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Ultra-long Read Sequencing for Whole Genomic DNA Analysis
Published on: March 15, 2019
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Engineering psychrophilic polymerase for nanopore long-read sequencing
Yaping Sun1, Danny Hsu Ko1, Jie Gao1
1Research Center of Molecular Diagnostics and Sequencing, Research Institute of Tsinghua University in Shenzhen, Shenzhen, China.
Frontiers in Bioengineering and Biotechnology
|July 16, 2024
Summary
Psychrophilic polymerases show promise for long-read sequencing, overcoming limitations of other enzymes. Fusion with Sso7d protein enhances salt tolerance and processivity, enabling advanced polymerase engineering.
Area of Science:
- Biochemistry
- Molecular Biology
- Genomics
Background:
- Conventional DNA polymerases like thermophilic *Bacillus* stearothermophilus (Bst) and mesophilic *Bacillus subtilis* phage (phi29) have limitations for long-read sequencing.
- Bst polymerases are temperature-limited, while phi29 polymerases exhibit strong exonuclease activity and poor salt tolerance.
Purpose of the Study:
- To explore psychrophilic polymerases as alternatives for polymerase-nanopore long-read sequencing.
- To engineer fusion DNA polymerases combining psychrophilic (PB) and *Bacillus* stearothermophilus (Bst) components for improved performance.
Main Methods:
- Structural and biochemical analysis of PB-Bst fusion DNA polymerases.
- Targeted substitution of functional domains, including fusion with sulfolobus 7-kDa protein (Sso7d).
- Investigation of functional sites regulating exonuclease activity and processivity using artificial nucleotides.
Main Results:
- PB-Bst fusion polymerases demonstrate significantly enhanced salt tolerance and reduced exonuclease activity.
- Sso7d fusion domain substantially improves PB-Bst processivity.
- Specific functional sites (Asp43, Glu45, Glu266, Gln283, Leu334, Glu335, Ser426, Asp430) were identified as key regulators of exonuclease activity and processivity.
Conclusions:
- Psychrophilic polymerases, engineered via domain fusion, offer a viable alternative for long-read sequencing applications.
- Understanding the dynamics of exonuclease activity and processivity at specific sites provides a foundation for further polymerase engineering.
- This research advances the fundamental knowledge of psychrophilic polymerases and their potential in biotechnological applications.
