Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform.
Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform.
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DOI：
10.3389/fmicb.2023.1267196
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发表时间：
2023
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
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在Phi 29-α-溶血素（α-HL）纳米孔测序系统中，强电化学信号依赖于高浓度的盐。然而，高盐浓度不利地影响聚合酶活性。合成测序（SBS）需要使用没有核酸外切酶活性的phi 29聚合酶，以防止修饰的核苷酸标签的降解;然而，缺乏核酸外切酶活性也影响聚合酶的持续合成能力。本研究旨在优化phi 29聚合酶以提高耐盐性和持续合成能力，同时保持其缺乏核酸外切酶活性以满足纳米孔测序的要求。利用耐盐区室化自我复制（stCSR）和微流控平台，我们获得了11个突变位点与增强耐盐属性。测序和生化分析表明，保守氨基酸如G197 D，Y369 E，T372 N和I378 R的取代在高盐条件下维持核酸外切酶缺陷型phi 29聚合酶的合成能力中起着关键作用。此外，Y369 E和T372 N已被鉴定为DNA聚合酶结合亲和力的重要决定因素。这项研究为实时聚合酶纳米孔测序提供了在高盐条件下优化聚合酶可加工性的见解，为提高纳米孔测序技术的性能和应用铺平了道路。
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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DOI：
10.3390/jof8111215
发表时间：
2022-11-17
期刊：
Journal of fungi (Basel, Switzerland)
影响因子：
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作者：
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通讯作者：
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影响因子：
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影响因子：
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通讯作者：
Shen X
影响因子：
14.8
作者：
Fujii, Ryota;Kitaoka, Motomitsu;Hayashi, Kiyoshi
通讯作者：
Hayashi, Kiyoshi
DOI：
10.1107/s0907444904026721
发表时间：
2005-01-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
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作者：
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通讯作者：
Steitz, TA