Nanopore Sequencing Threading-Blocker Primers
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Solution Overview
Problem
Nanopore-based nucleic acid sequencing techniques face inefficiencies due to deleterious template threading, which interferes with the detection of tag moieties and results in abbreviated sequence reads and lower throughput.
Innovation Solution
The use of threading-blocker primers comprising a compound of formula (I) or (II), which include a Blocking Moiety with poly-cationic, bulky, or base-modified nucleoside groups attached to the 5′-end, preventing template threading by reducing interference with nanopore detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If wide-pore mutant α-HL nanopores are used to improve nanopore lifetime and read-length, then nanopore stability is improved, but template threading into the nanopore increases causing deleterious stoppage events
Solution Approach 1:
A blocker molecule is introduced as an intermediary component that selectively binds to the nanopore constriction site, preventing template DNA from threading into the pore while allowing tagged nucleotides to pass through for detection. The blocker acts as a molecular gatekeeper that discriminates between template strands and incoming nucleotides based on their structural differences.
Solution Approach 2:
The blocking moiety is designed with specific local properties (positive charge, hydrophobicity, molecular size) that match the negative charge and structural characteristics of the template DNA backbone, creating selective binding at the constriction site. This localized interaction prevents threading without affecting the overall nanopore function.
2Measurement precision
If template threading is reduced by using blocking moieties, then detection accuracy is improved, but the complexity of the sequencing system increases
Solution Approach 1:
The blocker is constructed as a composite molecule combining multiple functional groups (charged amino acid residues, hydrophobic aromatic rings, hydrogen-bonding capabilities) within a single polypeptide or peptide structure. This composite design allows multiple interaction mechanisms to work together, enhancing binding specificity and stability while maintaining a relatively simple overall structure.
Solution Approach 2:
The blocking moiety's binding affinity and selectivity are optimized by adjusting parameters such as charge density, hydrophobicity, and molecular size. By tuning these physical-chemical parameters, the blocker achieves high specificity for template DNA while maintaining solubility and appropriate dynamics for reversible binding.
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
These primers enhance the efficiency of nanopore-based nucleic acid detection and sequencing by reducing template threading, allowing for longer sequence reads and improved throughput with wide-pore mutant α-HL nanopores.
Implementation Method 1
Each added nucleotide monomer is detected by monitoring signals due to changes in ion flow through the nanopore that is located adjacent to the polymerase active site
Implementation Method 2
Blocking Moiety comprises a poly-cationic group, a bulky group, or a base-modified nucleoside
Data Source
AI summary
This application discloses compositions comprising primer compounds that reduce or block deleterious threading into a nanopore of nucleic acid strands displaced by a nanopore-linked polymerase, for example during the use of a nanopore device for nucleic acid sequencing. Also disclosed are methods for using the compositions to reduce deleterious threading events during nanopore-based nucleic acid detection techniques, such as nanopore sequencing.