SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly

Area of Science:

• Supramolecular chemistry
• Biological chemistry
• Nanotechnology

Background:

• Streptavidin-biotin interaction is crucial but lacks orthogonal partners.
• This limitation restricts the complexity of molecular assemblies.
• Need for robust orthogonal interactions in supramolecular chemistry.

Purpose of the Study:

• To create a versatile molecular assembly tool by combining streptavidin with an orthogonal irreversible interaction.
• To engineer chimeric tetramers with tunable biotin and SpyTag/SpyCatcher binding sites.
• To enable the construction of complex, multi-component nanoarchitectures.

Main Methods:

• Fusion of SpyTag or SpyCatcher to streptavidin variants (traptavidin).
• Generation of chimeric SpyAvidin tetramers with varying binding sites.
• Assembly of higher-order structures (octamers, eicosamers) via mixing chimeric tetramers.
• Validation using electrophoresis and native mass spectrometry.

Main Results:

• Successfully created chimeric SpyAvidin tetramers with precise numbers of binding sites.
• Demonstrated assembly of SpyAvidin octamers and eicosamers.
• Engineered eicosameric SpyAvidin to cluster major histocompatibility complex (MHC) class I complexes.
• Showcased enhanced T cell signaling stimulation with MHC eicosamers compared to tetramers.

Conclusions:

• SpyAvidins provide a simple and robust method for creating complex nanoarchitectures.
• The combination of noncovalent and covalent interactions offers enhanced stability and control.
• This platform enables the construction of large, functional molecular assemblies for biological applications.