<i> <b>De Novo</b> </i> Development of Fluorogenic RNA-Based Biosensors Using Capture-SELEX in Tandem with Droplet Microfluidics

The detection of small molecules (e.g., pesticides, pollutants, antibiotics, drug residues) is critical for human health and environmental surveys as well as for many industrial applications. Fluorogenic RNA-based biosensors (FRBs) are aptamer-based tools specifically reporting on the presence of such targets by converting a specific binding event into fluorescence emission, thus making them of particular interest for on-site sensing applications. However, new sensor aptamers suited for FRB development are tedious to develop. In this study, we show that Capture-SELEX used in tandem with our microfluidic-assisted screening technology (μIVC) accelerates <i>de novo</i> FRB development by reprogramming the specificity of an existing sensor aptamer, followed by fluorescence-based functional selection. Furthermore, we show that these new sensor aptamers can be repurposed into functional biotechnological tools, such as gene-regulating aptazymes, thus demonstrating the versatility of our pipeline for the development of sensor aptamers adapted for various applications.