Automated Assembly of Programmable RNA-Based Sensors

ABSTRACT Engineered programmable RNA sensors have been applied in low-cost diagnostics, endogenous RNA detection, and multi-input genetic circuits. However, designing, producing, and screening high-performance RNA sensors remains time consuming and labor intensive. Here, we present an automated plasmid assembly pipeline using liquid handling robotics to enable high-throughput construction of plasmids with arbitrary sequences. We compare automated and manual assembly methods using the NGS Hamilton Microlab STAR across two plasmid backbones to evaluate efficiency and reliability. As a proof of concept, we use this modular platform to construct 144 total plasmids encoding riboregulators targeting diverse viral targets along with their cognate trigger sequences. We further demonstrate that the assembled plasmids are functional in both bacterial and cell-free expression systems.