Optimization of RNA Pepper Sensors for the Detection of Arbitrary RNA Targets

Abstract

The development of fluorescent light-up RNA aptamers (FLAPs) has paved the way for the creation of sensors to track RNA in live cells. A major challenge with FLAP sensors is their brightness and limited signal-to-background ratio both in vivo and in vitro . To address this, we develop sensors using the Pepper aptamer, which exhibits superior brightness and photostability when compared to other FLAPs. The sensors are designed to fold into a low fluorescence conformation and to switch to a high fluorescence conformation through toehold or loop-mediated interactions with their RNA target. Our sensors detect RNA targets as short as 20 nucleotides in length with a wide dynamic range over 300-fold in vitro, and we describe strategies for optimizing the sensor’s performance for any given RNA target. To demonstrate the versatility of our design approach, we generated Pepper sensors for a range of specific, biologically relevant RNA sequences. Our design and optimization strategies are portable to other FLAPs and offer a promising foundation for future development of RNA sensors with high specificity and sensitivity for detecting RNA biomarkers with multiple applications.