Dosa: A method to covalently barcode proteins for high throughput biochemistry

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Deep mutational scanning typically uses indirect assays (e.g., growth) to assess protein activity.
• Direct, high-throughput biochemical assays for protein variants are needed.

Purpose of the Study:

• To develop a method for directly linking protein variants to RNA barcodes for high-throughput biochemical analysis.
• To demonstrate the versatility of this method across various biochemical assays.

Main Methods:

• Developed Dosa (DNA-barcoded protein activity assay) by fusing variant proteins to an RNA-barcoding enzyme (TrmA E358Q).
• Proteins are covalently linked to RNA barcodes in vivo.
• Variant proteins are separated in vitro based on biochemical properties and identified via their barcodes.

Main Results:

• Successfully analyzed FLAG epitope variants binding to an anti-FLAG antibody.
• Profiled cleavage preferences of enteropeptidase and human rhinovirus 3C protease variants.
• Measured solubility of hundreds of Aβ(1-42) variants.

Conclusions:

• Dosa provides a versatile platform for high-throughput biochemical assays of protein variants.
• The method is applicable to proteins produced in E. coli and mammalian cells.
• Enables direct assessment of diverse protein functions, overcoming limitations of indirect assays.