Polymerization-sensitive switch-on monomer for terminal transferase activity assay.

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Terminal transferase (TdT) is a key enzyme in DNA synthesis.
• Accurate TdT activity detection is crucial for various biological and diagnostic applications.
• Existing methods for TdT activity determination can be complex or lack sensitivity.

Purpose of the Study:

• To develop a simple, efficient, and sensitive method for determining TdT activity.
• To enable the synthesis of fluorescent oligonucleotides using TdT activity.
• To demonstrate the practical applicability of the developed method in biological samples.

Main Methods:

• Utilizing Fe(III)-quenched boron-dipyrromethene (BODIPY)-ATP as a switch-on monomer for polymerization.
• Leveraging the displacement of Fe(III) upon TdT-mediated monomer addition, releasing pyrophosphate and activating fluorescence.
• Synthesizing highly fluorescent long oligonucleotides in a single step.

Main Results:

• Achieved selective and highly sensitive detection of TdT activity.
• Demonstrated the facile synthesis of fluorescent oligonucleotides without post-processing.
• Successfully applied the method to determine TdT activities in human serum samples.

Conclusions:

• The developed method provides a simple and efficient way to measure TdT activity.
• This technique facilitates the creation of fluorescent DNA probes.
• The method shows promise for diagnostic applications, particularly in analyzing TdT levels in biological fluids.