Pol6 Polymerase Variants for Nanopore Sequencing
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Solution Overview
Problem
Current DNA polymerases face challenges in recombinant DNA applications, particularly in nanopore sequencing, due to rapid DNA strand movement through nanopores, which complicates recording and introduces background noise, and the need for improved enzymes that can efficiently incorporate modified nucleotides under high salt conditions.
Innovation Solution
Development of modified DNA polymerases through directed evolution to enhance their extension rate and nucleotide dissociation rates for polyphosphate nucleotides, incorporating specific amino acid alterations that improve enzyme activity and processivity, allowing for better incorporation of tagged nucleotides in high salt environments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If wild-type DNA polymerase is used in nanopore sequencing, then the sequencing process can be performed, but the DNA strand moves rapidly through the nanopore causing background noise and reducing measurement precision
Solution Approach 1:
The patent applies parameter changes by modifying the DNA polymerase enzyme through directed evolution to alter its kinetic parameters. Specifically, the engineered polymerases have modified extension rates and nucleotide dissociation rates, allowing control over the DNA strand translocation speed through the nanopore, thereby reducing background noise and improving single-nucleotide resolution
2Reliability
If wild-type DNA polymerase is used, then the enzyme can perform basic polymerization, but it fails to efficiently incorporate modified polyphosphate nucleotides under high salt conditions
Solution Approach 1:
The patent uses parameter changes by engineering the DNA polymerase to function under high salt conditions (e.g., 1M NaCl or 0.5M KCl). The directed evolution process selected for polymerase variants that maintain catalytic activity and nucleotide incorporation efficiency in high salt environments, enabling reliable sequencing under these conditions
Solution Approach 2:
The patent applies self-service by using the DNA polymerase's own catalytic activity to incorporate modified polyphosphate nucleotides during DNA synthesis. The engineered polymerase autonomously performs the incorporation of tagged nucleotides without requiring additional external enzymes or complex reaction systems, even under high salt conditions
3Productivity
If faster extension rate is achieved, then productivity increases, but nucleotide dissociation rate may increase leading to reduced measurement precision
Solution Approach 1:
The patent applies parameter changes by independently optimizing the extension rate and nucleotide dissociation rate through directed evolution. The engineered polymerases achieve faster extension rates for improved productivity while simultaneously maintaining low nucleotide dissociation rates to ensure measurement precision and single-nucleotide resolution
Data Source
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.