Integrated Helicase Nanopore for Double-Stranded DNA Sensing

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Solution Overview

Problem

Existing protein nanopores, such as α-HL and MspA, face challenges in accurately distinguishing single bases in nucleic acid sequencing due to their limited channel size and the need for external enzymes to unwind double-stranded nucleic acids, leading to reduced sensing resolution and interference from poor voltage-gated characteristics.

Innovation Solution

A novel protein nanopore is constructed using bovine papillomavirus double-strand DNA helicase proteins, engineered to integrate helicase activity with nanopore functionality, allowing for the unwinding and translocation of double-stranded nucleic acids without external enzymes, and optimized through mutagenesis to enhance capture and sensitivity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If the transmembrane section length of nanopore is reduced to improve sensing resolution, then the sensing resolution is improved, but the structural stability and voltage-gated characteristics deteriorate

Engineering Contradiction:

Improvesensing resolutionVSAvoidvoltage-gated characteristics

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent merges the nanopore function and helicase function into a single integrated protein structure. The nanopore domain provides the transmembrane channel with voltage-gated characteristics, while the helicase domain provides unwinding activity. This merging resolves the contradiction by eliminating the need for separate external enzymes, thereby maintaining structural stability and voltage-gated characteristics while achieving the unwinding function necessary for high-resolution sensing.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent creates a composite protein structure combining nanopore and helicase domains. This composite structure integrates the electrical properties of the nanopore (for voltage-gated characteristics and sensing resolution) with the mechanical unwinding capability of the helicase domain, thereby resolving the contradiction between maintaining structural stability and achieving high sensing resolution.

Inventive Principle:

Principle #40Composite materials

2Adaptability or versatility

If external enzymes are used to unwind double-stranded nucleic acids, then the unwinding function is achieved, but the device complexity increases

Engineering Contradiction:

Improveunwinding functionVSAvoidsystem complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent creates a multi-functional protein that simultaneously performs nanopore sensing and helicase unwinding activities. This single protein structure replaces what would otherwise require multiple separate components (nanopore + external helicase enzymes), thereby reducing device complexity while maintaining both unwinding function and sensing capability.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Measurement precision

If the channel diameter is reduced to improve single base distinction, then the sensing resolution is improved, but the translocation speed decreases

Engineering Contradiction:

Improvesingle base distinctionVSAvoidtranslocation speed

Core Design Contradiction:

Measurement precisionVSSpeed

Solution Approach 1:

The helicase domain performs preliminary unwinding action on double-stranded nucleic acids before they enter the nanopore. By pre-unwinding the dsDNA into ssDNA, the nucleic acid is prepared in a form that can more easily translocate through the narrow channel, thereby maintaining translocation speed while achieving high single base distinction capability through the reduced channel diameter.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent segments the nucleic acid translocation process into two stages: (1) unwinding of double-stranded regions by the helicase domain, and (2) translocation of single-stranded regions through the nanopore. This segmentation allows the narrow channel to focus on high-resolution sensing of individual bases in the ssDNA form, while the helicase handles the more complex dsDNA structure, thereby resolving the speed-resolution trade-off.

Inventive Principle:

Principle #1Segmentation

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

The engineered nanopore system enables simultaneous unwinding and analysis of double-stranded DNA, improving sensing resolution and sensitivity, and can be applied in biosensing and liposome drug delivery.

Implementation Method 1

By studying its conductance distribution under various conductivity buffer systems... The electrical signal from phi29 DNA packaged motor protein

Methodology Applied

Scientific EffectElectrical current detection: Conduction (electrical)

Implementation Method 2

bovine papillomavirus double-strand DNA helicase proteins, engineered to integrate helicase activity with nanopore functionality, allowing for the unwinding and translocation of double-stranded nucleic acids

Methodology Applied

Scientific EffectHelicase activity: Enzyme

Data Source

PatentEP3575408B1Transmembrane nucleic acid unwinding nanopore, construction method therefor, and use thereof

Publication Date: 2025.10.01 GUANGZHOU KONGQUE GENE TECH CO LTD

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AI summary

Provided are two truncations of bovine papillomavirus DNA helicase E1 and use thereof in the preparation of an electroconductive nanopore.