Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide

Area of Science:

• Molecular Biology
• Biotechnology
• Enzymology

Background:

• Native DNA polymerases possess stringent substrate recognition, limiting their applications.
• Expanding polymerase substrate specificity is crucial for advancing molecular biology tools.
• Existing methods for polymerase engineering face challenges with compromised catalytic activity.

Purpose of the Study:

• To introduce a novel strategy, short-patch compartmentalized self-replication (spCSR), for targeted expansion of polymerase substrate spectrum.
• To select for DNA polymerases capable of incorporating both ribonucleotides (NTPs) and deoxyribonucleotides (dNTPs).
• To engineer Thermus aquaticus (Taq) DNA polymerase variants with dual substrate specificity.

Main Methods:

• Development and application of the spCSR technique, focusing diversification on a short gene region.
• Selection of Taq polymerase variants under conditions that challenge full self-replication efficiency.
• Characterization of engineered polymerases for substrate incorporation and catalytic activity.

Main Results:

• Isolation of multiple Taq polymerase variants exhibiting dual NTP and dNTP substrate specificity.
• Identification of the AA40 mutant (E602V, A608V, I614M, E615G) with wild-type catalytic efficiency for dNTPs and ability to incorporate NTPs.
• Demonstration of mixed RNA-DNA amplification products, indicating DNA polymerase, RNA polymerase, and reverse transcriptase activities in a single polypeptide.
• AA40 displayed an expanded substrate spectrum for 2"-substituted nucleotides.

Conclusions:

• spCSR is a powerful strategy for generating polymerases with altered substrate specificity.
• Engineered polymerases like AA40 offer versatile enzymatic capabilities for synthesizing mixed nucleic acid polymers.
• Potential applications include nano- and biotechnology, and enzymatic synthesis of antisense and RNAi probes.