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MicroRNA-based Regulation of Picornavirus Tropism

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A Modular T7 RNA Polymerase Toolbox Linking Selective miRNA Detection to Signal Amplification and Protein Expression.

Maria Vonk-de Roy¹, Andreas Walther¹

¹Life-Like Materials and Systems, University of Mainz, Duesbergweg 10-14, 55128 Mainz, Germany.

Journal of the American Chemical Society

|November 19, 2025

Summary

Scientists developed a T7 RNA polymerase (T7 RNAP) toolbox for programmable synthetic biology. This system enables selective oligonucleotide detection, gated transcription, and protein expression with enhanced signal amplification and reduced leakage.

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Area of Science:

Synthetic Biology
Biochemistry
Molecular Biology

Background:

Cells integrate molecular signals via complex biochemical reaction networks.
Current synthetic chemical reaction networks (CRNs) lack parallel processing, modularity, and are prone to leakage.

Purpose of the Study:

To develop a versatile, leakage-free T7 RNA polymerase (T7 RNAP) toolbox for programmable information processing.
To bridge selective oligonucleotide detection with gated transcription and protein expression.

Main Methods:

Designed modular T7-Locks releasing T7 promoters upon specific oligonucleotide binding.
Incorporated site-specific transcription terminators via C12 spacers for signal amplification.
Established miRNA-triggered protein expression in a cell-free transcription-translation (TX-TL) system.

Main Results:

Achieved quantitative and orthogonal miRNA detection with ON/OFF ratios >100.
Demonstrated controlled signal amplification through positive feedback mechanisms.
Successfully gated protein expression at the transcriptional level using T7-Locks.

Conclusions:

The developed modular platform integrates input sensing, amplification, and output synthesis.
This provides a generalizable strategy for DNA or RNA-driven information processing in synthetic biology.
The T7 RNAP toolbox offers a versatile and leakage-free solution for complex synthetic systems.