RNA Aptamer-Based Biosensors for Multiplexed in Vitro Diagnostics and Circuit Engineering

The need for metabolic sensors spans both in vitro and in vivo applications, particularly in the design, evolution and optimization of pathways and genetic control systems. In addition, there is great demand for orthogonal sensors to detect and propagate signals independently while maintaining the ability to be coupled for complex diagnostics or pathway design. We are developing RNA aptamer-based sensors that process molecular inputs into programmable nucleic acid outputs, with the goal of generating a robust multiplexed small molecule sensing assay on common laboratory equipment such as a qPCR machine. These sensors are engineered using a multi-state RNA folding design strategy where transient but metastable intermediate states are engineered along the co-transcriptional folding trajectory. Binding of the small molecule input to the aptamer domain directs the folding pathway towards an active state and generates a fluorescent signal through a nucleic acid strand displacement reaction. We are currently expanding this work to include sensors for a broader panel of small molecules, while simultaneously optimizing the nucleic acid circuits for multi-signal detection in vitro, with the goal of generating complete biosensor devices for the eventual use in mammalian cell systems.