Nanopore Base Modification Detection Using Pre-Labeled Nucleobases
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Solution Overview
Problem
Existing nanopore sequencing technologies struggle with high false discovery rates and require extensive training data to accurately detect modified nucleotides, such as 5mC, 5hmC, and m6A, due to low contrast between modified and unmodified nucleobases during sequencing.
Innovation Solution
A method involving chemical manipulations to selectively attach a detectable moiety to modified nucleobases, utilizing enzymes like methyltransferases, followed by nanopore analysis to enhance the electrical signal contrast, allowing precise detection of single modified nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If chemical manipulations are used to attach detectable moieties to modified nucleobases, then detection sensitivity and accuracy are improved, but device complexity and process steps increase
Solution Approach 1:
The patent applies preliminary action by performing chemical manipulations to attach detectable moieties to modified nucleobases before nanopore sequencing. This pre-labeling step enhances the electrical contrast of modified nucleotides, enabling more accurate detection during subsequent nanopore analysis. The preliminary chemical modification prepares the sample in advance, improving measurement precision without complicating the core sequencing process.
2Measurement precision
If algorithms are used to detect modified nucleotides, then detection capability is improved, but false discovery rate increases and training data requirements increase
Solution Approach 1:
The patent employs color changes principle by attaching detectable moieties (analogous to fluorescent tags or chromophores) to modified nucleobases. These moieties create distinct electrical signals during nanopore passage, providing clear signal differentiation between modified and unmodified nucleotides. This physical signal enhancement reduces false discoveries by providing unambiguous detection criteria, eliminating the need for complex algorithms and extensive training data.
3Device complexity
If nanopore sequencing is used without chemical manipulation, then device simplicity is maintained, but electrical contrast between modified and unmodified nucleobases is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying the electrical properties of nucleobases through attachment of detectable moieties with different molecular weights and charges. This changes the electrical contrast parameter during nanopore passage, creating more pronounced current deviations for modified nucleotides. The parameter change enhances measurement precision while maintaining nanopore sequencing simplicity, as the core sequencing mechanism remains unchanged.
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
Enhances the sensitivity and accuracy of nanopore sequencing in detecting modified nucleotides, reducing false discovery rates and enabling accurate, simultaneous long-range epigenetic mapping without the need for PCR amplification.
Implementation Method 1
A bias voltage is applied across a membrane containing a nanopore in the presence of an electrolyte solution, such that a steady ionic current in the vicinity of the nanopore can be detected by electrodes near the membrane
Implementation Method 2
A DNA/RNA molecule passing through a nanopore partially restricts the flow of ions, which is observed as an ionic current drop
Implementation Method 3
contacting said polynucleotide molecule with one or more reagents capable of attaching a detectable moiety to at least one nucleobase of said polynucleotide molecule
Implementation Method 4
attaching a detectable moiety to at least one nucleobase of said polynucleotide molecule to form a labeled nucleobase
Data Source
AI summary
Provided herein is a method of detecting the presence or absence of a single naturally- or synthetically-modified nucleobase in a polynucleotide molecule, which is achieved by first contacting the polynucleotide molecule with one or more reagents capable of attaching a detectable moiety to at least one nucleobase of the polynucleotide molecule or to a nucleobase adjacent to the modified nucleobase, thereby forming a labeled nucleobase, while the presence or absence of the modified nucleobase is determined by the attachment. The polynucleotide molecule is then assayed using a nanopore device to detection the presence or absence of the labeled nucleobase, wherein the detectable moiety attached to at least one nucleobase in the polynucleotide molecule has a molecular weight that ranges from 40 to 1,000 Daltons, and the average pore diameter in the nanopore device is no more than 5 nanometers.