Automated Assembly of Programmable RNA-Based Sensors.

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 a diverse set of programmable RNA regulators, including toehold switch riboregulators targeting viral RNAs, single-nucleotide-specific programmable riboregulators for discrimination of SARS-CoV-2 spike gene mutations, and metal-responsive riboswitches. In total, we construct 174 plasmids and test the designed methods by comparing both manual and automated assembly. We further demonstrate that the assembled toehold switch plasmids are functional in both bacterial and cell-free expression systems.