# Real‐time assay of ribonucleotide reductase activity with a fluorescent RNA aptamer

## TL;DR

This paper introduces a new real-time assay called FLARE to measure ribonucleotide reductase activity using a fluorescent RNA aptamer, enabling faster and more sensitive monitoring of enzyme function and inhibition.

## Contribution

The novel FLARE assay enables real-time, single-tube monitoring of RNR activity with improved sensitivity and reduced readout time.

## Key findings

- FLARE successfully detects RNR activity under dNTP-limiting conditions, mimicking allosteric regulation.
- The assay enables dose- and time-dependent inhibition detection of RNR by hydroxyurea.
- FLARE allows isothermal tracking of RNR activity at nanomolar enzyme levels.

## Abstract

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 statementRibonucleotide 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.

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.

Ribonucleotide reductases (RNR) synthesize DNA building blocks de novo, making them crucial in DNA replication and drug targeting. FLARE introduces the first single‐tube real‐time coupled RNR assay, which enables isothermal tracking of RNR activity at nanomolar enzyme levels and allows the reconstruction of allosteric regulatory patterns and rapid screening of inhibitors.