Signal Amplification for Cell-Free Biosensors, an Analog-to-Digital Converter

Signal Amplification for Cell-Free Biosensors, an Analog-to-Digital Converter

Toehold switches are biosensors useful for the detection of endogenous and environmental RNAs. They have been successfully engineered to detect virus RNAs in cell-free gene expression reactions. Their inherent sequence programmability makes engineering a fast and predictable process. Despite improve...

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Bibliographic Details
Published in:ACS synthetic biology Vol. 12; no. 10; pp. 2819 - 2826
Main Authors: Franco, Rafael A. L., Brenner, Gabriel, Zocca, Vitória F. B., de Paiva, Gabriela B., Lima, Rayane N., Rech, Elibio L., Amaral, Danilo T., Lins, Milca R. C. R., Pedrolli, Danielle B.
Format: Journal Article
Language:English
Published: American Chemical Society 20-10-2023
Subjects:
cell-free system
SARS-CoV-2
biosensor
serine integrase
toehold switch
Dengue
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Summary:Toehold switches are biosensors useful for the detection of endogenous and environmental RNAs. They have been successfully engineered to detect virus RNAs in cell-free gene expression reactions. Their inherent sequence programmability makes engineering a fast and predictable process. Despite improvements in the design, toehold switches suffer from leaky translation in the OFF state, which compromises the fold change and sensitivity of the biosensor. To address this, we constructed and tested signal amplification circuits for three toehold switches triggered by Dengue and SARS-CoV-2 RNAs and an artificial RNA. The serine integrase circuit efficiently contained leakage, boosted the expression fold change from OFF to ON, and decreased the detection limit of the switches by 3–4 orders of magnitude. Ultimately, the integrase circuit converted the analog switches’ signals into digital-like output. The circuit is broadly useful for biosensors and eliminates the hard work of designing and testing multiple switches to find the best possible performer.
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ISSN:2161-5063
2161-5063
DOI:10.1021/acssynbio.3c00227