Co-evolution of a near-infrared aptamer:dye system for live-cell super-resolution RNA imaging

Despite their advantageous properties for live-cell imaging and super-resolution microscopy, high-performance silicon rhodamine (SiR) near-infrared (NIR) probes are still rarely employed in RNA imaging via fluorescent light-up aptamers (FLAPs). Here, we developed the SiRiuS:SiR-5 system through a combined evolutionary approach: evolving the aptamer via fluorescence-activated cell sorting (FACS), along with targeted mutations, truncations, and rational design, and improvement of the dye by systematic chemical derivatization. This resulted in an aptamer:dye pair with high fluorogenicity and photostability, specifically optimized for visualization of RNAs in mammalian live cells. Our system demonstrates strong fluorescence enhancement in live-cell imaging, enabling time resolved imaging of dynamic processes such as stress-granule formation. Notably, we validate its application in STED super-resolution microscopy, establishing it as a powerful NIR imaging platform for RNA structures below the refraction limit. Its orthogonality to existing FLAPs operating in the yellow-orange spectrum further broadens its versatility for exploring complex RNA dynamics in live cells.