Lipid-free anchoring of thermophilic bacteriophage G20c portal adapter into solid-state nanopores
Inventors
Wanunu, Meni • Antson, Alfred • Greive, Sandra • Cressiot, Benjamin
Assignees
University of York • Northeastern University Boston
Publication Number
US-12320796-B2
Publication Date
2025-06-03
Expiration Date
2039-05-17
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Abstract
Hybrid nanopores, comprising a protein pore supported within a solid-state membrane, which combine the robust nature of solid-state membranes with the easily tunable and precise engineering of protein nanopores. In an embodiment, a lipid-free hybrid nanopore comprises a water soluble and stable, modified portal protein of the Thermus thermophilus bacteriophage G20c, electrokinetically inserted into a larger nanopore in a solid-state membrane. The hybrid pore is stable and easy to fabricate, and exhibits low peripheral leakage, allowing sensing and discrimination among different types of biomolecules.
Core Innovation
The invention provides hybrid nanopores, consisting of a protein pore—specifically, a modified portal protein from Thermus thermophilus bacteriophage G20c—supported within a solid-state membrane. This configuration creates a lipid-free hybrid nanopore that is water soluble, stable, and capable of being electrokinetically inserted into a larger nanopore formed in a solid-state matrix such as silicon nitride. The invention enables the combination of the physical robustness characteristic of solid-state membranes with the precision and tunability a protein channel provides.
The core of the invention lies in the stable insertion fit of engineered, hydrophilic protein channels within a solid-state pore opening, with modifications to the portal protein ranging from changes in internal and external surface residues to alterations promoting binding to solid-state matrices. These modifications can target the electrostatic surface potential, the tunnel loop geometry, or promote chemical attachment via mutations such as cysteine insertions. Such engineered protein variants can serve as nanoscale adapters, adapting snugly and stably inside nanopores for sensing applications, without reliance on fragile lipid bilayers.
The background addresses the limitations of conventional nanopore-based sensors: solid-state nanopores lack reproducibility and precise geometry, while protein nanopore sensors embedded in lipid bilayers suffer from durability issues and poorly defined pore positions. The present invention solves these problems by providing a hybrid, lipid-free nanopore structure that is robust, reproducible, tunable, and easy to fabricate. Its low peripheral leakage and ability to sense multiple types of biomolecules address key challenges in single-molecule detection for molecular medicine and biotechnology.
Claims Coverage
The patent includes multiple independent claims covering inventive features related to nucleic acid modifications, vectors, and cells involving engineered portal proteins for hybrid nanopores. There are several inventive features across these claims.
Nucleic acid molecule encoding modified portal protein with specific residue modifications
A nucleic acid molecule is claimed that includes a modification of SEQ ID NO: 2, where the modification is one or more of: - Modifications in sequences encoding upper or lower internal surface residues of the protein, altering the electrostatic surface potential. - Insertion of a cysteine residue or substitution of an amino acid residue in the encoded protein with cysteine. - Changes producing a modification in a tunnel loop residue: expansion, restriction, or removal of the narrowest constriction of the tunnel loop. - Modifications changing electrostatic charge properties of the tunnel loop residue. - Modifications altering an external charge of the encoded protein. - Modifications promoting binding of the encoded protein to a solid-state matrix. - Modifications extending the N-terminus or C-terminus of the protein or cleaved portion thereof. - Deletion of an amino acid residue from at least one of a C-terminal or N-terminal region. This feature enables encoding of protein variants with characteristics necessary for stable insertion and function within a solid-state nanopore.
Nucleic acid molecule directly comprising specific modified sequences
A nucleic acid molecule is claimed that comprises one of several specifically enumerated SEQ ID NOs (such as SEQ ID NO: 28, SEQ ID NO: 12, SEQ ID NO: 38, SEQ ID NO: 16, etc.), each corresponding to a different engineered portal protein variant implementing defined modifications to residues for targeted structural and electrostatic properties.
Vector comprising a modified nucleic acid sequence encoding engineered portal protein
A vector is claimed that includes a modified nucleic acid sequence of SEQ ID NO: 2, where the modification is one or more of: - Altering surface residues (upper/lower, internal/external) for electrostatic or chemical property changes; - Altering tunnel loop residues (by constriction manipulation or charge modification); - Inserting or substituting cysteine residues; - Promoting solid-state matrix binding; - Modifying termini; - Deleting terminal residues. This enables cloning, expression, or delivery of engineered portal proteins for hybrid nanopore sensors.
Cell comprising a vector with a modified nucleic acid sequence encoding a portal protein variant
A cell is claimed that contains one of the vectors described above, thus enabling the generation, expression or use of the engineered portal protein constructs for assembling hybrid nanopores in living or cell-free systems.
In summary, the claims cover nucleic acid molecules, vectors, and cells comprising engineered sequences encoding portal proteins with precisely defined modifications (targeting residue selection, surface charge, tunnel geometry, binding promotion, and terminal sequences) that enable the formation and use of lipid-free hybrid nanopores in solid-state matrices.
Stated Advantages
Combines the robust nature of solid-state membranes with the easily tunable and precise engineering of protein nanopores.
Provides a stable, easy-to-fabricate, and lipid-free hybrid nanopore that exhibits low peripheral leakage.
Allows for rapid and stable insertion of a protein into a solid-state nanopore.
Enables atomic-precision engineering to chemically define the pore sensor properties.
Facilitates sensing and discrimination among different types of biomolecules.
Mutations of the protein can be used for sensing improvement.
Translocation of biopolymers (such as nucleic acids and polypeptides) through the hybrid sensor can be performed for sensing applications.
Comparable or reduced ion current noise relative to lipid bilayer-supported protein nanopores, improving signal-to-noise ratio for detection.
Hybrid pores remain stable for hours, allowing for extended experimental use.
Documented Applications
High-resolution mapping of DNA, RNA sequencing, and DNA sequencing.
Protein identification and monitoring protein conformational changes.
Polypeptide sequencing.
Small-molecule detection, biomolecular complex detection, and enzyme-ligand binding studies.
Sensing and discrimination of proteins, nucleic acids, biopolymers, and organic molecules at the single-molecule level.
Genomic mapping, oligonucleotide sequencing, and polypeptide sequencing.
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