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Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform
In Phi29-α–hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activi...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10661337/ https://www.ncbi.nlm.nih.gov/pubmed/38029082 http://dx.doi.org/10.3389/fmicb.2023.1267196 |
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author | Sun, Yaping Ko, Danny Hsu Gao, Jie Fu, Kang Gao, Yaping Zhang, Qiwen Baldi, Salem Hong, Tao Ivanov, Igor He, Yun Tian, Hui |
author_facet | Sun, Yaping Ko, Danny Hsu Gao, Jie Fu, Kang Gao, Yaping Zhang, Qiwen Baldi, Salem Hong, Tao Ivanov, Igor He, Yun Tian, Hui |
author_sort | Sun, Yaping |
collection | PubMed |
description | In Phi29-α–hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activity to prevent the degradation of modified nucleotide tags; however, the lack of exonuclease activity also affects polymerase processivity. This study aimed to optimize phi29 polymerase for improved salt tolerance and processivity while maintaining its lack of exonuclease activity to meet the requirements of nanopore sequencing. Using salt tolerance compartmentalized self-replication (stCSR) and a microfluidic platform, we obtained 11 mutant sites with enhanced salt tolerance attributes. Sequencing and biochemical analyses revealed that the substitution of conserved amino acids such as G197D, Y369E, T372N, and I378R plays a critical role in maintaining the processivity of exonuclease-deficient phi29 polymerase under high salt conditions. Furthermore, Y369E and T372N have been identified as important determinants of DNA polymerase binding affinity. This study provides insights into optimizing polymerase processability under high-salt conditions for real-time polymerase nanopore sequencing, paving the way for improved performance and applications in nanopore sequencing technologies. |
format | Online Article Text |
id | pubmed-10661337 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-106613372023-11-07 Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform Sun, Yaping Ko, Danny Hsu Gao, Jie Fu, Kang Gao, Yaping Zhang, Qiwen Baldi, Salem Hong, Tao Ivanov, Igor He, Yun Tian, Hui Front Microbiol Microbiology In Phi29-α–hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activity to prevent the degradation of modified nucleotide tags; however, the lack of exonuclease activity also affects polymerase processivity. This study aimed to optimize phi29 polymerase for improved salt tolerance and processivity while maintaining its lack of exonuclease activity to meet the requirements of nanopore sequencing. Using salt tolerance compartmentalized self-replication (stCSR) and a microfluidic platform, we obtained 11 mutant sites with enhanced salt tolerance attributes. Sequencing and biochemical analyses revealed that the substitution of conserved amino acids such as G197D, Y369E, T372N, and I378R plays a critical role in maintaining the processivity of exonuclease-deficient phi29 polymerase under high salt conditions. Furthermore, Y369E and T372N have been identified as important determinants of DNA polymerase binding affinity. This study provides insights into optimizing polymerase processability under high-salt conditions for real-time polymerase nanopore sequencing, paving the way for improved performance and applications in nanopore sequencing technologies. Frontiers Media S.A. 2023-11-07 /pmc/articles/PMC10661337/ /pubmed/38029082 http://dx.doi.org/10.3389/fmicb.2023.1267196 Text en Copyright © 2023 Sun, Ko, Gao, Fu, Gao, Zhang, Baldi, Hong, Ivanov, He and Tian. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Sun, Yaping Ko, Danny Hsu Gao, Jie Fu, Kang Gao, Yaping Zhang, Qiwen Baldi, Salem Hong, Tao Ivanov, Igor He, Yun Tian, Hui Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform |
title | Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform |
title_full | Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform |
title_fullStr | Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform |
title_full_unstemmed | Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform |
title_short | Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform |
title_sort | unraveling the salt tolerance of phi29 dna polymerase using compartmentalized self-replication and microfluidics platform |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10661337/ https://www.ncbi.nlm.nih.gov/pubmed/38029082 http://dx.doi.org/10.3389/fmicb.2023.1267196 |
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