Enzyme-Pore Constructs for Rapid DNA Sequencing

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Solution Overview

Problem

Current DNA or RNA sequencing technologies are slow and expensive due to reliance on amplification techniques and high quantities of specialist chemicals, necessitating the development of rapid and cost-effective methods.

Innovation Solution

A construct comprising a transmembrane protein pore subunit covalently attached to a nucleic acid handling enzyme, allowing for the formation of pores capable of sequencing nucleic acids through stochastic sensing by interacting with and distinguishing nucleotides.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If amplification techniques and high quantities of specialist chemicals are used for sequencing, then sequencing accuracy can be maintained, but sequencing speed decreases and cost increases

Engineering Contradiction:

Improvesequencing speedVSAvoidquantity of chemicals required

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

The invention extracts the essential sequencing function from complex amplification techniques and chemical processes, reducing it to a single-pore stochastic sensing system that detects nucleotides individually as they pass through the pore, eliminating the need for amplification and large quantities of chemicals

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention replaces the mechanical/chemical amplification system with an electrical detection system based on ionic current measurements through a single pore, where nucleotide identity is determined by characteristic current blockades rather than chemical reactions

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Quantity of substance

If a single pore is used for stochastic sensing, then chemical quantity is reduced, but detection precision must be maintained

Engineering Contradiction:

Improvequantity of chemicals requiredVSAvoidnucleotide detection accuracy

Core Design Contradiction:

Quantity of substanceVSMeasurement precision

Solution Approach 1:

The invention introduces an enzyme as an intermediary that processively translocates the nucleic acid through the pore and sequentially releases individual nucleotides, ensuring controlled interaction between each nucleotide and the pore for accurate detection

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The enzyme maintains continuous processive translocation of the nucleic acid through the pore, ensuring that each nucleotide is systematically presented to the detection zone without interruption, thereby maintaining high measurement precision throughout the sequencing process

Inventive Principle:

Principle #20Continuity of useful action

3Stability of the object's composition

If enzyme is covalently attached to pore subunit, then construct stability is improved, but pore formation ability may be compromised

Engineering Contradiction:

Improveconstruct stabilityVSAvoidpore formation ability

Core Design Contradiction:

Stability of the object's compositionVSReliability

Solution Approach 1:

The invention segments the construct into distinct functional domains: a pore-forming subunit and an enzyme subunit, connected by a linker region. This segmentation allows each domain to independently perform its function while maintaining overall construct stability through the covalent connection

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention applies local quality by designing the linker region with specific properties (flexibility, length, composition) that are optimized to connect the pore subunit and enzyme subunit without interfering with either's function, while the pore subunit itself is designed to reliably form pores despite the attachment

Inventive Principle:

Principle #3Local quality

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 cost-effective sequencing by ensuring a proportion of nucleotides interact with the pore, altering current flow in a distinctive manner, facilitating efficient nucleic acid sequencing.

Implementation Method 1

measuring voltage-driven ionic transport through the pore

Methodology Applied

Scientific EffectIonic transport: Electrophoresis

Implementation Method 2

a nucleic acid handling enzyme, wherein the subunit is covalently attached to the enzyme such that both the subunit and enzyme retain their activity

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 3

The frequency of occurrence of fluctuations in the current reveals the concentration of an analyte that binds within the pore

Methodology Applied

Scientific EffectStochastic sensing: Electrical Resistance

Data Source

PatentUS11859247B2Enzyme-pore constructs

Publication Date: 2024.01.02 OXFORD NANOPORE TECH LTD

AI summary

The invention relates to constructs comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme. The pore subunit is covalently attached to the enzyme such that both the subunit and enzyme retain their activity. The constructs can be used to generate transmembrane protein pores having a nucleic acid handling enzyme attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect its component nucleotides by stochastic sensing.