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Translational control of enzyme scavenger expression with toxin-induced micro RNA switches
Journal article   Open access   Peer reviewed

Translational control of enzyme scavenger expression with toxin-induced micro RNA switches

Nina Pollak, Justin Cooper-White and Joanne Macdonald
Scientific Reports, Vol.11, pp.1-12
2021
PMCID: PMC7844233
PMID: 33510250
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Translational control of enzyme scavenger expression with toxin-induced micro RNA switches2.05 MBDownloadView
Published Version Open Access CC BY V4.0
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https://doi.org/10.1038/s41598-021-81679-6View
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Abstract

Biological computation requires in vivo control of molecular behavior to progress development of autonomous devices. miRNA switches represent excellent, easily engineerable synthetic biology tools to achieve user‑defined gene regulation. Here we present the construction of a synthetic network to implement detoxification functionality. We employed a modular design strategy by engineering toxin‑induced control of an enzyme scavenger. Our miRNA switch results show moderate synthetic expression control over a biologically active detoxification enzyme molecule, using an established design protocol. However, following a new design approach, we demonstrated an evolutionarily designed miRNA switch to more effectively activate enzyme activity than synthetically designed versions, allowing markedly improved extrinsic user‑defined control with a toxin as inducer. Our straightforward new design approach is simple to implement and uses easily accessible web‑based databases and prediction tools. The ability to exert control of toxicity demonstrates potential for modular detoxification systems that provide a pathway to new therapeutic and biocomputing applications. Biocompatible information processing is critical for developing autonomous biological therapeutics or biosen-sors. Currently, biological computation can reprogram nucleic acids to store and process information 1–8 , modulate protein translation at transcriptional levels 9–13 , and perform biosensing functions 14–17. However, designing molecular systems to regulate behaviour also requires programming high degrees of functionality into biochemical networks. Such advancement of synthetic gene expression control is critical to progress synthetic biology tools 18. However, while the synthetic biology toolbox has made rapid progress using prokaryotes, the expansion of eukaryotic tools is still lacking. To date, only one type of riboswitch, thiamine pyrophosphate, has been identified for eukaryotes, in filamentous fungi and plants 19 compared to the identification of around 40 families of riboswitches for prokaryotes 20. At the eukaryotic transcriptional level, engineered promoter regulation has been reported 9,10,13 but is challenging 21 , as it requires pairing of a protein surface with a DNA binding motif, and transport of activators and repressors into the nucleus. At the post-transcriptional level, engineering networks have focused on regulation of miRNAs 22–26. miRNAs regulate several physiological relevant biological processes, such as cell growth, differentiation, development and apoptosis, and miRNA disfunction is implicated in various diseases 27. miRNAs thus make excellent candidates to design engineered switches as synthetic biology tools for gene regulation applicable in higher eukaryotes. Such engineered post-transcriptional control networks have been demonstrated using artificial miRNAs with embedded aptamers 23,26 , including engineered reversibility (7). Notably, drug-induced miRNA switches were used to demonstrate control of T cell signalling pathways, making progress towards the development of synthetic biology therapeutic tools for mammalian systems 25. However, while miRNA-based gene expression control systems produce ON states close to maximal gene expression, OFF states show leakage 23. This is a critical limitation when regulating enzyme turnover function, because only highly stringent switches OPEN

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