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Background <p>Human coronaviruses are pathogenic viruses that have the ability to cause serious diseases or even death in humans. The receptor binding domain (RBD) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the region of the spike protein, through which the virus directly interacts with the human Angiotensin-converting enzyme 2 receptor (ACE-2). Understanding the mutations that occur in the RBD and determining their influence on host cell binding plays an essential role in learning about the transmission mechanism of the disease and developing potential treatment.</p> Methods <p>We chose to apply the error-prone Polymerase Chain Reaction (ep-PCR), usually used in directed evolution and targeted mutagenesis applications, and to optimize it for the SARS-CoV-2 RBD sequence. This was followed by sequencing to analyze the mutation pattern. In this study, a novel ep-PCR protocol was developed specifically for RBD, optimized for dNTP ratios, magnesium ions, manganese ions, and cycle numbers.</p> Results <p>Distribution of mutations in the RBD sequence was determined. The effect of each parameter on the generation of mutations showed that MnCl<sub>2</sub> concentration had the strongest effect, followed by the number of cycles, the dNTP ratio, and MgCl₂ concentration. ep-PCR analysis revealed that 12 best conditions for generating mutations in the 13–17&#xa0;bp range.</p> Conclusion <p>Our results highlight the virulence-enhancing potential of mutations and enable researchers to evaluate potential vaccine candidates in the future. Furthermore, compared to earlier reports, the method described here requires only Sanger sequencing and a standard PCR cycler, therefore it is much more accessible. Most importantly, it yields combinatorial mutation diversity in one round, along the entire length of the sequence of interest.</p>

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Improving directed evolution strategies: error-prone PCR optimization for SARS-CoV-2 spike receptor binding domain

  • 期刊论文

  • 发表日期：

    2025年12月10日

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  • Tanay Uzgan,
  • Bianca Schulte,
  • Husniye Tansel Yalcin

摘要

Background

Human coronaviruses are pathogenic viruses that have the ability to cause serious diseases or even death in humans. The receptor binding domain (RBD) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the region of the spike protein, through which the virus directly interacts with the human Angiotensin-converting enzyme 2 receptor (ACE-2). Understanding the mutations that occur in the RBD and determining their influence on host cell binding plays an essential role in learning about the transmission mechanism of the disease and developing potential treatment.

Methods

We chose to apply the error-prone Polymerase Chain Reaction (ep-PCR), usually used in directed evolution and targeted mutagenesis applications, and to optimize it for the SARS-CoV-2 RBD sequence. This was followed by sequencing to analyze the mutation pattern. In this study, a novel ep-PCR protocol was developed specifically for RBD, optimized for dNTP ratios, magnesium ions, manganese ions, and cycle numbers.

Results

Distribution of mutations in the RBD sequence was determined. The effect of each parameter on the generation of mutations showed that MnCl2 concentration had the strongest effect, followed by the number of cycles, the dNTP ratio, and MgCl₂ concentration. ep-PCR analysis revealed that 12 best conditions for generating mutations in the 13–17 bp range.

Conclusion

Our results highlight the virulence-enhancing potential of mutations and enable researchers to evaluate potential vaccine candidates in the future. Furthermore, compared to earlier reports, the method described here requires only Sanger sequencing and a standard PCR cycler, therefore it is much more accessible. Most importantly, it yields combinatorial mutation diversity in one round, along the entire length of the sequence of interest.