Charged Alpha-Hemolysin Variants for Accurate Nanopore Sequencing
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Solution Overview
Problem
Wild-type alpha-hemolysin nanopores result in significant deletion errors during DNA sequencing due to high translocation rates and inherent noise in the ionic current signal, making it difficult to achieve accurate and efficient DNA detection.
Innovation Solution
Mutant alpha-hemolysin variants with specific amino acid substitutions, such as V149K, E287R, H35G, and combinations thereof, are introduced to reduce the time-to-thread nucleotides, improving sequencing accuracy and efficiency by decreasing the time required for nucleic acid molecules to pass through the nanopore.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If wild-type alpha-hemolysin nanopores are used for DNA sequencing, then the translocation rate is high, but deletion errors increase and sequencing accuracy deteriorates
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (V149K, E287R, H35G, and combinations) into the alpha-hemolysin nanopore structure. These substitutions modify the physical and chemical parameters of the pore interior, creating electrostatic interactions that reduce the translocation rate of DNA strands. The reduced speed allows for more accurate detection of ionic current signals, thereby decreasing deletion errors and improving sequencing accuracy while maintaining efficient translocation.
2Loss of time
If wild-type alpha-hemolysin nanopores are used, then translocation occurs quickly, but the time required for accurate nucleotide identification increases due to noise
Solution Approach 1:
The patent modifies the nanopore parameters through amino acid substitutions that create favorable electrostatic interactions between the pore interior and the DNA backbone. This changes the translocation dynamics to optimize the residence time of nucleotides within the detection zone. The modified parameters enable slower, more controlled translocation that improves signal-to-noise ratio for ionic current measurements, allowing accurate nucleotide identification without excessive threading time.
3Productivity
If the translocation rate is high, then sequencing throughput is maintained, but deletion errors occur more frequently
Solution Approach 1:
The patent achieves a balance between productivity and reliability by introducing amino acid substitutions (V149K, E287R, H35G) that moderately reduce the translocation rate. These parameter changes create optimal electrostatic conditions that slow DNA movement enough to improve detection accuracy and reduce deletion errors, while maintaining sufficient translocation speed to preserve sequencing throughput. The modifications optimize the trade-off between speed and accuracy inherent in nanopore sequencing.
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 mutant alpha-hemolysin variants significantly enhance DNA sequencing accuracy by reducing deletion errors and increasing threading rates, allowing for more precise nucleotide identification.
Implementation Method 1
The substitution of the alpha-hemolysin may also be a positive charge. The disclosed variants reduce the time to thread of the molecule of interest
Data Source
AI summary
Described herein are variants of alpha-hemolysin having at least one mutation, such as a mutation to a positive charge. In certain examples, the mutation is selected from V149K, E287R, H35G, T109K, P151K, K147N, E111N, M113A, or combinations thereof in the mature, wild-type 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 a decreased time-to-thread. The decreased time-to-thread, for example, increases DNA sequencing efficiency and accuracy.