Mutant CsgG Pores for Low-Variance Nucleotide Sequencing
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Solution Overview
Problem
Existing nanopore sequencing technologies face challenges in achieving rapid and cost-effective nucleic acid sequencing due to high variance in current states and difficulty in discriminating between nucleotides, requiring improvements in nucleic acid movement and nucleotide discrimination.
Innovation Solution
Utilization of mutant CsgG monomers, specifically with substitutions and deletions such as R192D, R97W, and K94Q, to enhance nucleotide discrimination and reduce current variance, resulting in improved signal-to-noise ratio and easier nucleotide capture.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mutant hemolysin pores are used to improve nucleotide discrimination, then current range is improved, but variance of current states increases
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues in the CsgG pore (such as R192D, R97W, K94Q substitutions) to optimize the electrical characteristics. These parameter changes in the pore structure directly affect the current range and variance, resolving the contradiction by finding optimal amino acid compositions that provide both wide current range for discrimination and low variance for reliability.
Solution Approach 2:
The patent implements local quality by making targeted mutations at specific locations within the pore structure rather than uniform modifications. By selectively changing amino acids at particular positions (e.g., residues in the selectivity filter or vestibule regions), the invention achieves localized optimization that improves nucleotide discrimination while controlling variance in specific functional regions of the pore.
2Measurement precision
If more nucleotides contribute to observed current, then signal strength increases, but direct relationship between current and polynucleotide sequence becomes challenging
Solution Approach 1:
The patent applies segmentation by designing the pore to have distinct functional regions that process different aspects of the polynucleotide. The mutant CsgG pore creates segmented current signatures where different nucleotide positions contribute differently to the overall signal, allowing the system to maintain strong signals while preserving sequence information through structured current patterns that can be deconvoluted.
Solution Approach 2:
The invention uses another dimension by transitioning from considering only the magnitude of current block to analyzing multiple dimensions of the current signature including duration, variance, and temporal dynamics. This dimensional expansion allows the system to extract sequence information even when multiple nucleotides contribute to the current, as each nucleotide position contributes unique information across different temporal and statistical dimensions.
3Ease of manufacture
If wild-type CsgG is used, then expression is straightforward, but nucleotide discrimination capability is insufficient
Solution Approach 1:
The patent maintains ease of manufacture by using the CsgG backbone which is already known to be expressible, while applying parameter changes through site-directed mutagenesis of specific amino acid residues. This approach preserves the favorable expression properties of wild-type CsgG while enhancing nucleotide discrimination through targeted mutations that optimize the pore's electrical characteristics without affecting overall protein folding or membrane insertion.
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 mutant CsgG pores provide enhanced nucleotide discrimination and reduced variance, facilitating more accurate and efficient nucleic acid sequencing with increased throughput and expression yield.
Implementation Method 1
measuring voltage-driven ionic transport through the pore in the presence of analyte molecules
Implementation Method 2
measuring voltage-driven ionic transport through the pore
Data Source
AI summary
The invention relates to mutant forms of CsgG. The invention also relates to analyte detection and characterisation using CsgG.