Nanopore Polypeptide Translocation Control Using Binding Enzymes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional methods for protein sequencing, such as Edman degradation and mass spectrometry, are costly and time-consuming, while nanopore sequencing lacks a suitable enzyme to control the speed of polypeptide passage through the nanopore, hindering efficient and cost-effective polypeptide sequencing.
Innovation Solution
Conjugate a polypeptide to a polynucleotide and use a polynucleotide binding enzyme, such as helicase, to control the movement of the polynucleotide and thereby control the speed of the polypeptide through a nanopore, reading electrical signals to determine the amino acid sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (Edman degradation, mass spectrometry) are used for protein sequencing, then sequencing accuracy is achieved, but cost and time consumption increase significantly
Solution Approach 1:
The patent replaces conventional mechanical/chemical sequencing methods (Edman degradation, mass spectrometry) with a nanopore-based electrical detection system. The polypeptide is driven through a nanopore by a polynucleotide binding enzyme, and its sequence is determined by measuring electrical current changes, achieving both speed and accuracy.
Solution Approach 2:
The patent changes the detection parameter from chemical/physical measurements (mass-to-charge ratio, chemical derivatives) to electrical current measurements. By monitoring current changes as the polypeptide passes through the nanopore, the system achieves rapid sequencing without the time-consuming steps of conventional methods.
2Productivity
If nanopore sequencing is used for polynucleotide, then cost-efficiency and speed are achieved, but lack of suitable enzyme for polypeptide control prevents effective sequencing
Solution Approach 1:
The patent uses a polynucleotide binding enzyme (such as helicase or polymerase) as an intermediary to control polypeptide translocation. The enzyme binds to the polynucleotide and moves it through the nanopore, thereby indirectly controlling the polypeptide's passage speed and enabling effective sequencing.
Solution Approach 2:
The patent adapts the nanopore sequencing system originally designed for polynucleotides to work with polypeptides by using the same enzyme-based translocation mechanism. This multi-functional approach allows the system to sequence both polynucleotides and polypeptides using comparable infrastructure.
3Ease of operation
If polypeptide passes through nanopore without speed control, then simple operation is maintained, but sequencing accuracy and signal quality deteriorate
Solution Approach 1:
The patent employs a self-service translocation mechanism where the polynucleotide binding enzyme automatically moves the polypeptide through the nanopore at a controlled rate. The enzyme's natural movement along the polynucleotide backbone provides inherent speed control without requiring external manipulation or complex control systems.
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 high-throughput, cost-effective, and label-free polypeptide sequencing by controlling the speed of polypeptide passage through a nanopore, allowing for accurate determination of amino acid sequences and identification of mutations or post-translational modifications.
Implementation Method 1
The nanopore sequencing method utilizes electrophoresis technology to drive individual DNA or RNA molecules through a nanopore one by one to implement the sequencing
Implementation Method 2
reading a nanopore current signal to acquire an electrical signal of the polypeptide
Data Source
AI summary
The present invention provides a method for controlling a speed of a polypeptide passing through a nanopore and use thereof in determining an amino acid sequence of a polypeptide. Specifically, the method comprises: conjugating a polynucleotide to the polypeptide to give a polynucleotide-polypeptide conjugate, and applying a voltage across the nanopore in the presence of a polynucleotide binding enzyme to move the conjugate through the nanopore. The polynucleotide binding enzyme controls the movement of the polynucleotide and thereby controls the movement of the conjugated polypeptide in the nanopore, thus controlling the speed of the polypeptide passing through the nanopore.