MspA Nanopore Trapping for Label-Free Conformation Profiling
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Solution Overview
Problem
Existing nanopore technologies face challenges in accurately resolving the tertiary structures of RNA and conformational changes in proteins due to limitations in resolution, reproducibility, and consistency, as well as the need for chemical labeling and sample pretreatment, which hinder high-resolution discrimination of structurally similar molecules.
Innovation Solution
Utilizing MspA protein nanopores with a vestibule that accommodates but does not translocate analytes, enabling high-resolution characterization through ionic current measurements and machine learning algorithms to distinguish different conformations and interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If solid state nanopores are used for RNA sensing, then the device is robust and easy to manufacture, but the thickness of the nanopore prohibits refined sensing information and limits resolution
Solution Approach 1:
The patent uses biological nanopores with specifically optimized local constriction dimensions (e.g., 1-5 nm diameter) to achieve high-resolution sensing of RNA structures, while maintaining overall pore robustness through protein shell structure
Solution Approach 2:
The patent varies nanopore parameters including pore diameter, constriction length, and surface charge density to optimize both resolution and manufacturing feasibility for different RNA sensing applications
2Reliability
If solid state nanopores are used, then manufacturing is simplified, but geometric reproducibility remains a technical bottleneck reducing sensing consistency
Solution Approach 1:
The patent uses biological templates (protein structures) that naturally self-assemble with atomic precision to create nanopores with highly reproducible geometries, copying the precise structural information from biological blueprints
Solution Approach 2:
The patent optimizes manufacturing parameters including insertion voltage, annealing temperature, and pH conditions to achieve consistent nanopore formation and orientation in membranes
3Adaptability or versatility
If large nanopores are developed to permit passage of large biomolecules, then molecule access is improved, but storage time decreases and spontaneous gating occurs
Solution Approach 1:
The patent employs dynamic control of pore properties through pH, ionic strength, and voltage adjustments to stabilize large nanopores during measurement while maintaining biomolecule passage capability
Solution Approach 2:
The patent modifies operational parameters including buffer composition, applied voltage, and temperature to prevent spontaneous gating and extend stable measurement duration for large biomolecule sensing
4Measurement precision
If chemical labeling or surface immobilization is used for single-molecule techniques, then detection capability is improved, but the requirement for labeling reduces ease of operation
Solution Approach 1:
The patent extracts the detection function from complex labeling procedures by using the nanopore itself as the detection element that directly senses unlabeled biomolecules through their intrinsic physical and chemical properties
Solution Approach 2:
The nanopore system performs self-detection by measuring ionic current blockades and translocation patterns that inherently encode structural information about passing biomolecules, eliminating the need for external labeling agents
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
MspA nanopores provide high-resolution, label-free discrimination of RNA tertiary structures and protein conformations, overcoming limitations of previous nanopore technologies by ensuring consistency and stability, and allowing for simultaneous sensing of proteins with conflicting charge properties.
Implementation Method 1
applying an electrical potential difference between the first conductive liquid medium and the second conductive liquid medium to drive the analyte into the nanopore
Implementation Method 2
measuring an ionic current through the protein nanopore to provide a tested current pattern that contains at least ionic current measured during the analyte is in the vestibule
Data Source
AI summary
A method of characterizing an analyte or the interaction between the analyte and an agent in a nanopore system, wherein the nanopore system comprises a protein nanopore disposed in a membrane that separates a first conductive liquid medium from a second conductive liquid medium, wherein the protein nanopore is MspA, MspA homolog or variant thereof, wherein the analyte has an conformation and the analyte with the conformation can be accommodated in the vestibule of the MspA, the MspA homolog or the variant thereof but cannot translocate through the MspA, the MspA homolog or the variant thereof, the method comprising: i) applying an electrical potential difference between the first conductive liquid medium and the second conductive liquid medium to drive the analyte into the nanopore, and optionally contacting the agent with the analyte; ii) measuring an ionic current through the protein nanopore to provide a tested current pattern that contains at least ionic current measured during the analyte is in the vestibule of the MspA, the MspA homolog or the variant thereof; iii) associating the tested current pattern with at least one characteristic of the analyte or the interaction between the analyte and an agent.