Nanopore Sequencing Tethers Prevent Rehybridization
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Solution Overview
Problem
Current nanopore sequencing technologies face challenges in achieving rapid and accurate sequencing of nucleic acids due to issues like sample preparation time, strand rehybridization, and reduced sequencing accuracy, particularly with hairpin-linked polynucleotides.
Innovation Solution
The use of adaptors with leaders and tethers in a nanopore system, where a concentration of tethers is added to a well to promote the sequential translocation of non-covalently bound single-stranded nucleic acids through a nanopore, enhancing follow-on sequencing events and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If hairpin-linked polynucleotides are used to ensure sequential translocation, then sequencing completeness is improved, but sample preparation time increases and analyte loss occurs
Solution Approach 1:
The patent extracts the hairpin structure from the polynucleotide and replaces it with a separate tether molecule. The tether is added to the well and binds to the polynucleotide, providing the sequential translocation function without requiring time-consuming hairpin formation during sample preparation. This separation of function reduces preparation time while maintaining sequencing completeness.
Solution Approach 2:
The tether acts as an intermediary molecule that mediates the sequential translocation of polynucleotide strands. Instead of modifying the polynucleotide itself with hairpin structures, the tether serves as a separate agent that binds to the polynucleotide and controls its translocation through the nanopore, simplifying sample preparation.
2Reliability
If hairpin-linked polynucleotides are used to ensure sequential translocation, then sequencing completeness is improved, but valuable analyte is lost
Solution Approach 1:
The patent extracts the sequential translocation function from the polynucleotide structure itself and assigns it to a separate tether molecule. This prevents analyte loss that would occur during hairpin formation and manipulation, as the polynucleotide remains in its native state throughout the process.
Solution Approach 2:
The tether molecules are added to the well and self-assemble with the polynucleotides through binding. This self-organizing process eliminates the need for complex sample preparation steps that could lead to analyte loss, allowing the system to automatically configure the sequential translocation structure.
3Productivity
If strands are separated for translocation through nanopore, then sequencing is enabled, but rehybridization occurs on the trans side altering translocation rate and reducing accuracy
Solution Approach 1:
The patent applies preliminary action by adding tethers to the well before translocation occurs. The tethers bind to the polynucleotides and pre-configures the system to prevent rehybridization on the trans side. This preliminary configuration ensures that when strands separate for translocation, they cannot rehybridize, maintaining constant translocation rates and high sequencing accuracy.
Solution Approach 2:
The tether acts as an intermediary that prevents direct interaction between complementary strands on the trans side of the nanopore. By binding to the polynucleotide and extending into the trans compartment, the tether physically blocks rehybridization while allowing sequential translocation to proceed at constant rates.
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 significantly increases the frequency of follow-on sequencing events, improving sequencing quality and accuracy by ensuring sequential translocation of nucleic acid strands, thereby enhancing the overall sequencing process.
Implementation Method 1
applying a potential to the membrane to promote translocation of the single stranded nucleic acids through the nanopore
Implementation Method 2
adding a plurality of tethers to a well comprising a nanopore disposed in a membrane wherein the concentration of tethers added to the well is at least 100 nM
Data Source
AI summary
Aspects of the disclosure relate to compositions and methods for characterizing nucleic acids using a nanopore. The disclosure is based, in part, on methods for increasing follow-on sequencing of nucleic acid strands. In some embodiments, the methods comprise increasing the concentration of a tethering agent. In some embodiments, the methods comprise use of adaptors having a rigid (or stiffened) leader section. Compositions and systems including, e.g., adaptors for attachment to double-stranded poly nucleotides and/or tethering agents, which can be used in the methods are also provided.