Enzymatic DNA Reaction Networks for Orchestrating Stimuli‐Dependent Temporal Molecular Pulse

ABSTRACT Temporal dynamics are a hallmark of cellular information processing, yet cell‐free biosensors still mainly rely on amplitude‐based fluorescence readouts that face multiplex limits from limited spectral channels, cross‐talk, and rising complexity. Here we present an enzymatic DNA reaction network (EDRN) that introduces time as an orthogonal coding dimension by translating stimuli into programmable temporal pulses. EDRN integrates a polymerase‐based concentration converter that normalizes inputs into a standardized universal strand (Us) at set doses with an exonuclease‐driven temporal decoder that converts Us dose into transient fluorescence pulses. This modular separation provides orthogonal enzymatic control over pulse amplitude and lifetime, enabling a wide programmable range without delicate structure‐dependent fine‐tuning. By tuning Us production through single‐ or double‐layer converters, pulse lifetimes can be programmed from ∼10 min to ∼5 h and temporal signatures assigned across targets. As a proof of concept, we demonstrate multiplex bacterial nucleic‐acid detection in one tube, where targets are resolved by time‐color encoding, achieving ten‐plex readout using four fluorophores with multiple temporal windows. Clinical validation on 32 specimens (22 positives and 10 healthy controls) showed consistency with sequencing. These results establish a general stimulus‐to‐time strategy for nucleic‐acid circuits and expand the multiplexing capacity of fluorescence‐based cell‐free biosensing.