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

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Deep mutational scanning typically relies on indirect assays to assess protein variants.
• Directly linking protein function to sequence information is challenging, especially under denaturing conditions.

Purpose of the Study:

• To develop a novel method for directly coupling protein variants to RNA barcodes in vivo.
• To enable high-throughput biochemical assays of protein variants under denaturing conditions.

Main Methods:

• Engineered a mutant TrmA enzyme from Escherichia coli to covalently link variant proteins to RNA barcodes.
• Developed the Dosa (Deep mutational scanning) method for in vitro separation and sequencing of linked protein-RNA complexes.
• Applied Dosa to analyze FLAG epitope variants, protease substrates, and Aβ(1-42) variants.

Main Results:

• Demonstrated successful covalent linkage of proteins to RNA barcodes via engineered TrmA.
• Showcased the ability to perform assays in 8 M urea, indicating robustness.
• Successfully profiled antibody binding, protease cleavage, and protein solubility for numerous variants.

Conclusions:

• Dosa provides a powerful tool for high-throughput functional profiling of protein variants.
• The covalent linkage enables assays under previously inaccessible denaturing conditions.
• This method is broadly applicable to proteins expressed in various cell types.