Modified Archaeal Family B Polymerases for Reversible Terminators
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Solution Overview
Problem
Current DNA polymerases have difficulty incorporating modified nucleotides with reversible terminators efficiently, leading to interference from residual spacer arms and low incorporation fidelity, necessitating a polymerase that is tolerant of these modifications and maintains stability and high fidelity.
Innovation Solution
Modified Archaeal family B polymerases, derived from Pyrococcus abyssi, with reduced exonuclease activity and specific amino acid sequences, are used to enhance the incorporation of modified nucleotides into nucleic acid sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA polymerases are modified to increase incorporation rates of nucleotide analogues, then incorporation efficiency improves, but misincorporation errors increase and fidelity decreases
Solution Approach 1:
The patent applies local quality by making specific amino acid changes only at the motif A region (positions 409-411 in P. abyssi polymerase) while maintaining the rest of the enzyme structure intact. This localized modification allows the polymerase to accommodate reversible terminators without compromising overall enzyme stability or increasing misincorporation errors, thus resolving the contradiction between incorporation rate and fidelity.
Solution Approach 2:
The patent changes specific parameters (amino acid sequences at motif A) to optimize the polymerase for incorporating modified nucleotides. By carefully selecting and mutating specific residues at positions 409-411, the enzyme's kinetics are adjusted to improve incorporation rates of nucleotide analogues while maintaining high fidelity, thereby resolving the trade-off between productivity and reliability.
2Productivity
If polymerases are engineered to lack 3'-5' exonuclease activity, then incorporation of modified nucleotides improves, but error correction capability is lost
Solution Approach 1:
The patent maintains exonuclease activity by making localized changes only at motif A while preserving the exonuclease domain's integrity. This allows the polymerase to efficiently incorporate modified nucleotides through the modified motif A region while retaining the ability to proofread and correct errors through the intact exonuclease activity, thus resolving the contradiction between incorporation efficiency and error correction.
3Adaptability or versatility
If modified nucleotides with reversible terminators are used, then sequencing by synthesis is enabled, but residual spacer arms interfere with subsequent nucleotide incorporation
Solution Approach 1:
The patent makes localized amino acid changes at motif A that specifically enhance the polymerase's ability to handle reversible terminators. This local modification allows the enzyme to accommodate the residual spacer arms from cleavable linkers without interference, enabling smooth continuation of nucleotide incorporation during sequencing by synthesis while maintaining adaptability to the methodology.
Solution Approach 2:
The patent modifies specific parameters (amino acid sequences at motif A) to optimize the polymerase for sequencing by synthesis applications. These parameter changes enable the enzyme to tolerate residual spacer arms and maintain high incorporation rates throughout the sequencing process, resolving the contradiction between adaptability to SBS and ease of operation.
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
These polymerases demonstrate improved incorporation rates and stability, reducing misincorporation errors and accommodating modified nucleotides with reversible terminators, suitable for sequencing by synthesis processes.
Implementation Method 1
DNA-polymerases add nucleotide triphosphate (dNTP) residues to the 3'-end of the growing DNA chain, using a complementary DNA as template
Implementation Method 2
DNA polymerases have been engineered such that they lack 3'-5' exonuclease activity (designated exo-)
Data Source
AI summary
Provided herein are modified Archaeal family B polymerases derived from the Archaeal microorganism Pyrococcus abyssi that exhibit improved incorporation of nucleotide analogues utilized in DNA sequencing.