Alpha-hemolysin variants for nanopore sequencing accuracy
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Solution Overview
Problem
Current alpha-hemolysin nanopores face challenges in accurately detecting single-stranded nucleic acids due to high deletion errors and difficulty in distinguishing ionic current signatures, particularly due to the translocation rate and noise in the ionic current signal.
Innovation Solution
Development of mutant staphylococcal alpha-hemolysin variants with specific amino acid substitutions, such as H35G, T12K, T12R, N17K, and N17R, which form heptameric pore complexes with reduced time-to-thread nucleotides, enhancing the detection efficiency by modifying the stoichiometry and oligomerization properties of the alpha-hemolysin monomers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type alpha-hemolysin is used for nanopore sequencing, then the pore provides high stability and self-assembly properties, but it results in significant deletion errors and difficulty in distinguishing ionic current signatures due to high translocation rate
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (H35G, T12K, T12R, N17K, N17R) at key positions in the alpha-hemolysin sequence. These substitutions modify the pore's physical and chemical parameters, including pore diameter, surface charge distribution, and interaction affinity with nucleic acids, thereby reducing translocation rate and improving detection accuracy while maintaining stability
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific local positions (residues 12, 17, and 35) rather than throughout the entire protein. These localized modifications create specific functional zones within the pore that enhance nucleotide interaction and discrimination without compromising the overall structural integrity and stability of the nanopore
2Productivity
If alpha-hemolysin translocates single-stranded DNA through the pore, then the wide pore diameter accommodates the nucleic acid, but the high translocation rate causes difficulty in analyzing ionic current signatures
Solution Approach 1:
The patent modifies the translocation rate parameter by introducing amino acid substitutions that increase the interaction affinity between the pore and nucleic acid. The H35G substitution at position 35, for example, alters the pore's chemical properties to create stronger interactions, thereby slowing down translocation and improving signal resolution
Solution Approach 2:
The patent addresses the rushing through problem by introducing substitutions that create temporary binding sites or interaction zones within the pore. These modifications cause the nucleic acid to pause or slow down at specific locations, allowing for better signal capture and analysis rather than rushing through too quickly
3Measurement precision
If mutant variants with amino acid substitutions are introduced, then the time-to-thread nucleotides is reduced and detection efficiency is enhanced, but the stoichiometry of subunits must be manipulated to provide functional heptameric pores
Solution Approach 1:
The patent applies segmentation by dividing the heptameric pore formation process into controlled stages. By introducing specific substitutions that affect subunit interaction properties, the patent enables controlled assembly where mutant and wild-type subunits can be incorporated in defined ratios, simplifying the complexity of forming functional heptamers with improved detection properties
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 3A~3B
AI summary
Described herein are engineered alpha-hemolysin subunits having mutated oligomerization domains for assembling into heptameric nanopores in lipid bilayers.