Mutant CsgG Nanopores for Sharper Nucleotide Discrimination
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Solution Overview
Problem
Existing nanopores for nucleic acid sequencing and molecular sensing face challenges in optimizing dimensions and characteristics, leading to high variance in current states and the need for improved nucleotide discrimination and reduced nucleotide contribution to the observed current.
Innovation Solution
The use of modified CsgG nanopores with specific modifications at positions Tyr51, Asn55, and Phe56 to enhance nucleotide discrimination, reduce variance, and facilitate single-stranded DNA translocation, featuring a more favorable aspect ratio and wider channel opening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nanopores (MspA, alpha-hemolysin, ClyA) are used for nucleic acid sequencing, then sequencing functionality is achieved, but high variance in current states and poor nucleotide discrimination occur
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues (Tyr51, Asn55, Phe56) in the CsgG pore structure to optimize the constriction zone dimensions. These parameter modifications to the pore's physical characteristics directly improve nucleotide discrimination capability while reducing current state variance, resolving the contradiction between measurement precision and reliability.
2Measurement precision
If pore dimensions are optimized for better nucleotide discrimination, then sequencing accuracy improves, but the number of nucleotides contributing to current increases
Solution Approach 1:
The patent applies local quality by creating a specifically optimized constriction zone within the pore structure through targeted mutations at positions 51, 55, and 56. This localized optimization ensures that only nucleotides within the narrowed constriction zone contribute significantly to the current signal, improving discrimination while minimizing the number of contributing nucleotides. The wider channel opening elsewhere maintains favorable aspect ratio without increasing the effective sensing zone.
3Ease of operation
If pore structure is modified to reduce constriction, then translocation of single-stranded DNA is facilitated, but nucleotide discrimination capability decreases
Solution Approach 1:
The patent applies parameter changes by precisely tuning the constriction zone dimensions through specific amino acid substitutions. The mutations create an optimal balance where the constriction is sufficiently narrow to provide good nucleotide discrimination (maintaining measurement precision) while still allowing efficient translocation of single-stranded DNA (maintaining ease of operation). The wider channel opening compensates for the constriction to maintain favorable aspect ratio.
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 CsgG nanopores provide improved nucleotide discrimination, reduced variance in current states, and increased throughput, making nucleic acid sequencing more efficient and cost-effective by minimizing the number of nucleotides contributing to the current.
Implementation Method 1
When an electrical potential is applied across a membrane-bound nanopore, ions flow through the channel. This flow of ions can be measured as an electrical current.
Implementation Method 2
The degree of reduction in ion flow, as measured by the reduction in electrical current, is indicative of the size of the obstruction within, or in the vicinity of, the pore.
Data Source
AI summary
The invention relates to mutant forms of CsgG. The invention also related to analyte detection and characterisation using CsgG.