Synthetic protein-binding DNA sponge as a tool to tune gene expression and mitigate protein toxicity

Xinyi Wan,Baojun Wang,Filipe Pinto,Luyang Yu

doi:10.1038/s41467-020-19552-9

Abstract

Versatile tools for gene expression regulation are vital for engineering gene networks of increasing scales and complexity with bespoke responses. Here, we investigate and repurpose a ubiquitous, indirect gene regulation mechanism from nature, which uses decoy protein-binding DNA sites, named DNA sponge, to modulate target gene expression in Escherichia coli. We show that synthetic DNA sponges can be designed to reshape the response profiles of gene circuits, lending multifaceted tuning capacities including reducing basal leakage by >20-fold, increasing system output amplitude by >130-fold and dynamic range by >70-fold, and mitigating host growth inhibition by >20%. Further, multi-layer DNA sponges for decoying multiple regulatory proteins provide an additive tuning effect on the responses of layered circuits compared to single-layer sponges. Our work shows synthetic DNA sponges offer a simple yet generalizable route to systematically engineer the performance of synthetic gene circuits, expanding the current toolkit for gene regulation with broad potential applications.

Highlights

Versatile tools for gene expression regulation are vital for engineering gene networks of increasing scales and complexity with bespoke responses
We demonstrate this using representative gene circuits with response to three different external input stimuli in E. coli, showing the incorporation of DNA sponges could significantly reduce the circuits’ basal leakages and increase their output induction folds, and strikingly improve host growth when sponging away sensitive transcription factors that tend to become burdensome at increased expression levels
We designed synthetic DNA sponges to sponge off the regulatory proteins within the input sensing module and/or the signal processing module to modulate the circuits’ output gene expression

Summary

Introduction

Versatile tools for gene expression regulation are vital for engineering gene networks of increasing scales and complexity with bespoke responses. Studies have suggested that the DNA sponges may have a role in buffering noise for target gene expression by reducing transcription factor fluctuations and may transform a typically graded dose-response into an ultrasensitive switch-like response[22,23,24] Owing to these regulatory functions, synthetic DNA sponges have been repurposed for therapeutic treatments in human cells in the past two decades[25,26,27], and more recently were trialed for obtaining a robust oscillatory gene circuit in Escherichia coli[28,29] and de-repressing silent biosynthetic gene clusters in Streptomycetes[30]. Beyond the immediate application to circuits in E. coli, the synthetic DNA sponge-mediated regulation could be applied to other prokaryotic and eukaryotic organisms

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