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

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.

Enhancing efficiency of protein language models with minimal wet-lab data through few-shot learning

“…For example, WGA and MDA reactions performed at elevated temperatures could benefit from expedited reaction kinetics. Additionally, increased reaction temperatures may mitigate the influence of C/G content on amplification bias, offering further advantages 49 , 50 . Consequently, the enhancement of thermostability in Phi29 has been a focal point of academic research.…”

Engineering psychrophilic polymerase for nanopore long-read sequencing

“…Phi29 polymerase from the Bacillus subtilis phage phi29 is widely used in nanopore sequencing systems due to its strong strand displacement activity and processivity at 30°C ( Stranges et al, 2016 ). In this electrochemical sequencing systems, high salt concentrations, such as 300 mM NaCl buffer, are required to reduce the overall solution resistance of the electrochemical cell, thereby improving the signal-to-noise ratio and enabling the quantification of small changes in current or potential ( Davis and Chen, 2013 ; Stranges et al, 2016 ; Bard et al, 2022 ; Sun et al, 2023 ). However, the processivity of phi29 polymerase is greatly compromised under high-salt conditions due to higher tendency of dissociation from DNA templates ( Sun et al, 2023 ).…”

“…In this electrochemical sequencing systems, high salt concentrations, such as 300 mM NaCl buffer, are required to reduce the overall solution resistance of the electrochemical cell, thereby improving the signal-to-noise ratio and enabling the quantification of small changes in current or potential ( Davis and Chen, 2013 ; Stranges et al, 2016 ; Bard et al, 2022 ; Sun et al, 2023 ). However, the processivity of phi29 polymerase is greatly compromised under high-salt conditions due to higher tendency of dissociation from DNA templates ( Sun et al, 2023 ). Fusing DNA binding modules such as helix–hairpin–helix (HhH) 2 domain can compensate the reduction of DNA binding in high salt condition ( Gao et al, 2021 ).…”

“…Fusing DNA binding modules such as helix–hairpin–helix (HhH) 2 domain can compensate the reduction of DNA binding in high salt condition ( Gao et al, 2021 ). Apart from its weak binding in high salt conditions, Phi29 polymerase possesses potent 3'–5′ exonuclease activity for proofreading polymerization errors, albeit this activity poses a challenge by degrading oligonucleotide-modified substrates and obstructing polymerase processivity ( Del Prado et al, 2019 ; Sun et al, 2023 ). Strong exonuclease activity and weak salt tolerance have impeded the widespread application of phi29 polymerase in polymerase-nanopore sequencing systems.…”