Nanopore Polypeptide Characterization with Controlled Conjugate Translocation
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Solution Overview
Problem
Existing methods for characterizing polypeptides, such as mass spectrometry and Edman degradation, are inefficient and unsuitable for single molecule analysis, and nanopore sensing techniques lack alternatives for improved polypeptide characterization.
Innovation Solution
Conjugating a target polypeptide to a polynucleotide and using a modified polynucleotide-handling protein to control the movement of the conjugate through a nanopore, allowing for single molecule characterization by measuring current signatures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If mass spectrometry is used to characterize polypeptides, then bulk information about the sample can be obtained, but it cannot distinguish differences within individual polypeptide molecules and is affected by contaminants
Solution Approach 1:
The patent extracts and isolates individual polypeptide molecules from the bulk sample for separate analysis. By using nanopore technology, each polypeptide molecule is translocated through the nanopore individually, allowing single-molecule characterization without interference from other molecules or contaminants in the sample.
Solution Approach 2:
The patent introduces a polynucleotide as an intermediary to facilitate the translocation of polypeptides through the nanopore. The polynucleotide acts as a handle or tether that enables controlled movement of the polypeptide through the nanopore, allowing for systematic single-molecule analysis.
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 patent replaces the chemical degradation process of Edman sequencing with a physical translocation method using nanopores. Instead of chemically cleaving and analyzing amino acids sequentially, the polypeptide is physically pulled through the nanopore, and its sequence is determined by measuring changes in ionic current as each residue passes through the pore.
Solution Approach 2:
The patent changes the measurement parameter from chemical detection of cleaved amino acids to electrical detection of ionic current blockage. By monitoring the electrical properties (current blockage patterns) as the polypeptide translocates through the nanopore, rapid sequence determination is achieved without slow chemical reactions.
3Productivity
If nanopore sensing is used to detect polypeptides, then rapid and cheap characterization is possible, but control of polypeptide movement through the nanopore is challenging
Solution Approach 1:
The patent uses a polynucleotide as an intermediary to control polypeptide translocation through the nanopore. The polynucleotide serves as a handle that can be grabbed by motor proteins or subjected to controlled forces, enabling systematic and controllable movement of the attached polypeptide through the nanopore at desired speeds and directions.
Solution Approach 2:
The patent performs preliminary conjugation of the polypeptide to a polynucleotide before translocation. This preliminary action creates a controllable system where the polynucleotide can be used to drive or control the subsequent translocation process through the nanopore, making the operation easier and more reliable.
4Ease of operation
If polynucleotide-handling protein is used to control conjugate movement, then controlled translocation through nanopore is achieved, but the protein may disengage when contacting polypeptide portions
Solution Approach 1:
The patent segments the polynucleotide into distinct functional regions: a control region that interacts with the polynucleotide-handling protein for translocation control, and a conjugation region that remains bound to the polypeptide. This segmentation ensures that when the polynucleotide-handling protein contacts and processes the polynucleotide, it does not cause disengagement from the polypeptide, maintaining binding stability while achieving controlled translocation.
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 by controlling the movement of polypeptide-polynucleotide conjugates through nanopores, providing high fidelity and avoiding amplification bias.
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
the polynucleotide-handling protein is modified by covalently closing a polynucleotide-unbinding opening through which a polynucleotide strand may unbind
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 3A~3D
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.