Engineered DNA Polymerases for Accurate Sequencing by Binding
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Solution Overview
Problem
Existing DNA sequencing technologies face challenges in accurately discriminating between cognate and non-cognate nucleotides during sequencing processes, leading to errors and reduced efficiency.
Innovation Solution
Engineered DNA polymerases with modifications in the Motif A region and optionally in the palm and/or exonuclease domains, enhancing accuracy and processivity, are developed to improve nucleotide pairing and reduce errors in sequencing methods like Sequencing By Binding™ and sequencing by synthesis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If natural DNA polymerases are used for sequencing, then the basic polymerization function is maintained, but the accuracy and discrimination between cognate and non-cognate nucleotides is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the polymerase structure (e.g., position 790 in Therminator polymerase, positions 408-410 in Motif A region) to alter the enzyme's binding characteristics. These point mutations change the physical-chemical parameters of the polymerase-nucleotide interaction, enabling improved discrimination between correct and incorrect nucleotides while maintaining polymerization activity.
Solution Approach 2:
The invention implements local quality by making targeted modifications to specific regions of the polymerase molecule, particularly the Motif A region and palm domain. Rather than altering the entire enzyme, localized amino acid substitutions are introduced to enhance nucleotide binding specificity at the active site, while preserving the overall polymerization function of the enzyme.
2Measurement precision
If polymerase modifications are introduced to improve nucleotide binding accuracy, then sequencing precision is enhanced, but the complexity of the polymerase structure increases
Solution Approach 1:
The patent maintains simplicity by applying local quality - only specific amino acid positions are modified (e.g., single point mutations at position 790 or small sets of residues in Motif A). This localized approach enhances nucleotide pairing accuracy without requiring complex structural changes throughout the entire polymerase molecule.
Solution Approach 2:
The invention uses parameter changes through simple amino acid substitutions rather than complex structural redesign. By changing specific residues (e.g., F790L in Therminator), the patent achieves improved nucleotide discrimination with minimal increase in structural complexity, maintaining the overall simplicity of the polymerase architecture.
3Reliability
If engineered polymerases with multiple modifications are used, then sequencing accuracy and processivity are improved, but the ease of manufacture and implementation becomes more difficult
Solution Approach 1:
The patent simplifies manufacture by applying local quality - introducing only essential point mutations at specific positions (e.g., position 790 or Motif A residues) rather than multiple scattered modifications. This focused approach reduces the complexity of genetic engineering, protein expression, and purification processes while achieving the desired improvement in sequencing efficiency.
Solution Approach 2:
The invention uses parameter changes through simple amino acid substitutions that can be easily introduced via site-directed mutagenesis. These minimal parameter changes (single or few residue substitutions) simplify the manufacturing process compared to more complex polymerase engineering approaches, while still achieving improved sequencing performance.
Data Source
AI summary
Provided herein are engineered DNA polymerases comprising modifications improving accuracy and processivity of the polymerase including modifications in the Motif A region, optionally, along with additional modifications in the palm and/or exonuclease domains of the polymerase. Also provided are nucleic acids encoding the engineered DNA polymerases comprising modifications in motif A of the polymerase, optionally, with additional modifications. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.