Abstract
Here we report ultrastable synthetic binding pairs between cucurbit[7]uril (CB[7]) and adamantyl- (AdA) or ferrocenyl-ammonium (FcA) as a supramolecular latching system for protein imaging, overcoming the limitations of protein-based binding pairs. Cyanine 3-conjugated CB[7] (Cy3-CB[7]) can visualize AdA- or FcA-labeled proteins to provide clear fluorescence images for accurate and precise analysis of proteins. Furthermore, controllability of the system is demonstrated by treating with a stronger competitor guest. At low temperature, this allows us to selectively detach Cy3-CB[7] from guest-labeled proteins on the cell surface, while leaving Cy3-CB[7] latched to the cytosolic proteins for spatially conditional visualization of target proteins. This work represents a non-protein-based bioimaging tool which has inherent advantages over the widely used protein-based techniques, thereby demonstrating the great potential of this synthetic system.
Highlights
We report ultrastable synthetic binding pairs between cucurbit[7]uril (CB[7]) and adamantyl- (AdA) or ferrocenyl-ammonium (FcA) as a supramolecular latching system for protein imaging, overcoming the limitations of protein-based binding pairs
The ultrastable host–guest interaction between CB[7] and AdA has not been exploited for cellular imaging, despite its own unique features that make it useful for cell biology including: (1) a stable chemical structure that is robust in a cellular environment and not affected by enzymatic degradation; (2) their bio-orthogonality in binding to biomolecules; (3) their small size compared to other protein-based binding pairs such as SA–BT enabling for efficient cellular uptake; (4) controllable binding affinity by treatment with competing guests allowing for selective dissociation of the host–guest assembly, on demand; and (5) scalability using known chemical synthetic methods for costeffective and convenient uses
Prior to imaging with Cy3-CB[7], using the concept of supramolecular latching, the proteins of interest were targeted by labeling with AdA, a process we term adamantylation, using various chemical and biological approaches as follows: (1) ferrocenylation of plasma membrane proteins using EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride) coupling; (2) adamantylation of proteins in specific organelles using proximitydependent enzyme-assisted labeling using horseradish peroxidases (HRPs) and an engineered ascorbate peroxidase (APEX); and (3) adamantylation of specific target proteins through the use of a self-labeling proteins tag, namely SNAP-tag® (SNAP-tag) which can be expressed as a fusion with the target protein
Summary
We report ultrastable synthetic binding pairs between cucurbit[7]uril (CB[7]) and adamantyl- (AdA) or ferrocenyl-ammonium (FcA) as a supramolecular latching system for protein imaging, overcoming the limitations of protein-based binding pairs. The ultrastable host–guest interaction between CB[7] and AdA (or other tight binders) has not been exploited for cellular imaging, despite its own unique features that make it useful for cell biology including: (1) a stable chemical structure that is robust in a cellular environment and not affected by enzymatic degradation; (2) their bio-orthogonality in binding to biomolecules; (3) their small size compared to other protein-based binding pairs such as SA–BT enabling for efficient cellular uptake; (4) controllable binding affinity by treatment with competing guests allowing for selective dissociation of the host–guest assembly, on demand; and (5) scalability using known chemical synthetic methods for costeffective and convenient uses. Considering the synthetic nature, their bioorthogonality in binding and conditionally switchable visualization of cellular proteins of interest, we believe that the Cy3-CB[7]-guest pairs represent a general, non-protein-based and efficient cellular bioimaging tool for almost any target proteins which are labeled with AdA (or FcA)
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