Fluorimetric assay for terminal deoxynucleotidyl transferase activity

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Terminal deoxynucleotidyl transferase (TdT) is a key enzyme in DNA synthesis.
• Accurate measurement of TdT activity is crucial for diagnosing and monitoring certain leukemias.
• Existing methods for TdT detection have limitations.

Purpose of the Study:

• To develop a novel fluorimetric assay for quantifying terminal deoxynucleotidyl transferase (TdT) activity.
• To evaluate the utility of etheno-substituted deoxynucleotides as substrates for TdT.
• To assess the suitability of the assay for clinical applications, particularly in leukemia diagnostics.

Main Methods:

• Development of a fluorimetric assay using 1,N6-ethenodeoxyadenosine triphosphate (ε dATP) as a fluorescent substrate.
• Polymerization of ε dATP onto an oligodeoxynucleotide initiator under standard TdT reaction conditions.
• Quantification of incorporated ε dATP by fluorescence after isolation and nuclease digestion.
• Determination of enzymological properties of etheno substrates, including binding affinity and catalytic rates.

Main Results:

• The fluorimetric assay successfully measures TdT activity in both purified and crude enzyme preparations.
• Etheno-substituted deoxynucleotides function as substrates for TdT.
• ε dATP exhibits tighter binding to TdT than dATP but a lower catalytic rate, without affecting initiator binding.
• The assay demonstrates suitability for clinical analysis of TdT in human leukemias.

Conclusions:

• A novel, sensitive fluorimetric assay for TdT activity has been established using fluorescent nucleotide analogs.
• This assay provides a valuable tool for the clinical assessment of TdT, especially in the context of human leukemias.
• The fluorimetric method can serve as a complementary approach to immunochemical techniques for a comprehensive evaluation of TdT.