A QUENCHED SIZE-EXPANDED NUCLEOTIDE REPORTS ACTIVITY OF THE LEUKEMIA BIOMARKER 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 great selectivity over other polymerase enzymes. Importantly, TdT activity and its response to treatment with a promiscuous polymerase inhibitor could be monitored in human T-lymphocyte cell extract using a simple fluorescence assay. Finally, employing the probe in a high-throughput assay resulted in the identification of a nonnucleoside TdT inhibitor.