A genetic circuit on a single DNA molecule as autonomous dissipative nanodevice

Abstract

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcriptiontranslation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we used a genetically encoded fluorescent reporter system with improved spatiotemporal resolution and observed the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally designed a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibited enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that coexpressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.