Nanopore Polypeptide Characterization Using Polynucleotide-Guided Transport
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Solution Overview
Problem
Current methods for characterizing polypeptides, such as mass spectrometry and Edman degradation, are inefficient and unsuitable for single molecule analysis, particularly in distinguishing neighboring residues and processing fragile molecules without fragmentation.
Innovation Solution
Conjugating a target polypeptide to a polynucleotide and using a polynucleotide-handling protein to control the movement of the conjugate through a nanopore, allowing for single molecule characterization by measuring current signatures as the conjugate moves through the nanopore.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used to characterize polypeptides, then bulk information can be obtained, but single molecule analysis is not possible and neighboring residues cannot be distinguished
Solution Approach 1:
The patent uses a polynucleotide as an intermediary carrier to attach the polypeptide to a nanopore. The polynucleotide serves as a mediator that enables the polypeptide to be transported through the nanopore in a controlled manner, allowing single-molecule characterization while maintaining the ability to process multiple samples through parallel nanopore arrays.
Solution Approach 2:
The patent replaces traditional mechanical/chemical characterization methods (mass spectrometry, Edman degradation) with a physical nanopore sensing approach. The nanopore creates an ionic current that is modulated by the passing polypeptide, enabling direct electrical detection of single molecules without requiring ionization or chemical degradation steps.
2Measurement precision
If Edman degradation is used to sequence polypeptides, then residue-by-residue sequencing is achieved, but the process is slow and involves costly reagents
Solution Approach 1:
The nanopore sensing method enables continuous monitoring of the polypeptide as it passes through the nanopore. The ionic current modulation occurs in real-time as each residue interacts with the nanopore field, allowing sequential identification of amino acids without the time-consuming step-by-step chemical degradation required by Edman sequencing.
Solution Approach 2:
The patent changes the detection parameter from chemical residue identification (Edman degradation) to electrical current modulation (nanopore sensing). By measuring changes in ionic current frequency and amplitude, the system can identify amino acid sequences rapidly without requiring chemical reagents or prolonged incubation times.
3Measurement precision
If mass spectrometry is used, then polypeptide characterization is possible, but fragile molecules may fragment and contaminants affect results
Solution Approach 1:
The polynucleotide acts as a protective intermediary that attaches to the polypeptide and facilitates its transport through the nanopore. This intermediary connection allows fragile polypeptides to be handled and moved without the harsh ionization conditions that cause fragmentation in mass spectrometry, while the nanopore's physical structure filters out contaminants.
Solution Approach 2:
The patent replaces the violent ionization mechanism of mass spectrometry with a gentle electrical field interaction in the nanopore. The polypeptide is characterized through subtle modulations of ionic current caused by its passage through the nanopore, avoiding the high-energy processes that fragment fragile molecules and allowing contaminants to be physically excluded from the measurement.
4Measurement precision
If conventional nanopore sensing is used without polynucleotide conjugation, then polypeptide analysis is possible, but controlled movement through the nanopore is difficult
Solution Approach 1:
The polynucleotide serves as a navigational intermediary that guides the polypeptide through the nanopore. The polynucleotide's interaction with the nanopore's electrical field creates a controlled transport mechanism, allowing the polypeptide to pass through the nanopore in a regulated manner rather than randomly diffusing, thereby improving measurement precision.
Solution Approach 2:
The patent replaces uncontrolled passive diffusion with an active electrical field-based transport system. The nanopore's electrical field interacts with the polynucleotide's charge to create a directed force that moves the polypeptide through the nanopore in a controlled manner, improving ease of operation and measurement consistency.
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
Enables rapid and accurate characterization of polypeptides at the single molecule level, overcoming the limitations of existing techniques by providing high fidelity and avoiding amplification bias.
Implementation Method 1
measuring voltage-driven ion currents through the pore in the presence of analyte molecules. 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.
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.