Ion-Flow Amplifying Tagged Nucleotides for Nanopore Sequencing
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Solution Overview
Problem
Existing nanopore-based nucleic acid sequencing techniques face challenges in achieving high substrate concentrations and efficient polymerase extension rates due to the increased size and structural variability of tagged nucleotides, which reduces substrate concentrations and molecular concentrations, limiting the throughput and accuracy of sequencing-by-synthesis methods.
Innovation Solution
Development of ion-flow amplifying tagged nucleotides comprising a negatively-charged polymer moiety that increases positive ion flow through nanopores, allowing for improved sensitivity and dynamic range in detection and sequencing applications, and the use of nucleoside-5'-oligophosphate moieties as substrates for polymerases, enhancing the detection of nucleic acid sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bulky tags are attached to nucleotides for nanopore detection, then detection capability is improved, but substrate concentration decreases
Solution Approach 1:
The patent changes the physical-chemical parameters of the tag by using negatively charged polymer moieties with specific charge densities and molecular weights. This optimization allows the tags to generate sufficient detection signals while maintaining smaller effective sizes that do not excessively reduce substrate concentration in the reaction mixture.
Solution Approach 2:
The patent employs composite tag structures consisting of negatively charged polymer moieties attached to nucleotide substrates. These composite structures combine the detection functionality of polymer tags with the biochemical compatibility of nucleotides, achieving both detection capability and acceptable substrate concentration.
2Measurement precision
If larger tagged nucleotides are used for detection, then signal detectability is improved, but polymerase extension rate decreases
Solution Approach 1:
The patent optimizes parameters including polymer charge density, molecular weight, and attachment position on the nucleotide. These parameter optimizations ensure that the tags are sufficiently large for detection but not so large as to severely impede polymerase binding and catalytic activity.
3Measurement precision
If negatively charged polymer tags are used to increase ion flow, then sensitivity is improved, but tag size increases
Solution Approach 1:
The patent utilizes parameter changes by selecting polymer moieties with specific charge densities and molecular weights. The negative charge on the polymer attracts positive ions through the nanopore, amplifying the detection signal. By optimizing the charge-to-size ratio, the patent achieves high sensitivity without excessive tag size increase.
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 ion-flow amplifying tagged nucleotides provide increased ion flow through nanopores, resulting in higher sensitivity and dynamic range, enabling more accurate and efficient nanopore detection and sequencing-by-synthesis, with measured currents greater than the open channel current, thus improving the accuracy and throughput of nucleic acid sequencing.
Implementation Method 1
a negatively-charged polymer moiety which is capable of entering a nanopore and upon entering a nanopore in the presence of positive ions results in an increased flow of the positive ions through the nanopore
Implementation Method 2
applying an electric field across the nanopore to induce the nucleic acid to enter and partially block the nanopore
Data Source
Figure 1
AI summary
The present disclosure relates to compounds comprising a negatively-charged polymer moiety which is capable of entering a nanopore and upon entering a nanopore in the presence of positive ions results in an increased flow of the positive ions through the nanopore. The present disclosure provides methods of preparing the compounds and for their use as nanopore-detectable tags, in particular, for nanopore-based nucleic acid detection and sequencing.