A Quenched Size-Expanded Nucleotide Reports Activity of the Leukemia Biomarker Terminal Deoxynucleotidyl Transferase (TdT)

Terminal deoxynucleotidyl Transferase (TdT) is a template-independent DNA polymerase that plays an essential role in the human adaptive immune system and is upregulated in several types of leukemia. It has therefore gained interest as a leukemia biomarker and potential therapeutic target. Herein, we describe a FRET-quenched fluorogenic probe based on a size-expanded deoxyadenosine that reports directly on TdT enzymatic activity. The probe enables real-time detection of primer extension and de novo synthesis activity of TdT and displays selectivity over other polymerase and phosphatase enzymes. Importantly, TdT activity and its response to treatment with a promiscuous polymerase inhibitor could be monitored in human T-lymphocyte cell extract and Jurkat cells using a simple fluorescence assay. Finally, employing the probe in a high-throughput assay resulted in the identification of a non-nucleoside TdT inhibitor.

Automated summary

Terminal deoxynucleotidyl Transferase (TdT) is a template-independent DNA polymerase that has gained interest as a leukemia biomarker and potential therapeutic target. A FRET-quenched fluorogenic probe based on a size-expanded deoxyadenosine was developed to directly report on TdT enzymatic activity. The probe showed selectivity over other enzymes and its activity could be monitored in human T-lymphocyte cell extract and Jurkat cells using a simple fluorescence assay. A non-nucleoside TdT inhibitor was identified using the probe in a high-throughput assay.