Aromatic Nanopore Mutations for Peptide Discrimination
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Solution Overview
Problem
Current nanopore technologies face challenges in accurately characterizing and discriminating between peptides due to short dwell times and complex physico-chemical structures, especially under low pH conditions, leading to inaccurate detection and characterization of peptides.
Innovation Solution
Engineered proteinaceous nanopores with mutations in the lumen-facing recognition region, specifically introducing aromatic amino acids like tyrosine, phenylalanine, or tryptophan, to increase peptide capture frequency and dwell time, enabling improved peptide discrimination and label-free detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nanopores are used for peptide detection, then the detection system is simple, but the dwell time of peptides is short leading to poor discrimination accuracy
Solution Approach 1:
The patent applies parameter changes by modifying the chemical properties of the nanopore lumen through introduction of aromatic amino acids (tyrosine, phenylalanine, or tryptophan) at specific positions. These parameter changes in the nanopore composition create enhanced interactions with peptides, increasing dwell time from microseconds to milliseconds and improving discrimination accuracy between similar peptides.
Solution Approach 2:
The aromatic amino acids introduced in the nanopore lumen act as intermediaries that facilitate stronger interactions with peptide analytes. These intermediary aromatic residues provide pi-stacking and hydrophobic interactions that temporarily retain peptides in the detection zone, enabling more accurate characterization without requiring complex external trapping mechanisms.
2Productivity
If engineered nanopores with aromatic amino acids are used, then peptide capture frequency and dwell time increase, but the nanopore structure becomes more complex
Solution Approach 1:
The patent applies local quality by introducing aromatic amino acids only at specific lumen-facing positions within the nanopore structure, rather than modifying the entire nanopore. This localized modification at key interaction sites (such as positions 10, 13, 17, and 20 in actinoporins) enhances peptide capture frequency and dwell time while maintaining the overall simplicity and natural folding of the nanopore structure.
Solution Approach 2:
The invention changes specific compositional parameters of the nanopore by substituting certain amino acid residues with aromatic variants at defined positions. This targeted parameter change approach increases peptide capture efficiency without requiring complete redesign of the nanopore architecture, thus improving productivity with minimal increase in structural complexity.
3Measurement precision
If conventional nanopores are used under low pH conditions, then the system is simple to operate, but peptide detection accuracy deteriorates due to fast translocation
Solution Approach 1:
The patent converts the harmful effect of low pH conditions (which normally cause fast peptide translocation and poor detection) into a beneficial interaction regime. By introducing aromatic amino acids in the lumen, the system exploits low pH to enhance electrostatic and hydrophobic interactions between the aromatic residues and peptides, transforming the fast translocation problem into an opportunity for enhanced specific interactions that slow down peptides naturally.
Solution Approach 2:
The invention changes the chemical parameter composition of the nanopore lumen by incorporating aromatic amino acids that have enhanced interaction capabilities under low pH conditions. This parameter change allows the system to maintain slow, detectable translocation speeds at low pH where conventional nanopores would exhibit fast, undetectable translocation, thereby improving detection accuracy without requiring pH adjustment.
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 nanopores enhance peptide recognition and capture, allowing for real-time, single-molecule analysis and identification, with improved accuracy and resolution, particularly under low pH conditions.
Implementation Method 1
The lumen-facing recognition region of the pore comprises one or more aromatic amino acid residues, such as tyrosine, phenylalanine, or tryptophan, which are known to interact with peptide analytes through pi-stacking and hydrophobic interactions
Implementation Method 2
The lumen-facing recognition region of the pore comprises one or more aromatic amino acid residues, such as tyrosine, phenylalanine, or tryptophan, which are known to interact with peptide analytes through pi-stacking and hydrophobic interactions
Implementation Method 3
passing the polypeptide through the pore, wherein increasing the net aromaticity of the pore decreases the translocation speed of the polypeptide through the pore
Implementation Method 4
subjecting the nanopore to an electric field such that the analyte electrophoretically and/or electroosmotically translocates through the nanopore
Implementation Method 5
increasing the net aromaticity of the pore by substituting one or more non-aromatic amino acids with one or more aromatic amino acids... decreases the translocation speed of the polypeptide through the pore
Data Source
AI summary
The invention relates to the field of genetically engineered nanopores and the use thereof in analyzing biopolymers and other (biological) compounds. Provided is a proteinaceous nanopore comprising a mutant pore-forming toxin, or a pore-forming fragment thereof, wherein the lumen-facing recognition region of the pore-forming protein or fragment thereof comprises one or more substitution(s) of lumen-facing amino acid(s) in the recognition region corresponding to amino acids 10-20 of Fragaceatoxin C (FraC), to a natural or non-natural aromatic amino acid residue.