Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR

Area of Science:

• Molecular Biology
• Enzyme Engineering
• Biochemistry

Background:

• Thermostable DNA polymerases are essential for polymerase chain reaction (PCR).
• Beyond thermostability, enzyme fidelity, processivity, and nucleotide compatibility are critical for advanced molecular biology.
• Existing polymerases may have limitations in amplifying challenging DNA templates or tolerating inhibitors.

Purpose of the Study:

• To engineer and characterize a novel fusion DNA polymerase with enhanced properties.
• To assess the performance of the engineered enzyme in amplifying difficult DNA targets and its tolerance to inhibitors.
• To evaluate the fidelity and processivity trade-offs in the engineered enzyme.

Main Methods:

• Engineering a fusion protein combining a DNA polymerase from Nanoarchaeum equitans and a DNA binding domain from Sulfolobus solfataricus.
• Characterizing the enzyme's activity on long and GC-rich DNA templates.
• Assessing tolerance to PCR inhibitors and compatibility with dUTP.
• Evaluating enzyme fidelity using a magnification via nucleotide imbalance fidelity assay.

Main Results:

• The engineered enzyme, Neq2X7, exhibits high activity and efficiently amplifies long and GC-rich DNA.
• Neq2X7 is tolerant to various PCR inhibitors and can utilize dUTP instead of dTTP.
• Fidelity assays indicate an error rate lower than 2 x 10^-5 bp^-1, with a notable trade-off between fidelity and processivity compared to the parental enzyme.
• The enzyme is readily producible and its expression plasmid is available.

Conclusions:

• Neq2X7 is a versatile DNA polymerase suitable for applications like uracil excision DNA assembly and contamination-free diagnostics.
• The observed fidelity-processivity trade-off is an important consideration for engineered DNA polymerases.
• The enzyme's robustness and ease of production make it valuable for molecular biology laboratories.