Nanopore Protein Conjugates Enhance Polymerase Processivity
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Solution Overview
Problem
Nanopore-based nucleotide sequencing faces challenges in maintaining polymerase processivity and accurate signal detection, with conventional methods either reducing processivity or compromising sequencing accuracy due to high salt concentrations.
Innovation Solution
The development of nanopore protein conjugates, specifically combining an α-hemolysin monomer with a DNA binding domain like Sso7d, which enhances polymerase processivity by maintaining the DNA template strand's proximity to the nanopore assembly, allowing for improved sequencing even in higher salt conditions without sacrificing processivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If conventional methods mutate DNA polymerase to bind more stringently to DNA template strand, then polymerase processivity is improved, but polymerase discrimination between mismatched and matched primer/template decreases
Solution Approach 1:
The invention separates the functions of processivity enhancement and discrimination into distinct components: a DNA-binding domain is separated from the polymerase enzyme and placed on the nanopore assembly, while the polymerase retains its native sequence and discrimination capability. This segmentation allows the polymerase to maintain high discrimination accuracy while the separate DNA-binding domain on the nanopore provides processivity enhancement through stringent binding to the template strand.
Solution Approach 2:
The invention introduces an intermediary DNA-binding domain that acts as a mediator between the nanopore assembly and the template DNA strand. This intermediary domain binds stringently to the template strand and physically tethers it to the nanopore assembly, thereby enhancing polymerase processivity without requiring modifications to the polymerase enzyme itself, thus preserving its discrimination function.
2Measurement precision
If higher salt concentrations are used to improve ionic current signal strength, then signal detection is improved, but polymerase processivity is reduced
Solution Approach 1:
The DNA-binding domain on the nanopore assembly acts as an intermediary that maintains template strand tethering through salt-resistant binding interactions. This intermediary mechanism allows the system to operate in higher salt concentrations for improved signal detection while the DNA-binding domain continues to hold the template strand firmly, preventing polymerase dissociation and maintaining processivity despite the harsher ionic conditions.
Solution Approach 2:
The invention enables operation in higher salt concentration environments by introducing a DNA-binding domain with appropriate binding characteristics that remain effective under these conditions. The DNA-binding domain's binding affinity and specificity are optimized to function in higher salt concentrations, allowing simultaneous improvement of signal detection (through enhanced ionic current) and maintenance of polymerase processivity (through stable template tethering).
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 nanopore protein conjugates significantly reduce the time between polymerase cessation and nanopore inactivity, increase sequencing end time, and maintain high polymerase processivity in higher salt concentrations, enhancing sequencing accuracy and reliability.
Implementation Method 1
The nanopore protein monomer is conjugated to a DNA binding protein, such as a protein having an Sso7d domain or Sso7d-like domain
Implementation Method 2
Inaccurate signals, for example, can lead to the misidentification of one or more nucleotides during the sequencing reaction, which in turn negatively impacts the reliability of the determined sequence. Unfortunately, inherent noise in the ionic current signal can make accurate signal detection difficult.
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3B
AI summary
Described herein are nanopore protein conjugates that can be used in DNA sequencing reactions. The nanopore protein conjugates includes a nanopore protein monomer that is joined to a DNA binding domain. The nanopore protein monomer is available to oligomerize with other nanopore protein monomers, while the DNA binding domain is available to bind to a template DNA strand. In certain examples, the nanopore protein monomer is an alpha-hemolysin monomer or variant thereof and the DNA binding domain is an Sso7d protein or variant thereof, such as an Sso7d-like protein. Also provided are nanopore protein assemblies incorporating the nanopore protein conjugates, along with methods of using the nanopore protein assemblies in sequencing reactions.