Compositions comprising a dehalogenase substrate and a contrast agent and methods of use
View Patent ↗A mutant hydrolase optionally fused to a protein of interest is provided. The mutant hydrolase is capable of forming a bond with a substrate for the corresponding nonmutant (wild-type) hydrolase which is more stable than the bond formed between the wild-type hydrolase and the substrate. Substrates for hydrolases comprising one or more functional groups are also provided, as well as methods of using the mutant hydrolase and the substrates of the invention. Also provided is a fusion protein capable of forming a stable bond with a substrate and cells which express the fusion protein.
1. A compound of formula (I): R-linker-A-X, wherein R comprises a metal ion chelator, wherein the linker is a multiatom straight or branched chain including C, N, S and/or O and comprising no more than 30 carbon atoms, wherein A-X is a substrate for a dehalogenase, wherein A is (CH 2 ) n and n=2-10, and wherein X is a halogen.
2. The compound of claim 1 , which is a substrate for a Rhodococcus dehalogenase.
3. The compound of claim 1 , wherein X is Cl or Br.
4. The compound of claim 1 , wherein the linker separates R and A by at least 11 atoms.
5. The compound of claim 1 , wherein the metal ion chelator is separated from A-X by up to 500 angstroms.
6. The compound of claim 1 , wherein the linker comprises—C(O)NH(CH 2 CH 2 O) y , wherein y=2-8.
7. A complex of a gadolinium metal ion and a compound of formula (I): R-linker-A-X, wherein R comprises a metal ion chelator, wherein the linker is a multiatom straight or branched chain including C, N, S and/or O and comprising no more than 30 carbon atoms, wherein A-X is a substrate for a dehalogenase, wherein A is (CH 2 ) n and n=2-10, and wherein X is a halogen.
8. The compound of claim 7 , which is a substrate for a Rhodococcus dehalogenase.
9. The compound of claim 7 , wherein X is Cl or Br.
10. A complex of a manganese metal ion and a compound of formula (I): R-linker-A-X, wherein R comprises a metal ion chelator, wherein the linker is a multiatom straight or branched chain including C, N, S and/or O and comprising no more than 30 carbon atoms, wherein A-X is a substrate for a dehalogenase, wherein A is (CH 2 ) n and n=2-10, and wherein X is a halogen.
11. The compound of claim 10 , which is a substrate for a Rhodococcus dehalogenase.
12. The compound of claim 10 , wherein X is Cl or Br.
13. A complex of the compound of claim 1 and a metal ion.
14. The complex of claim 13 , wherein the metal ion is a contrast agent.
15. The complex of claim 14 , wherein the contrast agent is an MRI contrast agent.
16. The complex of claim 14 , wherein the metal ion is gadolinium.
17. The complex of claim 14 , wherein the metal ion is manganese.
18. The complex of claim 14 , wherein the metal ion is iron oxide.
19. A method to label a cell, comprising:
(a) contacting a cell comprising a mutant dehalogenase with the complex of claim 14 , wherein the mutant dehalogenase comprises at least one amino acid substitution relative to the corresponding wild-type dehalogenase, wherein the at least one amino acid substitution results in the mutant dehalogenase forming a bond with the substrate which is more stable than the bond formed between the corresponding wild-type dehalogenase and the substrate, wherein the at least one amino acid substitution in the mutant dehalogenase is a substitution (i) at an amino acid residue in the corresponding wild-type dehalogenase that is associated with activating a water molecule which cleaves the bond formed between the corresponding wild-type dehalogenase and the substrate or (ii) at an amino acid residue in the corresponding wild-type dehalogenase that forms an ester intermediate with the substrate; and
(b) incubating the cell with the complex, wherein the incubation results in the cell being labeled with the contrast agent.
20. The method of claim 19 , wherein the substrate is a substrate for a Rhodococcus dehalogenase.
21. The method of claim 19 , wherein Xis Cl or Br.
22. The method of claim 19 , wherein the contrast agent is an MRI contrast agent.
23. The method of claim 22 , wherein the MRI contrast agent comprises gadolinium.
24. The method of claim 22 , wherein the MRI contrast agent comprises manganese.
25. The method of claim 22 , wherein the contrast agent comprises iron oxide.
26. The method of claim 19 , wherein the linker comprises—C(O)NH(CH 2 CH 2 O) y , wherein y=2-8.
27. A method to detect or determine the presence or amount of a mutant dehalogenase, comprising:
a) contacting a mutant dehalogenase with the complex of claim 14 , wherein the mutant dehalogenase comprises at least one amino acid substitution relative to the corresponding wild-type dehalogenase, wherein the at least one amino acid substitution results in the mutant dehalogenase forming a bond with a dehalogenase substrate which is more stable than the bond formed between the corresponding wild-type dehalogenase and the dehalogenase substrate, wherein the at least one amino acid substitution in the mutant dehalogenase is a substitution (i) at an amino acid residue in the corresponding wild-type dehalogenase that is associated with activating a water molecule which cleaves the bond formed between the corresponding wild-type dehalogenase and the substrate or (ii) at an amino acid residue in the corresponding wild-type dehalogenase that forms an ester intermediate with the substrate; and
b) detecting or determining the presence or amount of the contrast agent, thereby detecting or determining the presence or amount of the mutant dehalogenase.
28. The method of claim 27 , which is the substrate is a substrate for a Rhodococcus dehalogenase.
29. The method of claim 27 , wherein X is Cl or Br.
30. The method of claim 27 , wherein the contrast agent is an MRI contrast agent.
31. The method of claim 30 , wherein the MRI contrast agent comprises gadolinium.
32. The method of claim 30 , wherein the MRI contrast agent comprises manganese.
33. The method of claim 30 , wherein the contrast agent comprises iron oxide.
34. The method of claim 27 , wherein the linker comprises—C(O)NH(CH 2 CH 2 O) y , wherein y=2-8.