SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• Protein display systems like phage and ribosome display are crucial for directed evolution.
• Existing systems lack multivalent formats to leverage avidity effects for improved selection.

Purpose of the Study:

• To develop a novel in vitro display system enabling multivalent display of proteins.
• To utilize dendrimer-like DNA as a scaffold for constructing tailor-made valencies.
• To enhance affinity panning and protein binder selection through avidity effects.

Main Methods:

• Construction of dendrimer-like DNA scaffolds with benzylguanine (BG) docking points.
• In vitro expression of SNAP-tag fused proteins within emulsion droplets.
• Covalent linkage of protein-coding DNA to the DNA scaffold via BG reaction.
• Comparison of mono- and multivalent display constructs in model selections.

Main Results:

• The novel multivalent SNAP-display system achieved up to 0.86% recovery and 400-fold enrichment.
• Avidity effects enhanced enrichment by up to fivefold and recovery by up to 25-fold compared to monovalent constructs.
• Demonstrated successful construction and application of tailor-made DNA nanostructures for protein display.

Conclusions:

• The developed multivalent SNAP-display system based on DNA dendrimers is the first in vitro display with defined, tailor-made valencies.
• This system offers a significant advantage for selecting protein binders, particularly in early rounds of directed evolution.
• DNA nanostructures provide a versatile platform for advancing in vitro display technologies and protein engineering.