Multiplex Enzyme Variant Screening Using Nanopore Sequencing
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Solution Overview
Problem
Existing methods for screening enzyme variants, such as DNA polymerases, are time-consuming and costly, and there is a need for efficient and rapid multiplex screening to determine properties like efficacy, stability, and fidelity.
Innovation Solution
A method using nanopore-based sequencing that allows for the simultaneous screening of multiple enzyme variants by incorporating unique molecular barcodes into polynucleotides, enabling classification and derivation of kinetic parameters for each variant.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used to screen enzyme variants, then measurement precision can be achieved, but productivity is low and loss of time is high
Solution Approach 1:
The patent divides the screening process into parallel independent units by using multiple nanopores, each capable of simultaneously evaluating different enzyme variants. This segmentation enables concurrent processing of multiple samples, transforming a sequential screening process into a parallel one, thereby dramatically improving productivity and reducing time loss.
Solution Approach 2:
The patent transitions from single-molecule sequencing to multi-molecule simultaneous sequencing by introducing spatial dimensionality through nanopore arrays. This dimensional expansion allows multiple enzyme variants to be screened at the same time across different nanopores, converting a one-dimensional sequential process into a multi-dimensional parallel process.
2Productivity
If multiple enzyme variants are screened simultaneously, then productivity increases, but device complexity increases
Solution Approach 1:
The patent designs nanopores with universal functionality to handle multiple enzyme variants through a standardized interface and detection mechanism. Each nanopore can accommodate different enzyme variants using the same basic structural and operational framework, allowing the system to scale from single to multi-variant screening without proportionally increasing complexity.
Solution Approach 2:
The patent employs identical nanopore structures replicated multiple times to create an array, where each nanopore serves as a copy capable of independent operation. This replication strategy allows the system to screen multiple enzyme variants using the same proven nanopore design, avoiding the need to develop unique nanopores for each variant and thus controlling device complexity.
3Measurement precision
If unique molecular barcodes are incorporated into polynucleotides, then measurement precision improves for variant identification, but device complexity and manufacturing difficulty increase
Solution Approach 1:
The patent incorporates unique molecular barcodes into polynucleotides during the synthesis or preparation stage, before the enzymes are introduced. This preliminary action ensures that the barcodes are already in place to enable precise variant identification during the screening process, eliminating the need for subsequent barcode addition steps and simplifying the overall manufacturing workflow.
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 determination of enzyme variant properties, facilitating the engineering of enzymes with novel functions and tailormade needs, and allowing for the identification of desirable nanopore-enzyme pairs.
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
a nanopore sequencing by synthesis (SBS) approach in which identifiable polymer tags are attached to nucleotides and registered in nanopores during enzyme-catalyzed DNA synthesis.
Implementation Method 3
Akeson et. al. (see, e.g., PCT Publication No. WO/20150344945) disclose methods for characterizing polynucleotides in a nanopore that utilize an adjacently positioned molecular motor to control the translocation rate of the polynucleotide through or adjacent to the nanopore aperture.
Data Source
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.