Template-independent enzymatic functionalization of DNA oligonucleotides with environment-sensitive nucleotide probes...

Given the emerging use of terminal deoxynucleotidyl transferase (TdT) in biotechnology and its clinical potential as a cancer marker and target, the development of a versatile probe system to study its processivity, substrate properties, and inhibition is highly desired. Here, we demonstrate a multilayered application of a series of environment-sensitive fluorescent 2'-deoxynucleotide probes that harness the activity of TdT in accessing site-specifically functionalized DNA oligonucleotides and devising a real-time fluorescence platform to monitor the enzyme activity and identify potential inhibitors. The nucleotides constructed by coupling heterocycles of progressively increasing chemical modifications (selenophene, benzothiophene, benzofuran, and fluorobenzofuran) at the C5 position of 2'-deoxyuridine serve as suitable substrates for TdT, albeit differences in incorporation efficiency. A battery of experiments provided valuable insights into the scope of this functionalization method. It revealed how a fine balance between steric hindrance and stacking interaction between the heterocycle moiety and primer 3'-end nucleobase in the active site modulates the recognition and processing of nucleotides based on their size. Remarkably, the excellent responsiveness of benzofuran-modified dUTP enabled the design of fluorescence assays to estimate TdT activity, and detect nucleotide and non-nucleotide inhibitors. The findings obtained using our probes should significantly advance TdT-based functionalization, diagnostic, and therapeutic strategies.