Polymerase Conjugate Binding Stabilization for Longer Sequencing Reads
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Solution Overview
Problem
Conventional sequencing-by-synthesis methods suffer from 'molecular scarring' due to the incorporation of reversibly terminated nucleotides, limiting the length of template sequences that can be sequenced, and the stability of polymerase-nucleotide complexes is inherently unstable in stochastic binding-based methods.
Innovation Solution
The use of a polymerase conjugate with a heterologous polynucleotide-binding moiety enhances the stability of the complex by binding to the priming strand and template nucleic acid, allowing for a reversibly terminated nucleotide at the 3′ end without incorporation, and detecting the presence of complementary nucleotides to improve signal detection and enable longer sequence reads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing-by-synthesis incorporates reversibly terminated nucleotides into the priming strand, then sequencing can be performed with signal detection, but molecular scarring is created that destabilizes the extended priming strand and limits the length of template sequence that can be sequenced
Solution Approach 1:
The invention extracts the harmful incorporation step by using a polymerase-conjugate system where the nucleotide is delivered bound to the polymerase but not incorporated into the priming strand. The reversibly terminated nucleotide remains on the 3′ end of the priming strand without being incorporated, eliminating molecular scarring while maintaining signal detection capability.
Solution Approach 2:
The polymerase-conjugate acts as an intermediary that delivers the fluorescently labeled, reversibly terminated nucleotide to the priming strand 3′ end without incorporating it. This intermediary system allows signal detection while preventing the destabilizing incorporation that causes molecular scarring.
2Length of stationary object
If stochastic binding of polymerase and fluorescent nucleotide is used to avoid molecular scarring, then longer template sequences can be sequenced, but the complex formation is inherently unstable upon rinsing away unbound polymerase and fluorescent nucleotides
Solution Approach 1:
The invention merges the polymerase and the polynucleotide-binding moiety into a single conjugate. This combined structure ensures that when the polymerase binds to the priming strand-template complex, the polynucleotide-binding moiety simultaneously provides stable anchoring, preventing complex dissociation during rinsing steps while enabling long sequence reads.
Solution Approach 2:
The polymerase-conjugate is a composite structure combining polymerase enzyme activity with polynucleotide-binding capability. This composite design provides both the catalytic function for nucleotide delivery and the stable binding function needed to maintain complex integrity during washing steps.
3Reliability
If a polymerase-conjugate with heterologous polynucleotide-binding moiety is used to stabilize the complex, then complex stability is enhanced, but device complexity increases
Solution Approach 1:
The polymerase-conjugate is designed to perform multiple functions: the polymerase provides nucleotide delivery and catalytic activity, while the heterologous polynucleotide-binding moiety provides stable binding to the priming strand or template. This multi-functional design stabilizes the complex without requiring separate components for each function.
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
This approach minimizes 'molecular scarring' and stabilizes the complex, enabling longer sequence reads by maintaining a stable optical signal through enhanced complex stability.
Implementation Method 1
the polynucleotide-binding moiety of the polymerase conjugate enhances stability of the complex (through binding to the primed template nucleic acid molecule)
Implementation Method 2
a fluorescent, reversibly terminated nucleotide into an extended priming strand, where the incorporated nucleotide is complementary to a nucleotide at the position of the template nucleic acid molecule that is being probed
Data Source
AI summary
The present disclosure relates in some aspects to methods, systems, and kits for sequencing a template nucleic acid molecule, where the methods comprise: (i) contacting a priming strand bound to the template nucleic acid molecule with a first plurality of nucleotide molecules and a polymerase coupled to a heterologous polynucleotide-binding moiety to form a complex comprising a 3′ terminus of the priming strand, the template nucleic acid molecule, the polymerase, and a nucleotide molecule of the first plurality of nucleotide molecules, wherein the polynucleotide-binding moiety enhances stability of the complex, and wherein the priming strand comprises a reversibly-terminated nucleotide at its 3′ end such that the nucleotide molecule of the transient complex is not incorporated; and (ii) detecting a presence of the nucleotide molecule in the complex to identify a complementary nucleotide in the template nucleic acid molecule.