Engineered Polymerase Motif Modifications for Sequencing Accuracy
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Solution Overview
Problem
Current sequencing technologies face challenges in accurately discriminating between cognate and non-cognate nucleotides during DNA sequencing, particularly in reducing binary complex formation and stabilizing ternary complexes, which affects the accuracy and efficiency of sequencing methods like Sequencing By Binding™.
Innovation Solution
Engineered DNA polymerases with modifications in specific motifs (A and B) are developed, which exhibit improved enzymatic properties such as increased accuracy, stability, and reduced polymerization error rates, enhancing the discrimination between cognate and non-cognate nucleotides by forming stable ternary complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If naturally occurring DNA polymerases are used for sequencing, then the basic polymerization function is maintained, but the discrimination between cognate and non-cognate nucleotides is insufficient leading to higher error rates
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the polymerase enzyme structure (particularly in motifs A and B) to alter the enzyme's binding affinity and catalytic properties. These parameter changes in the enzyme's molecular structure enable improved discrimination between cognate and non-cognate nucleotides while maintaining polymerization function, directly resolving the contradiction between measurement precision and reliability
Solution Approach 2:
The patent applies local quality by making targeted modifications to specific regions (motifs A and B) of the polymerase enzyme rather than altering the entire enzyme structure. This localized approach allows optimization of nucleotide binding and discrimination functions in specific areas while preserving the overall polymerization capability, thereby improving nucleotide discrimination accuracy without compromising polymerization reliability
2Strength
If binary complex formation is increased to enhance polymerase-nucleotide binding, then binding affinity is improved, but specificity for cognate nucleotides decreases leading to higher error rates
Solution Approach 1:
The patent modifies parameters of the polymerase enzyme (amino acid sequences in motifs A and B) to achieve optimal binding affinity while maintaining high specificity. The engineered polymerases exhibit altered binding parameters that favor cognate nucleotide recognition, resolving the contradiction between binding strength and specificity by optimizing molecular interaction parameters
Solution Approach 2:
The patent employs feedback mechanisms where the engineered polymerase structure provides steric and electrostatic feedback during nucleotide binding. The modified motifs create a binding environment that naturally favors cognate nucleotides through structural feedback, ensuring that high binding affinity correlates with high specificity rather than reducing it
3Measurement precision
If ternary complex stability is increased to improve sequencing accuracy, then cognate nucleotide identification is enhanced, but the complexity of the sequencing process increases
Solution Approach 1:
The patent extracts and optimizes the critical ternary complex formation step by engineering polymerases with enhanced stability specifically for this intermediate state. By focusing modifications on motifs involved in ternary complex stabilization, the patent improves cognate nucleotide identification without requiring complex additional process steps, thereby enhancing accuracy while minimizing process complexity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The engineered polymerases improve the accuracy and efficiency of DNA sequencing by reducing binary complex formation and increasing the stability of ternary complexes, leading to higher specificity and lower error rates in nucleotide pairing, thereby enhancing the overall sequencing process.
Implementation Method 1
Naturally occurring DNA polymerizing enzymes are responsible for accurately replicating DNA within the cells of an organism. This process involves catalysis at the 3'-end of a growing DNA strand
Data Source
AI summary
Provided are nucleic acids encoding engineered polymerases comprising at least one modification in a motif A and/or at least one modification in a motif B of the polymerase and engineered polymerases encoded by the nucleic acids. Also provided are engineered DNA polymerases comprising a variant of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the variant being at least 80% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and comprising an amino acid substitution at one or more positions selected from the group consisting of L408, Y409, P410, R484, A/L485, and I486. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.