Modified SSB C-Terminal Charge for Nanopore Sequencing Flow
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Solution Overview
Problem
Existing polynucleotide sequencing technologies are slow and expensive due to reliance on amplification techniques and require high quantities of fluorescent chemicals, and nanopore sequencing is hindered by pore blockage that reduces duty cycle and data output.
Innovation Solution
Utilizing single-stranded binding proteins (SSBs) with modified or uncharged C-terminal regions to prevent secondary structure formation and act as molecular brakes, allowing polynucleotides to pass through nanopores without blocking, enabling high-throughput sequencing by maintaining a high duty cycle.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If SSB with negatively charged C-terminal region is used to bind polynucleotide, then secondary structure formation is prevented, but pore blockage occurs reducing duty cycle
Solution Approach 1:
The invention modifies only the C-terminal region of the SSB protein to reduce its net negative charge, while preserving the DNA-binding functionality of the rest of the protein. This localized modification allows the SSB to maintain its ability to prevent secondary structure formation while eliminating the harmful pore blockage effect caused by the overly negative C-terminal region.
Solution Approach 2:
The invention changes the charge parameter of the C-terminal region of the SSB protein by modifying amino acid residues (e.g., replacing negatively charged residues with neutral or positively charged ones). This parameter change transforms the SSB from a pore-blocking molecule to a pore-friendly molecule that still performs its stabilizing function.
2Ease of operation
If conventional SSB is used to control polynucleotide movement, then sequencing is enabled, but pore blockage reduces sequencing efficiency
Solution Approach 1:
The modification is localized to the C-terminal region of the SSB, leaving the DNA-binding and movement-control functionalities intact. This allows the SSB to continue controlling polynucleotide movement through the pore while the modified C-terminal region no longer causes pore blockage, thereby maintaining ease of operation while improving productivity.
3Measurement precision
If amplification techniques are used for polynucleotide sequencing, then signal detection is improved, but cost and time increase
Solution Approach 1:
The invention extracts and eliminates the unnecessary amplification step from the sequencing workflow by enabling direct nanopore sequencing. The modified SSB allows sufficient signal detection without amplification, removing the time-consuming and costly amplification process while maintaining measurement precision.
4Measurement precision
If high quantity of fluorescent chemicals are used for signal detection, then detection sensitivity is improved, but cost increases
Solution Approach 1:
The invention eliminates the need for large quantities of fluorescent chemicals by using the modified SSB-enhanced nanopore method for direct detection. The electrical signal changes detected during nanopore sequencing provide sufficient sensitivity without requiring expensive fluorescent labeling reagents.
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
Prevents pore blockage, ensuring high data output and effective sequencing by maintaining a high duty cycle, thereby improving the efficiency and cost-effectiveness of nanopore-based sequencing.
Implementation Method 1
certain SSBs may be used, for example, to prevent a target polynucleotide from forming secondary structure or as a molecular brake when the polynucleotide is characterized
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
Nanopore detection of the nucleotide gives a current change of known signature and duration
Implementation Method 4
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 method of characterising a target polynucleotide using a single-stranded binding protein (SSB). The SSB is either an SSB comprising a carboxy-terminal (C-terminal) region which does not have a net negative charge or a modified SSB comprising one or more modifications in its C-terminal region which decreases the net negative charge of the C-terminal region.