Mutant Porin Monomer for Nanopore Sequencing Precision
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Solution Overview
Problem
Current nanopore sequencing technologies face limitations in sequencing precision due to the stability and consistency of interactions between porin and rate-controlling proteins, leading to errors in homopolymeric oligonucleotide regions and the need for improved nanoporins to enhance sequencing accuracy and resolution.
Innovation Solution
A mutant porin monomer with specific amino acid mutations at positions corresponding to A74, P75, G76, N77, A78, T79, N80, and F81 is developed, forming a protein pore that improves the interaction interface with rate-controlling proteins, enhancing sequencing data consistency and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If wild-type porin is used for nanopore sequencing, then the sequencing can be performed with basic functionality, but the sequencing precision and consistency are insufficient due to unstable interaction interface with rate-controlling proteins
Solution Approach 1:
The patent applies local quality by introducing specific amino acid mutations at positions 74-83 in the porin sequence, which corresponds to the interaction interface region. These localized mutations (such as A74P, P75Y, G76F, N77P, A78A, T79P, N80A, F81P) specifically enhance the interaction stability with rate-controlling proteins without altering the overall porin structure or function, thereby resolving the contradiction between maintaining basic functionality and improving sequencing precision through enhanced reliability.
Solution Approach 2:
The patent employs parameter changes by modifying the amino acid sequence parameters at specific positions in the porin molecule. By changing the chemical properties and spatial configuration of amino acids at the interaction interface (positions 74-83), the patent optimizes the binding affinity and stability with rate-controlling proteins, leading to improved sequencing precision and data consistency while preserving the fundamental nanopore sequencing capability.
2Measurement precision
If conventional porin is used, then the nanopore sequencing can be implemented, but errors occur in homopolymeric oligonucleotide regions due to insufficient resolution
Solution Approach 1:
The patent applies local quality by introducing specific amino acid mutations at positions 74-83 in the porin sequence, which corresponds to the interaction interface region. These localized mutations (such as A74P, P75Y, G76F, N77P, A78A, T79P, N80A, F81P) specifically enhance the interaction stability with rate-controlling proteins without altering the overall porin structure or function, thereby resolving the contradiction between maintaining basic functionality and improving sequencing precision through enhanced reliability.
Solution Approach 2:
The patent employs parameter changes by modifying the amino acid sequence parameters at specific positions in the porin molecule. By changing the chemical properties and spatial configuration of amino acids at the interaction interface (positions 74-83), the patent optimizes the binding affinity and stability with rate-controlling proteins, leading to improved sequencing precision and data consistency while preserving the fundamental nanopore sequencing capability.
3Reliability
If the porin structure is optimized for better protein interaction, then sequencing data consistency improves, but the complexity of porin design and characterization increases
Solution Approach 1:
The patent applies local quality by introducing specific amino acid mutations at positions 74-83 in the porin sequence, which corresponds to the interaction interface region. These localized mutations (such as A74P, P75Y, G76F, N77P, A78A, T79P, N80A, F81P) specifically enhance the interaction stability with rate-controlling proteins without altering the overall porin structure or function, thereby resolving the contradiction between maintaining basic functionality and improving sequencing precision through enhanced reliability.
Solution Approach 2:
The patent employs parameter changes by modifying the amino acid sequence parameters at specific positions in the porin molecule. By changing the chemical properties and spatial configuration of amino acids at the interaction interface (positions 74-83), the patent optimizes the binding affinity and stability with rate-controlling proteins, leading to improved sequencing precision and data consistency while preserving the fundamental nanopore sequencing capability.
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 porin monomer and protein pore demonstrate improved sequencing precision and resolution, effectively distinguishing between different nucleotides, including methylated and unmethylated nucleotides, with enhanced signal characteristics and separation efficiency.
Implementation Method 1
utilizes a nanopore capable of providing an ion current channel
Implementation Method 2
enable the single-stranded nucleic acid molecule to pass through the nanopore driven by electrophoresis
Data Source
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AI summary
The present invention belongs to the technical field of characterization of target analyte properties, and particularly provides a mutant of a porin monomer, a protein pore comprising same, and use thereof in the detection of a target analyte, wherein an amino acid of the mutant of the porin monomer comprises mutations at one or more of positions corresponding to A74, P75, G76, N77, A78, T79, N80, and F81 of SEQ ID NO: 1.