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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...

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Autores principales: Sun, Yaping, Ko, Danny Hsu, Gao, Jie, Fu, Kang, Gao, Yaping, Zhang, Qiwen, Baldi, Salem, Hong, Tao, Ivanov, Igor, He, Yun, Tian, Hui
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
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.
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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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