Methods and kits for quantifying adenosine-containing molecules

A method for quantifying adenosine-containing compounds within an aqueous composition comprising the steps of:

• a. providing a fluorescent probe comprising:
• i. an adenosine scaffold comprising an adenine moiety and a ribosyl moiety, and
• ii. a TAMRA fluorophore,
• wherein the adenosine scaffold is covalently bound to the fluorophore by a linking portion comprising a linear chain of six or more atoms extending from the 5′-carbon of the ribosyl moiety to the 5-carboxyl functional group within TAMRA;
• b. providing a mutated MTAN enzyme, wherein the mutated MTAN enzyme comprises:
• i. an amino acid sequence selected from the group consisting of SEQ ID NO:
• 4, SEQ ID NO:
• 6, and SEQ ID NO:
• 8, and
• ii. a cysteine residue that is conjugated to a secondary fluorophore comprising a fluorescently-active moiety and a thiol-reactive crosslinker that bridges the fluorescently-active moiety to the cysteine residue;
• c. forming a protein-probe complex between the mutated MTAN enzyme and the fluorescent probe;
• d. measuring the fluorescence intensity of the fluorescent probe within the protein-probe complex;
• e. combining an adenosine-containing compound with the protein-probe complex to form an adenosine quantification mixture;
• f. measuring the increase of fluorescence intensity of the fluorescent probe in the presence of the adenosine quantification mixture;
• and
• g. calculating the amount of the adenosine-containing compound within the adenosine quantification mixture using the increase of fluorescence intensity of the fluorescent probe in the presence of the adenosine quantification mixture.

An isolated nucleic acid molecule comprising a nucleotide sequence that encodes for a mutated MTAN enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

• 1, SEQ ID NO:
• 4, SEQ ID NO:
• 6, and SEQ ID NO:
• 8.

A kit for quantifying adenosine-containing compounds within an aqueous composition or reaction mixture, the kit comprising:

• a. a fluorescent probe comprising:
• i. an adenosine scaffold comprising an adenine moiety and a ribosyl moiety, and
• ii. a TAMRA fluorophore,
• wherein the adenosine scaffold is covalently bound to the fluorophore by a linking portion comprising a linear chain of six or more atoms extending from the 5′-carbon of the ribosyl moiety to the 5-carboxyl functional group within TAMRA;
• and
• b. a mutated MTAN enzyme, wherein the mutated MTAN enzyme comprises a mutation within the active site from an aspartic acid residue to an asparagine residue.