Compositions comprising a dehalogenase substrate and a fluorescent label and methods of use

A method to detect or determine the presence or amount of a mutant dehalogenase, comprising:

• contacting a mutant dehalogenase with a dehalogenase substrate which comprises a fluorophore, wherein the mutant dehalogenase comprises at least one amino acid substitution relative to a corresponding wild-type dehalogenase, wherein 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 at least one amino acid substitution in the mutant dehalogenase is a substitution 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 at an amino acid residue in the corresponding wild-type dehalogenase that forms an ester intermediate with the substrate, wherein the substrate is a compound of formula (I):
• R-linker-A-X, wherein R comprises the fluorophore, the linker is branched or unbranched carbon chain comprising no more than 30 carbon atoms and —C(O)NH(CH2CH2O)y, wherein y=2-8, A is (CH2)n and n=2-10, A-X is a substrate for the dehalogenase, and X is a halogen;
• and
• detecting or determining the presence or amount of the fluorophore, thereby detecting or determining the presence or amount of the mutant dehalogenase.

A method for detecting the presence or amount of a molecule in a cell, comprising:

• a) contacting a cell comprising a mutant hydrolase with a hydrolase substrate which comprises one or more functional groups, wherein the mutant hydrolase comprises at least one amino acid substitution relative to a corresponding wild-type hydrolase, wherein at least one amino acid substitution results in the mutant hydrolase forming a bond with the substrate which is more stable than the bond formed between the corresponding wild-type hydrolase and the substrate, wherein at least one amino acid substitution in the mutant hydrolase is a substitution at an amino acid residue corresponding to residue 272 in a Rhodococcus dehalogenase encoded by SEQ ID NO:51 that is associated with activating a water molecule which cleaves the bond formed between the corresponding wild-type hydrolase and the substrate or at an amino acid residue corresponding to residue 106 in a Rhodococcus dehalogenase encoded by SEQ ID NO:51 that forms an ester intermediate with the substrate, wherein the cell is further contacted with a fixative or a permabilization reagent, wherein the substrate is a compound of formula (I):
• R-linker-A-X, wherein R comprises the fluorophore, wherein the linker is branched or unbranched carbon chain comprising no more than 30 carbon atoms and comprises —C(O)NH(CH2CH2O)y, wherein y=2-8, A is (CH2)n and n=2-10, A-X is a substrate for the dehalogenase, and X is a halogen;
• and
• b) detecting or determining the presence or amount of the functional group in the cell.