Cationic Nucleobase Modifications for Nanopore Sequencing Noise Reduction
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Solution Overview
Problem
Nanopore-based sequencing methods face challenges due to positively charged nanopores attracting negatively charged nucleic acids, leading to increased background and loss of active sites, which hampers sequencing accuracy and efficiency.
Innovation Solution
Development of base-modified nucleoside-5′-oligophosphates (bm-N5OP) with a positively charged moiety at the base position, which reduces the net negative charge of nucleic acids, mitigating attraction to positively charged nanopores and minimizing background interference during sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional negatively charged nucleic acids are used in nanopore sequencing, then the nanopore can detect nucleic acid translocation, but the positively charged nanopore attracts the negatively charged nucleic acids causing increased background noise and loss of active sites
Solution Approach 1:
The patent modifies the charge parameter of nucleic acids by incorporating base-modified nucleoside-5'-oligophosphates with cationic nucleobases. This changes the net charge from negative to positive or neutral, fundamentally altering the electrostatic interaction with the nanopore and reducing non-specific attraction that causes background noise
Solution Approach 2:
The patent converts the harmful effect of electrostatic attraction between oppositely charged surfaces into a beneficial feature. By making nucleic acids cationic, the same electrostatic principle that caused attraction now creates repulsion or neutral interaction, reducing background noise while maintaining detectable translocation signals
2Productivity
If conventional nucleic acids are used, then sequencing can proceed, but active sites on the nanopore are lost due to attraction and binding of negatively charged nucleic acids
Solution Approach 1:
By changing the charge parameter of nucleic acids to positive or neutral through cationic base modifications, the patent prevents electrostatic binding to the nanopore surface, thereby preserving active sites and maintaining high sequencing throughput over extended periods
3Reliability
If base-modified nucleoside-5'-oligophosphates with cationic bases are used, then background noise is reduced and active sites are preserved, but the complexity of nucleotide synthesis and modification increases
Solution Approach 1:
The patent applies local quality modification by introducing cationic groups at specific positions on the nucleobase structure (such as the 7-position of purines or 5-position of pyrimidines) while maintaining the overall nucleotide structure and function. This localized modification achieves the desired charge reversal without requiring complete restructuring of the nucleotide
Solution Approach 2:
The patent creates composite nucleotide structures by combining conventional nucleoside frameworks with cationic base modifications. This composite approach allows the molecule to retain recognition by polymerases and nanopores while acquiring new electrostatic properties that reduce background noise
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
The use of bm-N5OPs in nanopore-based sequencing systems reduces template threading and background noise, enhancing sequencing accuracy and throughput by neutralizing the negative charge of nucleic acids and reducing attraction to the nanopore, thereby improving the reliability of nucleotide identification.
Implementation Method 1
positively charged nanopores attracting negatively charged nucleic acids
Implementation Method 2
reduces the net negative charge of nucleic acids, mitigating attraction to positively charged nanopores
Data Source
AI summary
Disclosed herein are base-modified nucleoside-5′-oligophosphates (bm-N5OP) that include a positively charged moiety at least at one position of the base, compositions comprising the same, compositions made from the same, methods of making the same, and methods of using the same. The bm-N5OP disclosed herein are useful, for example, as tagged nucleotides for use in nanoSBS methods and for generating primers and/or templates for use in nanoSBS methods. When incorporated into a polynucleotide, the disclosed bm-N5OPs can neutralize at least a portion of the negative charge of the overall polynucleotide molecule.