A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

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. Here, using aptamer-coupled ribozyme libraries and a ribozyme regeneration method, de novo rapid in vitro evolution of RNA biosensors (DRIVER) enables multiplexed discovery of biosensors. With DRIVER and high-throughput characterization (CleaveSeq) fully automated on liquid-handling systems, we identify and validate biosensors against six small molecules, including five for which no aptamers were previously found. DRIVER-evolved biosensors are applied directly to regulate gene expression in yeast, displaying activation ratios up to 33-fold. DRIVER biosensors are 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.

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

“…However, as mentioned previously, selecting large aptazyme libraries in cells is challenging. A recent publication by Townshend et al presents a novel, automated method for selecting functional aptazymes from 10 12 -10 14 -member libraries followed by screening for function in live yeast [173]. In the DRIVER selection technique, iterative cleavage reactions in either the presence of the target ligand (positive selections) or structurally-similar small molecule decoys (negative selections) are performed in vitro.…”

Engineering synthetic RNA devices for cell control

“…Similarly, affinity can be increased by lowering the ligand concentration as the selection progresses to place selective pressure for higher-affinity aptamers 75,78,79 . Finally, once an aptamer is generated, further tuning of specificity and affinity is possible through directed mutagenesis or rational engineering [80][81][82][83] . Despite advances in RNA aptamer selection and engineering, the total number of small molecules that can be sensed remains limited, and the imminent need for increased numbers of genetically encoded sensors requires an exponential expansion of our current throughput.…”

“…In the original demonstration of the DRIVER method, small molecule-responsive RNA biosensors with nanomolar and micromolar affinities were generated against six small molecules, five of which had no previously reported aptamers 81 . Aptamers created via DRIVER can be used in the HHRz framework directly as genetic controllers or can be isolated and used separately.…”

“…RNA sequences that bind the target molecules and undock from the biotinylated oligonucleotides are subsequently eluted from the beads, creating the starting library for the next selection cycle. b | In the DRIVER (de novo rapid in vitro evolution of RNA biosensors) method 81 , libraries are designed based on a modified hammerhead ribozyme (HHRz) from satellite RNA of tobacco ringspot virus (sTRSV) -a small, naturally occurring self-cleaving ribozyme -with the two loops replaced by either a randomized 30-60mer or a randomized 4-8mer. Sequences are mixed with target molecules.…”

“…Inspired by coordinated efforts in the protein and enzymology fields, RNA foundries should be established to focus on scaling high-throughput methods to generate open-source libraries of standard, well-characterized RNA components that can be used widely by the field in support of new device generation 23,81,88 . Scaling such efforts will ensure a critical open resource to advance the broader adoption of RNA devices in research, commercial and clinical settings.…”