Modified Alpha Hemolysin Polypeptides for Polymer Sequencing
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Solution Overview
Problem
Wild-type alpha hemolysin polypeptides facilitate polymer translocation too quickly for accurate analysis, posing a technological challenge in polymer sequencing and analysis.
Innovation Solution
Modified alpha hemolysin polypeptides with specific amino acid substitutions, particularly in the beta barrel region, are designed to slow down polymer translocation, allowing for more efficient and accurate analysis by increasing the translocation time of polymers through the modified protein pores.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If wild-type alpha hemolysin polypeptides are used for polymer translocation, then translocation speed is high, but measurement accuracy deteriorates due to insufficient translocation time
Solution Approach 1:
The patent applies parameter changes by substituting specific amino acid residues (such as E111Q, E111A, D127N, D127K) in the alpha hemolysin polypeptide sequence to modify the physical-chemical properties of the pore. These substitutions change the electrostatic environment and interaction characteristics within the pore, thereby altering the translocation dynamics of polymers to achieve optimal measurement conditions.
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific positions (1-109 and 149-293) within the polypeptide sequence, particularly in regions that form the pore structure. These localized modifications affect specific interaction sites within the pore without altering the overall structure, enabling precise control over polymer translocation behavior.
2Measurement precision
If amino acid substitutions are made to slow polymer translocation, then measurement accuracy improves, but protein structure complexity increases
Solution Approach 1:
The patent systematically modifies specific parameters (amino acid identities at defined positions) to achieve the desired translocation slowing effect while maintaining a relatively simple modification strategy that involves only a few targeted substitutions rather than comprehensive sequence redesign.
3Duration of action of moving object
If multiple amino acid substitutions are introduced in the beta barrel region, then polymer translocation time increases, but manufacturing complexity increases
Solution Approach 1:
The patent focuses on changing specific parameters (amino acid residues) at strategically important positions in the beta barrel region that have the greatest impact on translocation time. By selecting key positions for modification rather than uniformly modifying all residues, the patent achieves effective translocation control while minimizing manufacturing complexity.
Data Source
AI summary
Provided herein are alpha hemolysin polypeptides comprising modified amino acid sequences that can reduce the rate of translocation of a polymer. Also provided herein are apparatuses and devices comprising modified hemolysin polypeptides. Also provided herein are methods of using modified alpha hemolysin proteins for use in characterizing and/or sequencing a polymer or for use as molecular sensors.