Comprehensive and Facile Strategy for Enhanced Visualization of Sialylated RNA via Dual Bioorthogonal Labeling

Metabolic oligosaccharide engineering advancements in 2021 facilitated the discovery of glycoRNA, a glycan-modified RNA found on cell membranes, exhibiting sialic acid-terminated glycosylation across various cell types and mammals. However, the current imaging process is complex, cumbersome, and restricted to the recognition of specific sequences only. To overcome this limitation, we developed an enhanced visualization strategy for sialylated RNA, employing a dual bioorthogonal approach that integrates metabolic labeling of sialic acid (Sia) and RNA with orthogonal reactions. This method facilitates the recognition of total sialylated RNA through the use of tunable Sia and RNA probes, which can be regulated to initiate proximity-induced hybridization chain reaction amplification, effectively enhancing the fluorescence signal without the requirement for supplementary proteases. Our strategy offers high versatility, simplifies visualization steps, and broadens the recognition scope. It has been successfully employed in comparative analyses of RNA glycosylation states across breast, lung, and leukemic cell lines, demonstrating its potential as a robust tool for exploring the roles of sialylated RNA in biological processes and disease progression.