Pol6 Polymerase Variants for Nanopore Sequencing Stability
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Solution Overview
Problem
Nanopore sequencing technologies face limitations in processivity, leading to short read lengths and instability of the polymerase-DNA template complex at high salt concentrations, which affects the accuracy and length of DNA sequencing reactions.
Innovation Solution
A variant Pol6 enzyme with increased processivity is developed, featuring amino acid modifications such as E585K, D44A, S366A, T529M, and A547F, which decreases the rate of template dissociation and enhances read length, allowing for longer continuous reads and improved sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high salt concentrations are used in nanopore sequencing, then sequencing accuracy is improved, but polymerase processivity decreases and template dissociation increases
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the polymerase enzyme through site-directed mutagenesis. Specific amino acid residues are changed to alter the enzyme's biochemical properties, enabling it to maintain stable binding to the DNA template under high salt conditions. This molecular-level parameter change allows the system to simultaneously achieve high sequencing accuracy and maintain polymerase processivity.
2Length of moving object
If read length is increased, then more sequence information is obtained, but polymerase processivity must be enhanced to maintain stability
Solution Approach 1:
The patent modifies the polymerase enzyme's amino acid parameters to enhance its processivity. By changing specific amino acid residues, the enzyme's ability to remain bound to the DNA template during extended polymerization reactions is improved, enabling longer read lengths while maintaining complex stability throughout the sequencing process.
3Reliability
If template dissociation rate is decreased, then processivity is improved, but enzyme modification complexity increases
Solution Approach 1:
The patent applies local quality by making targeted modifications at specific amino acid positions within the polymerase enzyme rather than attempting global optimization. By focusing mutations on particular residues that are critical for template binding and processivity, the patent achieves improved enzyme performance with minimal modification complexity, allowing the enzyme to function effectively in nanopore sequencing applications.
Data Source
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AI summary
The present disclosure provides variant Pol6 polymerase polypeptides, compositions comprising the Pol6 variant polypeptides, and methods for using the variant Pol6 polypeptides for determining the sequencing of nucleic acids, for example, by nanopore sequencing. The variant Pol6 polymerases possess decreased rates of dissociation of template from the polymerase-template complex, which result in increased processivity relative to the parental Pol6 polypeptides from which they are derived.