Suitability Evaluation of Toehold Switch and EXPAR for Cell-Free MicroRNA Biosensor Development

ABSTRACT The development of a robust and cost-effective sensing platform for microRNA (miRNA) is of paramount importance in detecting and monitoring various diseases. Current miRNA detection methods are marred by low accuracy, high cost, and instability. The toehold switch riboregulator has shown promising results in detecting viral RNAs integrated with the cell-free system (CFS). This study aimed to leverage the toehold switch technology to detect miRNA in the CFS and to incorporate the exponential amplification reaction (EXPAR) to bring the detection to clinically relevant levels. We assessed various EXPAR DNA templates under different temperatures and additives to enhance the accuracy of the sensing platform. Furthermore, different structures of toehold switches were tested with either high-concentration synthetic miRNA or EXPAR product to assess sensitivity. Herein, we elucidated the mechanisms of the toehold switch and EXPAR, presented the findings of these optimizations, and discussed the potential benefits and drawbacks of their combined use.