Alpha-hemolysin nanopore variants for extended sequencing lifetime

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Solution Overview

Problem

Nanopores using wild-type alpha-hemolysins have a short sequencing lifetime and result in significant deletion errors during DNA sequencing reactions.

Innovation Solution

Mutant staphylococcal alpha-hemolysin (αHL) polypeptides with specific substitutions, such as E111N, M113A, K147N, H35G, and glycine residues at positions 126-131, are incorporated into a nanopore to improve sequencing lifetime and efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Duration of action of moving object

If wild-type alpha-hemolysin is used to form nanopore, then the nanopore can be assembled and function for DNA sequencing, but the sequencing lifetime is short

Engineering Contradiction:

Improvesequencing lifetimeVSAvoidsequencing accuracy

Core Design Contradiction:

Duration of action of moving objectVSReliability

Solution Approach 1:

The patent applies parameter changes by modifying specific amino acid residues in the alpha-hemolysin protein sequence. Mutations at positions E111, M113, K147, H35, and residues 126-131 alter the biochemical parameters of the nanopore, resulting in extended sequencing lifetime and improved reliability without sacrificing the fundamental DNA sequencing function.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If wild-type alpha-hemolysin is used, then the nanopore structure is stable and self-assembling, but deletion errors occur during sequencing

Engineering Contradiction:

Improvesequencing accuracyVSAvoiddeletion errors

Core Design Contradiction:

ReliabilityVSLoss of information

Solution Approach 1:

The patent modifies the amino acid sequence parameters of alpha-hemolysin at specific positions (E111N, M113A, K147N, H35G, and glycine residues at 126-131) to reduce deletion errors. These parameter changes maintain the self-assembling capability while improving sequencing accuracy and reducing information loss.

Inventive Principle:

Principle #35Parameter changes

3Duration of action of moving object

If the nanopore lifetime is extended through mutations, then more sequencing data can be generated, but the protein structure is modified

Engineering Contradiction:

Improvesequencing lifetimeVSAvoidprotein structure

Core Design Contradiction:

Duration of action of moving objectVSStability of the object's composition

Solution Approach 1:

The patent applies local quality by making targeted mutations at specific amino acid positions (E111, M113, K147, H35, and 126-131) rather than throughout the entire protein. This localized modification approach extends sequencing lifetime while preserving the overall structural stability and self-assembling properties of the alpha-hemolysin nanopore.

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS20250034637A1Long lifetime alpha-hemolysin nanopores

Publication Date: 2025.01.30 ROCHE SEQUENCING SOLUTIONS INC

AI summary

Described herein are variants of alpha-hemolysin having at least one amino acid substitution at H35G, E111N, M113A, and/or K147N in the mature, wild-type alpha-hemolysin amino acid sequence. In certain examples, the variant may have a substitution at E111S, M113S, T145S, K147S, or L135I in the mature alpha-hemolysin amino acid sequence. The α-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having an increased nanopore lifetime. Further, provided are variants that, in addition to providing increased lifetime, provide a decreased time-to-thread. Hence, the variants provided herein both increase nanopore lifetime and improve efficiency and accuracy of DNA sequencing reactions using nanopores comprising the variants.