Real‐time assay of ribonucleotide reductase activity with a fluorescent <scp>RNA</scp> aptamer

Ribonucleotide reductases (RNRs) convert all four ribonucleotides to deoxyribonucleotides, providing essential building blocks for DNA biosynthesis and repair through radical-based catalysis. These functions are key to cellular proliferation and have made RNRs well established targets for antimicrobial and antiviral drugs and combination chemotherapies. Here, we describe a novel highly sensitive one-pot enzymatic assay, which amplifies RNR activity by coupling it to the synthesis of a fluorogenic RNA aptamer. We validated this approach by testing RNR activity under dNTP-limiting conditions to emulate RNR's complex allosteric regulatory patterns and by detecting the dose- and time-dependent inhibition of RNR by hydroxyurea. This unique assay builds on previous high-throughput screening assays for investigation of RNR's catalytic mechanisms by improving sensitivity and reducing readout timeframes. Impact statement Ribonucleotide reductases (RNRs) are essential for controlling cellular dNTP supply and are major targets in cancer, antiviral, and antimicrobial therapy. FLARE is a novel single-tube, real-time RNR assay, coupling dNTP synthesis to the transcription of a fluorogenic aptamer for continuous monitoring of activity, regulation, and inhibition using standard microplate readers.