Nanopore Sequencing With Structural Locks for Controlled Translocation
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Solution Overview
Problem
Existing nanopore sequencing techniques suffer from insufficient robustness, reproducibility, accuracy, sensitivity, and throughput, and are not cost-effective for practical implementation due to stochastic enzyme behaviors and fast translocation speeds that exceed electronic measurement capabilities.
Innovation Solution
The method involves using a polymerase to synthesize a complementary strand on a template polynucleotide, which is then removed after each nucleotide incorporation, and utilizing structural locks to maintain the template-complementary strand complex near the nanopore for controlled translocation and multiple readings under varying voltages to enhance signal-to-noise ratio and sequence identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If processive enzymes are used to aid translocation of polynucleotides through nanopores, then translocation can be achieved, but the translocation time per nucleotide becomes highly variable due to stochastic enzyme behaviors
Solution Approach 1:
The patent replaces enzyme-driven translocation with direct electrical manipulation of the nanopore or polynucleotide. By applying controlled voltage pulses or electric fields to the nanopore itself, the system achieves deterministic translocation speeds without relying on stochastic enzyme behavior, thereby improving both speed consistency and measurement accuracy
Solution Approach 2:
The patent dynamically adjusts electrical parameters (voltage, current, pulse duration) applied to the nanopore to control translocation speed. By changing these electrical parameters in real-time, the system can slow down translocation to match electronic measurement capabilities while maintaining precise control over the process
2Productivity
If translocation speed is increased, then throughput is improved, but the speed exceeds the rate that can be accurately measured with electronics and detectors
Solution Approach 1:
The patent employs periodic voltage pulses applied to the nanopore to create a rhythm of translocation that synchronizes with electronic detection capabilities. By using pulsed rather than continuous electrical driving, the system ensures that each nucleotide passage occurs at measurable intervals, maintaining both throughput and measurement precision
Solution Approach 2:
The system uses real-time feedback from electronic detectors to adjust the electrical parameters applied to the nanopore. When detection accuracy begins to deteriorate due to excessive speed, the feedback mechanism automatically reduces translocation velocity, ensuring that measurement precision is maintained while maximizing throughput within operational limits
3Measurement precision
If multiple readings are performed on the same template strand, then sequence identification accuracy is improved, but the time required for sequencing increases
Solution Approach 1:
The patent performs preliminary alignment and positioning of the polynucleotide template in the nanopore before actual sequencing begins. By pre-positioning the template correctly and using structural locks to maintain alignment, the system enables rapid repeated readings without the time penalty of realignment, thus improving accuracy while minimizing time loss
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 improves the confidence and accuracy of polynucleotide sequencing by allowing multiple readings and utilizing structural locks to stabilize the complex, enabling better detection of nucleotide identities and modifications, including non-natural bases and epigenetic markers, with potential for de novo sequencing without a reference genome.
Implementation Method 1
a nanopore disposed in a membrane provides a path for ionic current
Implementation Method 2
As a single-stranded DNA (ssDNA) subject to an electrical driving force traverses through the nanopore
Implementation Method 3
each passing nucleotide of the single ssDNA or each series of nucleotides of the ssDNA that passes through the nanopore yields a characteristic ionic electrical current
Data Source
AI summary
Systems and methods for sequencing polynucleotides using nanopores are disclosed. In some embodiments, a polynucleotide including a single-stranded region and a double-stranded region, in which the single-stranded region is disposed through a nanopore. The polynucleotide can be moved relative to the nanopore by electric forces while one or more structural locks keep the polynucleotide close to the nanopore. A characteristic signal based on nanopore ionic current blockade and associated with the regions of the polynucleotide at or near the nanopore recognition zone is measured and used to infer the nucleobase sequence of the polynucleotide. In some examples, the double-stranded region is extended by a polymerase, and the polymerase is removed from the polynucleotide. In some examples, signals measured under different applied voltages provide nonredundant information regarding the polynucleotide sequence.