Abstract

Biosensors are key components in engineered biological systems, providing a means of measuring and acting upon the large biochemical space in living cells. However, generating small molecule sensing elements and integrating them into in vivo biosensors have been challenging. Using aptamer-coupled ribozyme libraries and a novel ribozyme regeneration method, we developed de novo rapid in vitro evolution of RNA biosensors (DRIVER) that enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identified and validated biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors were applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors were also applied in detecting metabolite production from a multi-enzyme biosynthetic pathway. This work demonstrates DRIVER as a scalable pipeline for engineering de novo biosensors with wide-ranging applications in biomanufacturing, diagnostics, therapeutics, and synthetic biology.