Molecular modification assays
View Patent ↗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.
1. A method of detecting nucleotide cyclization or decyclization in a sample, comprising:
contacting a nucleotide with a binding partner that binds specifically to a noncyclized form but not to a cyclized form of the nucleotide, the binding partner including at least one metal ion required for specific binding of the binding partner to the noncyclized form of the nucleotide;
contacting the nucleotide with at least one enzyme selected from the group consisting of cyclases and phosphodiesterases;
detecting a response indicative of the extent of binding between the noncyclized form of the nucleotide and the binding partner; and
correlating the response with a cyclization or decyclization activity of the at least enzyme on the nucleotide.
2. The method of claim 1 , 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.
3. The method of claim 1 , 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.
4. The method of claim 1 , wherein the step of contacting a nucleotide with a binding partner includes (1) a step of placing a metal salt in a liquid, the metal salt including the metal ion, and (2) a step of contacting the nucleotide with the metal salt after the step of placing.
5. The method of claim 1 , wherein the step of contacting a nucleotide with a binding partner 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 metals ions are required for specific binding of the binding partner to the noncyclized form of the nucleotide.
6. The method of claim 1 , wherein the step of contacting a nucleotide with a binding partner 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 selective binding of the binding partner to the noncyclized form of the nucleotide.
7. The method of claim 1 , wherein the step of contacting a nucleotide with a binding partner includes a step of contacting the nucleotide with gallium, the gallium being required for specific binding of the binding partner to the noncyclized form of the nucleotide.
8. The method of claim 1 , wherein the steps of contacting a nucleotide with a binding partner and detecting a response are performed a plurality of times for a plurality of samples disposed in different wells of a microplate.
9. The method of claim 1 , further comprising a step of associating the metal ion with a distinct solid support that does not include the metal ion.
10. The method of claim 9 , wherein the step of associating includes associating the metal ion with beads that do not include the metal ion.
11. The method of claim 1 , wherein the step of detecting is performed after the steps of contacting without separation of bound and unbound species of the nucleotide.
12. 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, wherein the luminescence response is indicative of the extent of binding between the noncyclized form of the nucleotide and the binding partner.
13. The method of claim 12 , wherein the nucleotide is luminescent.
14. The method of claim 12 , wherein the step of detecting a luminescence response includes a step of detecting luminescence polarization, luminescence intensity, luminescence lifetime, luminescence resonance energy transfer, or a combination thereof.
15. The method of claim 1 , wherein the step of contacting a nucleotide with a binding partner includes a step of contacting the nucleotide with a binding partner including a tricationic metal ion required for specific binding of the binding partner to the noncyclized form of nucleotide.
16. The method of claim 15 , wherein the step of contacting a nucleotide with a binding partner includes a step of contacting the nucleotide with a binding partner including a tricationic metal ion selected from the group consisting of Al 3+ , Fe 3+ , and Ga 3+ .
17. The method of claim 16 , wherein the step of contacting a nucleotide with a binding partner includes a step of contacting the nucleotide with a binding partner including Ga 3+ .
18. The method of claim 1 , wherein the step of contacting a nucleotide with a binding partner includes a step of contacting the nucleotide with a binding partner that includes a dicationic metal ion required for specific binding of the binding partner to the noncyclized form of nucleotide.
19. The method of claim 1 , wherein the step of detecting a response includes a step of exposing the nucleotide to a condition capable of inducing luminescence from the nucleotide, and wherein the condition is light capable of inducing photoluminescence or is electrochemical energy capable of inducing electrochemiluminescence.
20. The method of claim 1 , wherein the step of contacting a nucleotide with a binding partner includes a step of contacting the nucleotide with a binding partner that includes a macromolecule, a nanoparticle, or both.
21. The method of claim 1 , wherein the step of contacting a nucleotide with a binding partner includes a step of contacting the nucleotide with a binding partner that includes a quencher, an energy transfer partner, or both.
22. The method of claim 1 , wherein the specific binding to the noncyclized form is characterized by a dissociation constant of no larger than about 10 −8 M.
23. The method of claim 1 , further comprising (1) a step of providing a sample holder having a plurality of sample sites supporting a corresponding plurality of samples, and (2) a step of repeating the steps of contacting and detecting for the plurality of samples.
24. The method of claim 1 , where the nucleotide includes an adenine or a guanine moiety.
25. The method of claim 1 , further comprising a step of washing the sample prior to the step of detecting to remove any nucleotide not bound to the binding partner.