Polymerase Variants for Nanopore Sequencing Accuracy
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Solution Overview
Problem
Current DNA polymerases used in recombinant DNA technologies face challenges in sequencing accuracy due to rapid DNA strand movement through nanopores, leading to background noise and difficulty in achieving single-nucleotide resolution, particularly when incorporating modified nucleotides under high salt concentrations.
Innovation Solution
Development of modified DNA polymerases with specific amino acid alterations identified through directed evolution, which enhance enzyme activity, fidelity, processivity, elongation rate, stability, and solubility, allowing for improved incorporation of polyphosphate nucleotides and enhanced sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If DNA polymerase is used in nanopore sequencing, then DNA sequencing can be performed, but the DNA strand moves rapidly through the nanopore causing background noise and difficulty in achieving single-nucleotide resolution
Solution Approach 1:
The patent introduces detectable tags on nucleotides as intermediary elements that slow down the DNA strand movement through the nanopore. These tags act as mediators between the polymerase and the nanopore detection system, allowing sufficient time for detection while maintaining sequencing functionality. The tags create a physical barrier that reduces the transit speed of the DNA strand through the pore.
Solution Approach 2:
The patent modifies the physical and chemical parameters of the nucleotides by adding detectable tags, which changes the interaction dynamics between the DNA strand and the nanopore. This parameter change slows down the translocation rate, allowing for improved measurement precision. The tags alter the electrical, steric, or chemical properties that govern nanopore detection.
2Productivity
If wild-type polymerase is used, then basic polymerization function is maintained, but incorporation of modified polyphosphate nucleotides is inefficient under high salt concentrations
Solution Approach 1:
The patent applies directed evolution to change the biochemical parameters of the polymerase enzyme, specifically optimizing its activity under high salt concentrations. The evolved polymerase variants have modified amino acid sequences that enhance their ability to incorporate modified polyphosphate nucleotides in high salt conditions, where wild-type enzymes fail.
Solution Approach 2:
The polymerase enzyme is engineered to self-optimize its function for specific applications through directed evolution. The enzyme naturally evolves to preferentially incorporate modified nucleotides under the desired reaction conditions (high salt), making the system self-adapting to the required performance characteristics.
3Measurement precision
If detectable tags are added to nucleotides to improve sequencing detection, then single-nucleotide resolution can be achieved, but the complexity of the sequencing system increases
Solution Approach 1:
The detectable tags are designed to serve multiple functions simultaneously: they slow down DNA translocation through the nanopore, provide detectable signals for sequencing, and maintain compatibility with the polymerase incorporation process. This multi-functionality reduces the need for separate components, thereby managing system complexity while achieving high measurement precision.
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 modified DNA polymerases demonstrate increased sequencing accuracy and stability, enabling efficient incorporation of modified nucleotides and improved read capabilities under high salt conditions, addressing the limitations of wild-type enzymes in nanopore sequencing.
Implementation Method 1
DNA polymerases are a family of enzymes that use single-stranded DNA as a template to synthesize the complementary DNA strand
Implementation Method 2
Many DNA polymerases have 3′→5′ exonuclease activity. These polymerases can recognize an incorrectly incorporated nucleotide and the 3′→5′ exonuclease activity of the enzyme allows the incorrect nucleotide to be excised (this activity is known as proofreading)
Data Source
AI summary
Described herein is a variant pol6 polymerase having at least one mutation selected from H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, 1550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.