Elucidating the Determinants of Polymerase Specificity by Microfluidic-Based Deep Mutational Scanning

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Engineering polymerases for novel genetic polymers is challenging due to limited understanding of substrate specificity determinants.
• Current methods for mapping polymerase sequence-function relationships are often inefficient and lack high-throughput capabilities.

Purpose of the Study:

• To develop a high-throughput method for mapping sequence-function relationships in polymerases.
• To identify structural determinants governing polymerase substrate specificity for artificial genetic polymers.

Main Methods:

• A microfluidic-based approach combining droplet-based optical polymerase sorting and deep mutational scanning.
• Application of the method to map the finger subdomain of a Thermococcus kodakarensis replicative DNA polymerase.
• Utilizing threose nucleic acid as a model non-natural genetic polymer.

Main Results:

• The enrichment profile provided an unbiased view of mutant polymerase activity.
• Identified two instances of positive epistasis in polymerase mutants.
• Demonstrated near inversion of substrate specificity in a double mutant variant.

Conclusions:

• Deep mutational scanning is effective for elucidating polymerase specificity determinants.
• A small number of specific residues can govern polymerase substrate specificity.
• This method bypasses the need for iterative rounds of directed evolution for engineering polymerases.