Research Assistant Professor The University of Hong Kong, Hong Kong
Abstract: Fluorescent light-up aptamer/fluorogen pairs are crucial in tracking RNA within cells, although they face challenges in terms of thermostability and fluorescence intensity. Current in vitro selection techniques struggle to replicate the intricate intracellular environments, which results in limitations in biomolecule functionality in vivo. Inspired by the microenvironment-dependent RNA folding observed within cells and droplets that mimic organelles, we developed a system using microscale heated water droplets to imitate intracellular conditions. This system successfully replicates the intracellular RNA folding landscape. We combined this system with microfluidic droplet sorting to evolve RNA aptamers. Through this method, we engineered an RNA aptamer with enhanced fluorescence activity by exploring the chemical fitness landscape under conditions that mimic biological environments. We named our improved RNA aptamer eBroccoli, which demonstrated increased fluorescence intensity and thermal stability, both in vitro and in vivo in bacterial and mammalian cells. In the future, tracking of RNA in cells in this way could be used investigate cell physiology. Expanding upon this technology offers opportunities for non-destructive intracellular biomarker sensing, that could be used to characterize cell differentiation.
Funding Source: HKU Grants Council GRF [No. 17303123, 17306221, 17317322, 17163416, 17127515, 17125920], HKU Grants Council Theme-based Research Scheme [T12-201/19-R], Health@InnoHK program of the ITC and HKU Seed Funding [No. 102009959].
