RecD Helicase Nanopore Sequencing High Salt Tolerance
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current polynucleotide sequencing and identification technologies are slow and expensive due to reliance on amplification techniques and high quantities of specialist fluorescent chemicals, and they lack efficient methods for characterizing polynucleotides at high salt concentrations.
Innovation Solution
A method utilizing a RecD helicase to control the movement of a target polynucleotide through a transmembrane pore, allowing for characterisation and sequencing by measuring characteristics as the polynucleotide moves through the pore, particularly effective at high salt concentrations and capable of moving in both directions relative to the applied voltage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amplification techniques and fluorescent chemicals are used for polynucleotide sequencing, then detection sensitivity is improved, but cost and complexity increase
Solution Approach 1:
The invention extracts and eliminates the need for amplification techniques and fluorescent chemicals by using a nanopore-based direct detection method. The RecD helicase controls polynucleotide translocation through the pore, enabling direct electrical detection of nucleotides without requiring complex amplification or fluorescent labeling systems.
Solution Approach 2:
The invention replaces the chemical-based detection system (fluorescent chemicals and amplification reactions) with an electrical measurement system. The nanopore detects nucleotides through changes in ionic current as they pass through the pore, substituting chemical fluorescence detection with electrical signal measurement.
2Measurement precision
If high salt concentration is used for characterizing polynucleotide, then signal-to-noise ratio is improved, but helicase function is typically inhibited
Solution Approach 1:
The invention changes the operational parameters of the RecD helicase to enable function at high salt concentrations (up to 1M NaCl). By optimizing the helicase-pore system and adjusting operational conditions, the helicase maintains its ability to control polynucleotide translocation even in high salt environments that would normally inhibit its activity.
3Productivity
If nanopore detection is used for sequencing, then speed and cost are improved, but control of polynucleotide movement becomes challenging
Solution Approach 1:
The invention introduces the RecD helicase as an intermediary protein that mediates between the applied voltage and the polynucleotide. The helicase binds to the polynucleotide and controls its translocation through the nanopore, preventing uncontrolled movement while enabling steady, measurable passage that maintains high sequencing speed.
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, cost-effective characterization and sequencing of polynucleotides with high salt tolerance, providing a high signal-to-noise ratio and allowing for single-base resolution, thus overcoming the limitations of existing technologies.
Implementation Method 1
The helicase is capable of moving a target polynucleotide in a controlled and stepwise fashion against or with the field resulting from the applied voltage
Implementation Method 2
When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time
Implementation Method 3
When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel
Data Source
AI summary
The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a RecD helicase. The helicase controls the movement of the target polynucleotide through the pore.