Abstract

The imaging characterization of spatial proximity of covalently linked structural motifs (e.g., protein-specific glycoform) is essential for thorough understanding of cellular chemistry and biology. The current imaging formats rely on gating-based mechanisms for generating correct closed-loop signaling topology, and they can suffer from low signal intensity, restricted applicability, and complicated design. We report herein the development of a mechanistically distinct filter beacon architecture for protein-specific glycoform imaging on the cell surface. The elaborate structuring of molecular beacon segment, nicking restriction site, and docking moiety lays out a general nongated design principle for passing through intended closed-loop signaling topology and sifting out false-positive open-loop leakage topology, furnishing a straightforward imaging format with high signal intensity and broad applicability. Proof-of-concept protocols have been developed for the imaging of MUC1-bound terminal sialic acid and fucose. The versatile adaptability of the protocols also enables dynamic monitoring of protein-specific glycosylation pattern changes in response to the alteration of cellular physiological states. Given the convenience for achieving multiplexed encoding and decoding, through fluorescence signals alone or together with filter beacon sequences, the filter beacon architecture should permit comprehensive imaging of diverse-structured carbohydrates on a given glycoprotein.