Nanopore Polypeptide Characterization via Protein Control
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Solution Overview
Problem
Current methods for characterizing polypeptides, such as mass spectrometry and Edman degradation, are limited by contamination issues, fragmentation of fragile molecules, and the inability to provide single molecule-level data, leading to a need for more accurate and efficient techniques.
Innovation Solution
The method involves forming a conjugate of a target polypeptide with a polynucleotide and using a polynucleotide-handling protein to control the movement of the conjugate with respect to a nanopore, allowing for repeated measurements of the polypeptide as it moves in controlled directions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used to characterise polypeptides, then bulk information can be obtained, but single molecule-level data is not provided and contamination affects results
Solution Approach 1:
The patent segments the polypeptide characterization process by using individual nanopore sensing events to analyze single molecules, rather than bulk analysis. Each nanopore measurement represents an independent segmentation of the sample, allowing single-molecule resolution while maintaining the ability to process multiple molecules through sequential measurements.
Solution Approach 2:
The patent replaces the mechanical/bulk-based mass spectrometry system with an electrical sensing system using nanopores. The nanopore detects polypeptide characteristics through ionic current changes, substituting the mechanical ionization and mass analysis process with an electrical measurement approach that enables single-molecule sensitivity.
2Measurement precision
If Edman degradation is used to sequence polypeptides, then residue-by-residue sequencing is achieved, but the process is slow and requires costly reagents
Solution Approach 1:
The patent substitutes the chemical Edman degradation process with an electrical nanopore sensing system. Instead of using chemical reagents to sequentially cleave and detect amino acids, the system uses electrical current measurements through nanopores to detect polypeptide sequences, dramatically increasing speed while eliminating costly reagent requirements.
Solution Approach 2:
The patent changes the detection parameter from chemical identification (via chromatography or electrophoresis of cleaved residues) to electrical current measurement. This parameter change enables rapid, real-time sequencing without the slow step-by-step chemical degradation process, achieving both high precision and high productivity.
3Measurement precision
If multiple polypeptides are characterised and data aggregated to improve accuracy, then overall accuracy improves, but heterogeneity in samples causes loss of useful information
Solution Approach 1:
The patent segments the analysis by maintaining separate tracking of individual polypeptide molecules through sequential nanopore measurements. Each molecule's characteristics are recorded independently, allowing aggregation of data while preserving heterogeneity information. The segmentation enables both accuracy through multiple measurements and retention of individual molecule differences.
Solution Approach 2:
The patent implements feedback by repeatedly measuring the same polypeptide molecule multiple times through sequential nanopore passages. This repeated measurement provides feedback that improves accuracy for each individual molecule while maintaining the ability to compare differences between molecules, eliminating the need to aggregate and lose heterogeneity data.
4Productivity
If nanopore sensing is used for polypeptide characterisation, then rapid and cheap analysis is enabled, but controlled movement of the conjugate through the nanopore is required for repeated measurements
Solution Approach 1:
The patent introduces a polynucleotide-handling protein as an intermediary to control conjugate movement. This protein mediator binds to the polynucleotide portion of the conjugate and uses its own movement (driven by ATP hydrolysis) to controllably transport the conjugate through the nanopore, enabling repeated measurements without directly controlling the polypeptide itself.
Solution Approach 2:
The patent substitutes direct mechanical control of polypeptide movement with a biochemical approach using polynucleotide-handling proteins. Instead of applying external mechanical force or voltage control to move the conjugate, the system uses enzymatic activity (ATP hydrolysis) to drive protein movement, which in turn controls conjugate translocation through the nanopore.
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
This approach enables accurate characterization of polypeptides at the single molecule level, improving data accuracy and efficiency by allowing multiple readings of the polypeptide as it is moved through the nanopore.
Implementation Method 1
The presence of an analyte inside or near the nanopore will alter the ionic flow through the pore, resulting in altered ionic or electric currents being measured over the channel
Implementation Method 2
A polynucleotide-handling protein is used to control movement of the conjugate with respect to the nanopore
Data Source
AI summary
Provided herein are methods of characterising a target polypeptide as it moves with respect to a nanopore. Also provided are related kits, systems and apparatuses for carrying out such methods.