A modular T7 RNA polymerase toolbox linking selective miRNA detection to signal amplification and protein expression

Abstract

Cells process and integrate diverse molecular signals through complex biochemical reaction networks combining sensing, computation, and response. Synthetic analogs in the form of chemical reaction networks (CRNs) still fall short in parallel and selective processing and often suffer from limited modularity, incompatibilities, or leakage. Here, we present a versatile and leakage-free T7 RNA polymerase (T7 RNAP) toolbox that bridges from selective oligonucleotide detection to gated transcription and finally protein expression in a programmable manner. Central to our system is the design of modular T7-Locks, that release active T7 promoters only upon specific oligonucleotide binding to trigger transcription. This enables quantitative and orthogonal miRNA detection with ON/OFF ratios exceeding 100. We further demonstrate that incorporation of site-specific transcription terminators via C12 spacers inside T7 templates enables controlled signal amplification through positive feedback. Finally, we establish miRNA-triggered protein expression in a cell-free TX-TL system using specifically engineered sticky-end genes that are gated through T7-Locks on a transcriptional level. This modular platform connects input sensing, amplification, and output synthesis, providing a generalizable strategy for DNA or RNA-driven information processing in synthetic biology.