Mutant Alpha-Hemolysin Pore for Stochastic Nucleotide Sequencing
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Solution Overview
Problem
Current DNA or RNA sequencing technologies are slow and expensive due to reliance on amplification techniques and high quantities of specialist chemicals, necessitating the development of rapid and cost-effective methods for nucleotide detection.
Innovation Solution
A mutant α-HL pore with a molecular adaptor covalently attached near residue 139 is used for stochastic sensing, allowing for the detection and discrimination of nucleotides, enabling efficient sequencing of nucleic acids like DNA or RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If amplification techniques are used for DNA sequencing, then the quantity of nucleic acid can be increased, but the cost and time required increase significantly
Solution Approach 1:
The invention extracts and detects individual nucleotides directly without requiring amplification. The stochastic sensor detects single nucleotide binding events, eliminating the need for amplification techniques while maintaining detection sensitivity. This directly resolves the contradiction by removing the time-consuming amplification step while preserving the ability to detect nucleic acids.
Solution Approach 2:
The invention replaces the mechanical amplification process with a direct stochastic sensing mechanism. Instead of using amplification to increase signal strength, the system uses single-molecule detection with high sensitivity to detect individual nucleotides, substituting the amplification mechanism with direct detection.
2Quantity of substance
If amplification techniques are used for DNA sequencing, then the quantity of nucleic acid can be increased, but the cost increases due to high quantities of specialist chemicals required
Solution Approach 1:
The invention extracts and detects individual nucleotides directly without requiring amplification. The stochastic sensor detects single nucleotide binding events, eliminating the need for amplification techniques while maintaining detection sensitivity. This directly resolves the contradiction by removing the time-consuming amplification step while preserving the ability to detect nucleic acids.
Solution Approach 2:
The invention uses a reusable stochastic sensor that can detect multiple nucleotides sequentially without requiring expensive amplification reagents. The sensor maintains sensitivity for single-molecule detection, replacing expensive consumable chemicals with a durable, reusable detection platform.
3Measurement precision
If non-covalently attached molecular adaptors are used, then the pore can detect nucleotides, but the attachment stability and positioning precision are reduced
Solution Approach 1:
The invention uses a covalently attached molecular adaptor as an intermediary between the pore and the nucleotide. This adaptor maintains the detection capability while providing stable, permanent attachment to the pore structure, resolving the contradiction between detection functionality and attachment stability.
4Measurement precision
If the adaptor is positioned precisely at residue 139, then the nucleotide detection sensitivity is maximized, but the manufacturing complexity increases
Solution Approach 1:
The invention incorporates the adaptor attachment site into the pore structure during the protein folding process. The hydrophobic interior of the pore provides a pre-formed binding site that naturally positions the adaptor at residue 139, eliminating the need for post-translational positioning and reducing manufacturing complexity while maintaining precision.
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 α-HL pore demonstrates high sensitivity in distinguishing between nucleotides, facilitating rapid and cost-effective sequencing by reducing the need for large volumes of nucleic acid and reagents, and functioning under various conditions including low salt concentrations.
Implementation Method 1
Stochastic detection is an approach to sensing that relies on the observation of individual binding events between nucleotide molecules and a receptor. Stochastic sensors can be created by placing a single pore of nanometer dimensions in an insulating membrane and measuring voltage-driven ionic transport through the pore in the presence of nucleotide molecules. The frequency of occurrence of fluctuations in the current reveals the concentration of an nucleotide that binds within the pore.
Implementation Method 2
measuring voltage-driven ionic transport through the pore in the presence of nucleotide molecules
Implementation Method 3
a different strategy was discovered, which utilized non-covalently attached molecular adaptors, notably cyclodextrins (Gu, L.-Q., Braha, O., Conlan, S., Cheley, S., and Bayley, H. (1999) Nature 398, 686-690)
Implementation Method 4
A mutant α-HL pore with a molecular adaptor covalently attached near residue 139 is used for stochastic sensing
Data Source
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AI summary
The invention relates to a mutant a-hemolysin (a-HL) pore which is useful for detecting one or more nucleotides by stochastic sensing. The pore is particularly useful for sequencing DNA or RNA. A molecular adaptor that allows detection of the nucleotide(s) is covalently attached to the pore. The pore is specifically modified to facilitate positioning of the adaptor and may be modified to facilitate covalent attachment.