the nucleotide.

27. The method of claim 24, wherein the steps of contacting and detecting are performed a plurality of times for a plurality of samples disposed in different wells of a microplate.

28. The method of claim 24, wherein the step of detecting is performed after the step of contacting without separation of bound and unbound species of the nucleotide.

29. The method of claim 24, wherein the step of detecting includes (1) a step of exposing the nucleotide to light;

and (2) a step of detecting a luminescence response produced by the step of exposing.

30. The method of claim 29, wherein the step of detecting a luminescence response includes a step of detecting at least one of luminescence polarization, luminescence intensity, luminescence lifetime, and luminescence resonance energy transfer.

31. The method of claim 30, wherein the nucleotide is luminescent.

32. A kit for performing the method of claim 24, 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, substantially without regard to the base portion the nucleotide.

33. A method of detecting a cyclized or noncyclized form of a nucleotide in a sample, comprising:

contacting a nucleotide with a binding partner that specifically binds to one of a cyclized form and a noncyclized form of the nucleotide, but not both;

exposing the nucleotide to light without separation of bound and unbound species of the nucleotide produced by the step of contacting; and

detecting a luminescence response produced by the step of exposing, the luminescence response being indicative of the extent of binding between the one form of the nucleotide and the binding partner.

34. The method of claim 33, wherein the nucleotide is a luminescent nucleotide, and wherein the step of contacting includes a step of contacting the luminescent nucleotide with the binding partner.

35. The method of claim 33, wherein the step of contacting includes a step of contacting a luminescent reporter and a nonluminescent nucleotide with the binding partner, and wherein detecting a luminescence response detects displacement of at least a portion of the luminescent reporter from the binding partner by the nonluminescent nucleotide through competition.

36. The method of claim 33, wherein the nucleotide is nonradioactive.

37. A method of detecting the activity of a cyclase or a phosphodiesterase that operates on a nucleotide substrate to form a nucleotide product in a sample, comprising:

contacting the nucleotide substrate with a binding partner that specifically binds to the nucleotide substrate or to the nucleotide product but not to both, where the binding partner includes a metal required for binding between the binding partner and the nucleotide substrate or product;

contacting the nucleotide substrate with at least one of a cyclase and a phosphodiesterase;

detecting a response indicative of the extent of binding between the nucleotide substrate or product and the binding partner without separating bound and unbound fractions of such nucleotide substrate or product; and

correlating the response with the activity of the enzyme.

38. A method of detecting, in a sample, a first analyte or a second analyte related to the first analyte by a molecular modification, comprising:

contacting the sample with a metal that forms a covalent coordination complex with one of the first and second analytes, but not both; and

detecting a response indicative of the extent of binding between metal and the one analyte without separating bound and unbound species of the one analyte produced by the step of contacting.

39. A method of detecting, in a sample, a first analyte or a second analyte related to the first analyte by a molecular modification, comprising:

contacting the sample with gallium, the gallium specifically binding to one of the first and second analytes, but not both; and

detecting a response indicative of the extent of binding between the gallium and the one analyte.