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      Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform

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          Abstract

          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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              Synthetic genetic polymers capable of heredity and evolution.

              Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.
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                Author and article information

                Contributors
                URI : https://loop.frontiersin.org/people/2328574/overviewRole: Role: Role: Role: Role: Role: Role: Role:
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                URI : https://loop.frontiersin.org/people/2391297/overviewRole: Role:
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                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                07 November 2023
                2023
                : 14
                : 1267196
                Affiliations
                [1] 1Research Center of Molecular Diagnostics and Sequencing, Research Institute of Tsinghua University in Shenzhen , Shenzhen, China
                [2] 2Research Center of Molecular Diagnostics and Sequencing, Axbio Biotechnology (Shenzhen) Co., Ltd. , Shenzhen, China
                Author notes

                Edited by: Mohammad Ali Amoozegar, University of Tehran, Iran

                Reviewed by: Ida Helene Steen, University of Bergen, Norway; Xi-Peng Liu, Shanghai Jiao Tong University, China

                *Correspondence: Yun He, hej@ 123456tsinghua-sz.org
                Article
                10.3389/fmicb.2023.1267196
                10661337
                38029082
                a15bfb77-1c3f-4f03-9812-547828b0604c
                Copyright © 2023 Sun, Ko, Gao, Fu, Gao, Zhang, Baldi, Hong, Ivanov, He and Tian.

                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.

                History
                : 26 July 2023
                : 24 October 2023
                Page count
                Figures: 6, Tables: 2, Equations: 0, References: 29, Pages: 11, Words: 7229
                Funding
                Funded by: China Postdoctoral Science Foundation, doi 10.13039/501100002858;
                Award ID: 2020M682977
                Funded by: National Natural Science Foundation of China, doi 10.13039/501100001809;
                Award ID: 32001043
                The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants from the China Postdoctoral Science Foundation (2020M682977) and National Natural Science Foundation of China (grant no. 32001043).
                Categories
                Microbiology
                Original Research
                Custom metadata
                Microbiotechnology

                Microbiology & Virology
                compartmentalized self-replication (csr),sequencing by synthesis (sbs),salt tolerance polymerase,droplet-based microfluidics,conservative amino acids

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