Modified Dda Helicases for Stepwise Nanopore Sequencing
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Solution Overview
Problem
Existing polynucleotide sequencing technologies are slow and expensive due to reliance on amplification techniques and the need for high quantities of fluorescent chemicals, limiting their applicability across various applications.
Innovation Solution
The use of modified Dda helicases with specific mutations in domains such as the tower, pin, and RecA-like motor domains to control the movement of polynucleotides through a pore, enabling direct electrical biosensing and sequencing without the need for amplification and fluorescent chemicals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If amplification techniques and fluorescent chemicals are used for polynucleotide sequencing, then sequencing accuracy can be maintained, but the process becomes slow and expensive
Solution Approach 1:
The invention extracts and eliminates the requirement for amplification techniques and fluorescent chemicals from the sequencing process. By using nanopore technology with modified Dda helicases, the system directly sequences polynucleotides in their native state, removing the need for these additional substances and steps, thereby achieving faster and more cost-effective sequencing.
Solution Approach 2:
The invention replaces the chemical-based detection system (fluorescent chemicals) with an electrical detection system (nanopore current measurement). The modified Dda helicase controls polynucleotide movement through the nanopore, and nucleotide identity is determined by electrical current changes, substituting mechanical/electrical principles for chemical methods.
2Manufacturing precision
If modified Dda helicases with multiple domain mutations are used to control polynucleotide movement, then movement control precision is improved, but protein structure complexity increases
Solution Approach 1:
The invention applies local quality by introducing specific mutations only in certain domains of the Dda helicase protein (tower domain, pin domain, RecA-like motor domains) while leaving other domains unchanged. This targeted approach improves polynucleotide movement control precision in specific functional regions without unnecessarily complicating the entire protein structure.
Solution Approach 2:
The helicase protein is segmented into functional domains (tower domain, pin domain, RecA-like motor domains), and mutations are independently introduced into specific segments. This allows precise control of polynucleotide movement by modifying only the necessary domains while maintaining the overall protein architecture and reducing unnecessary complexity.
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 allows for rapid and cost-effective polynucleotide sequencing by controlling the movement of polynucleotides through nanopores, providing a more efficient and economical method for characterizing target polynucleotides.
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
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
Data Source
AI summary
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Dda helicase. The helicase controls the movement of the target polynucleotide through the pore. The invention also relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.