Engineering Fluorescent Light-Up Aptamer Scaffolds for Multi-input Sensing

Abstract

In the fields of synthetic biology and molecular diagnostics, the development of adaptable and responsive molecular systems is crucial. Fluorescent light-up aptamers (FLAPs), gaining prominence recently, are powerful tools widely utilized across cell biology, molecular biology, and biomedicine. These aptamers can detect specific molecules or emit signals and have been increasingly used to stabilize fluorescence signals in complex biological environments. This study advances the field by engineering FLAPs designed for multi-input sensing, innovatively expanding RNA-based logic gates and sensing modules. We developed various RNA scaffold gates that enable the construction of complex genetic circuits, effectively implementing 2-input NOR, NAND, and IMPLY gates, as well as more intricate 3-input NAND gate configurations. Unlike traditional systems that primarily use an OFF to ON mechanism, the methods employed in our experiments leverage a unique ON to OFF transition mechanism that responds independently of sequence constraints. This innovative approach allows for the detection of subtle genetic variations, proving highly effective in applications requiring high specificity and sensitivity. This includes the precise detection of fusion genes and single nucleotide polymorphisms (SNPs), which holds vast potential for cancer diagnostics. Additionally, we have extended these RNA-based gates for tetracycline detection, showcasing the potential of these molecular systems as tools for environmental monitoring that can sense various molecules. The programmability and orthogonality of our RNA scaffold gates pave the way for further advancements, indicating flexibility across diverse environments. Notably, our engineered RNA scaffold gates have been proven to operate effectively using tap water at room temperature, highlighting their applicability outside conventional laboratory settings. This adaptability underscores the innovative potential of RNA-based devices to address global health challenges, opening new avenues for advanced applications in molecular diagnostics and synthetic biology.