Hierarchical Fluorescence Imaging Strategy for Assessment of the Sialylation Level of Lipid Rafts on the Cell Membrane

Abstract

Glycosylation is one of the most ubiquitous and complicated modifications of proteins and lipids. The revelation of glycosylation-mediated regulation mechanisms of biological processes relies critically on the tools that can reflect the spatial heterogeneity of cell surface glycans, for example, distinguishing glycans exhibited in lipid raft or nonraft domains. To achieve simultaneous visualization of raft and raft-harbored glycans on the cell surface, we combine specific raft recognition, glycan chemoselective labeling, and DNA dynamic hybridization techniques to develop a hierarchical fluorescence imaging strategy using N-acetyl-neuraminic acid (Sia) as the model sugar. We fabricate a raft probe and Sia probe for rafts and Sia, respectively. After specifically anchoring the two probes on the cell surface, the raft probe can be cyclically utilized to turn on the fluorescence of the Sia probe, only residing in rafts, via a proximity cascade DNA reaction. The duplex imaging capability for spatially relevant levels of biological structures enables the revelation of the reason for raft-confined Sia variation in different biological processes. Thus, this work provides an elegant and powerful tool for interrogation of the glycan regulation mechanisms on raft composition, organization, and functions and also contributes to the development of raft-carried glycoconjugate-based theranostic techniques.