Bistability of an In Vitro Synthetic Autoregulatory Switch

TL;DR

This paper reports the design and analysis of an in vitro synthetic autoregulatory DNA switch with bistability, demonstrating how specific enzyme interactions enable stable state control and potential applications in biochemical networks.

Contribution

It introduces a modular DNA-based autoregulatory switch with bistability, combining experimental validation and mathematical modeling to understand its dynamics and control mechanisms.

Findings

RNase H alone achieves switch bistability

RNase R maintains stable RNA levels and prevents degradation artifacts

Mathematical model accurately predicts switch behavior

Abstract

The construction of synthetic biochemical circuits is an essential step for developing quantitative understanding of information processing in natural organisms. Here, we report construction and analysis of an in vitro circuit with positive autoregulation that consists of just four synthetic DNA strands and three enzymes, bacteriophage T7 RNA polymerase, Escherichia coli ribonuclease (RNase) H, and RNase R. The modularity of the DNA switch template allowed a rational design of a synthetic DNA switch regulated by its RNA output acting as a transcription activator. We verified that the thermodynamic and kinetic constraints dictated by the sequence design criteria were enough to experimentally achieve the intended dynamics: a transcription activator configured to regulate its own production. Although only RNase H is necessary to achieve bistability of switch states, RNase R is necessary to maintain stable RNA signal levels and to control incomplete degradation products. A simple mathematical model was used to fit ensemble parameters for the training set of experimental results and was then directly applied to predict time-courses of switch dynamics and sensitivity to parameter variations with reasonable agreement. The positive autoregulation switches can be used to provide constant input signals and store outputs of biochemical networks and are potentially useful for chemical control applications.