Modified Dda Helicase and CsgG Pore for Stable Nanopore Sequencing
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Solution Overview
Problem
Existing nanopore sequencing technologies face challenges in accurately controlling the movement of analytes, particularly polynucleotides, leading to errors in sequencing due to variability in dwell times and signal noise, which affects the precision of nucleotide identification.
Innovation Solution
Modified Dda helicases and transmembrane pores, such as CsgG, with specific mutations at positions 55, 114, 156, 177, 210, 221, 350, and 358, are used to control the movement of analytes through the pore, reducing variability in dwell times and enhancing sequencing accuracy by minimizing errors to less than 10%.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If wild-type CsgG pore and Dda helicase are used for nanopore sequencing, then the system can detect and characterise analytes, but the variability in dwell times causes statistical noise and sequencing errors
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at positions 55, 114, 156, 177, 210, 221, 350, and 358 in the Dda helicase sequence. These mutations alter the enzymatic properties of the helicase, specifically reducing variability in dwell times while maintaining translocation speed. The mutant helicases exhibit more consistent kinetic parameters during polynucleotide translocation through the nanopore, thereby reducing statistical noise and improving sequencing accuracy
2Productivity
If the polynucleotide moves quickly through the pore, then sequencing speed is improved, but dwell times become too short to accurately identify nucleotides
Solution Approach 1:
The patent modifies the Dda helicase parameters through targeted amino acid substitutions that optimize the balance between translocation speed and dwell time consistency. The mutant helicases maintain efficient polynucleotide translocation while ensuring that each nucleotide spends a sufficient and consistent time interval in the pore, enabling accurate base calling even at high sequencing speeds
3Measurement precision
If mutations are introduced to improve pore properties, then sequencing accuracy is enhanced, but the complexity of the system increases
Solution Approach 1:
The patent applies local quality by introducing mutations at specific, strategically selected positions (55, 114, 156, 177, 210, 221, 350, and 358) within the Dda helicase structure. Rather than random mutagenesis, these targeted positions were identified to optimize dwell time consistency while minimizing overall structural changes. This localized approach achieves improved sequencing accuracy with a controlled and manageable number of mutations
Data Source
AI summary
The present invention relates to modified Dda helicases which can be used to control the movement of analytes such as polynucleotides. The modified Dda helicases are used in analyte detection and characterisation. The present invention also relates to novel protein pores and their uses in analyte detection and characterisation. The invention particularly relates to an isolated pore complex formed by a CsgG-like pore and a modified CsgF peptide, or a homologue or mutant thereof, thereby incorporating an additional channel constriction or reader head in the nanopore.