Nanopore Enzyme Screening with Molecular Barcodes for Multiplex Kinetics
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Solution Overview
Problem
Current nanopore sequencing methods for enzyme screening, such as DNA polymerase variants, are time-consuming and costly, requiring individual screening of each enzyme variant, and struggle with distinguishing between similar nucleotides and controlling reaction rates for accurate base identification.
Innovation Solution
A method and system for multiplex screening of enzyme variants using nanopore-based sequencing, involving a biochip with unique molecular barcodes and automated alignment algorithms to classify and derive parameters for enzyme variants, enabling rapid and accurate determination of enzyme properties like efficacy, stability, and fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If individual screening of each enzyme variant is performed using nanopore sequencing, then measurement precision of enzyme properties can be obtained, but productivity and time efficiency deteriorate
Solution Approach 1:
The patent divides the enzyme screening process into multiple parallel nanopore sequencing complexes, each capable of independently screening different enzyme variants. This segmentation allows simultaneous processing of multiple variants, transforming a sequential individual screening approach into a parallel multiplexed approach, thereby improving productivity while maintaining measurement precision through dedicated sequencing complexes for each variant.
Solution Approach 2:
The patent introduces a molecular barcode dimension to enable multiplexing of enzyme variants in a single nanopore sequencing complex. By assigning unique barcodes to different enzyme variants and using computational methods to distinguish and analyze signals from barcoded molecules, the system achieves high-throughput screening without sacrificing measurement precision, effectively adding a dimensional layer of identification that enables parallel processing.
2Productivity
If molecular barcodes are used for multiplex screening, then productivity is improved, but device complexity increases
Solution Approach 1:
The patent uses molecular barcodes as informational copies that encode the identity of enzyme variants. Instead of physically separating each variant in different complexes, the system creates digital copies of variant information through barcode sequences that can be read and distinguished by computational algorithms. This copying approach enables multiplexing without requiring proportional increases in physical device complexity, as the barcode information is processed through software rather than requiring separate hardware channels for each variant.
3Ease of operation
If reaction rates are not precisely controlled, then ease of operation is improved, but measurement precision of base identification deteriorates
Solution Approach 1:
The patent implements feedback control through real-time monitoring of ionic current signals during nanopore sequencing. The system continuously analyzes current changes to detect nucleotide incorporation events and adjusts the sequencing process accordingly. This feedback mechanism ensures that reaction rates are precisely controlled during critical measurement phases while maintaining ease of operation through automated regulation, thereby achieving both operational simplicity and measurement precision.
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 rapid and accurate screening of multiple enzyme variants, allowing for the identification of enzyme variants with unique properties and nanopore variants, facilitating the engineering of enzymes with tailored functions, such as incorporating unnatural substrates or temperature-sensitive mutants.
Implementation Method 1
It was demonstrated that during polynucleotide translocation partial blockage of the nanopore aperture could be measured as a decrease in ionic current
Implementation Method 2
DNA polymerases are enzymes that duplicate genetic information by synthesizing a new complementary DNA strand from the parent template
Data Source
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AI summary
The present disclosure is directed to compositions and methods for deriving a plurality of kinetics parameters (240) for at least two different enzyme variants in a multiplex manner using nanopore-based sequencing. In some embodiments, the systems and methods may be used to screen different nanopore variants, or different combinations of both nanopore variants and enzyme variants.