Nanopore Sequencing Feedback Control for Stable Polymer Translocation
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Solution Overview
Problem
Existing nanopore sequencing methods struggle with controlling the rate at which target polynucleotides are analyzed, leading to variable translocation rates and challenges in resolving nucleotide composition and spatial relationships within polynucleotides.
Innovation Solution
The use of thin film devices with cis and trans chambers connected by an electrical communication means, featuring a thin film with a pore or channel, and controlled electric fields to manage the passage of polymers like polynucleotides, allowing for precise control of enzyme binding and sequencing through voltage feedback control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nanopore sequencing is performed without controlled enzyme binding, then sequencing speed increases, but sequencing resolution and accuracy deteriorate due to variable translocation rates
Solution Approach 1:
The patent implements feedback control by monitoring the translocation rate of the polynucleotide through the nanopore and dynamically adjusting the enzyme binding conditions. The system measures the actual translocation speed and uses this information to regulate enzyme activity, ensuring that nucleotides are added at optimal intervals for both speed and accuracy. This closed-loop control resolves the contradiction by allowing high throughput while maintaining consistent translocation rates for accurate sequencing.
Solution Approach 2:
The system dynamically adjusts enzyme binding affinity and activity based on real-time translocation conditions. By making the enzyme-polynucleotide interaction dynamic rather than static, the system can optimize binding strength to match the desired translocation speed, thereby achieving both high productivity and measurement precision simultaneously.
2Measurement precision
If enzyme binding is increased to improve sequencing accuracy, then translocation rate control improves, but device complexity increases due to additional control mechanisms
Solution Approach 1:
The nanopore sequencing system employs self-regulating mechanisms where the polynucleotide itself provides feedback on its translocation state. The system leverages the natural physical and chemical properties of the polynucleotide-enzyme interaction to automatically adjust binding conditions without requiring complex external control systems. This self-service approach improves sequencing accuracy while minimizing the addition of complex control mechanisms.
3Productivity
If translocation rate is increased for faster sequencing, then productivity improves, but the ability to resolve nucleotide composition and spatial relationships deteriorates
Solution Approach 1:
The system employs periodic enzyme binding and release cycles that synchronize with the translocation rate. By creating regular, periodic interactions between the enzyme and polynucleotide, the system ensures that nucleotide composition information is captured at consistent intervals, preventing information loss even at high translocation speeds. This periodic action maintains resolution while enabling high throughput sequencing.
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
Enables precise regulation of polymer interaction rates and improved sequencing resolution by controlling enzyme binding, facilitating accurate determination of nucleotide sequences and polymer characteristics.
Implementation Method 1
The use of membrane channels to characterize polynucleotides as the molecules pass through the small ion channels has been studied by Kasianowicz et al. (Proc. Natl. Acad. Sci. USA. 93:13770-13773, 1996, incorporate herein by reference) by using an electric field to force single stranded RNA and DNA molecules through a 1.5 nanometer diameter nanopore aperture
Implementation Method 2
As the polynucleotide traversed the nanopore aperture, the polynucleotide partially blocked the nanopore aperture, resulting in a transient decrease of ionic current. Since the length of the decrease in current is directly proportional to the length of the polynucleotide, Kasianowicz et al. (1996) were able to determine experimentally lengths of polynucleotides by measuring changes in the ionic current.
Data Source
AI summary
Devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore are provided. The devices and methods also determine (˜>50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore. The invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.