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US 12,320,796 B2Granted
Lipid-free anchoring of thermophilic bacteriophage G20c portal adapter into solid-state nanopores
AssigneesNortheastern University, The University of York
InventorsMeni Wanunu, Alfred Antson, Sandra Greive, Benjamin Cressiot
Life of this patent
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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.
Claims18
A nucleic acid molecule comprising a modification of a nucleic acid sequence of SEQ ID NO:
- 2, the modification comprising one or more of:
- (i) a modification in a portion of the nucleic acid sequence that encodes an upper internal surface residue or a lower internal surface residue of a protein encoded by the nucleic acid sequence, the modification producing an alteration of an electrostatic surface potential of an upper internal surface or a lower internal surface of the protein encoded by the nucleic acid sequence;
- (ii) a modification comprising an insertion of a cysteine residue into a protein encoded by the nucleic acid sequence or a replacement of an amino acid residue of a protein encoded by the nucleic acid sequence with a cysteine residue;
- (iii) a modification to produce a modification of a tunnel loop residue of a protein encoded by the nucleic acid sequence, the modification in the tunnel loop residue comprising an expansion of a narrowest constriction of a tunnel loop of the protein encoded by the nucleic acid sequence, a restriction of the narrowest constriction of the tunnel loop of the protein encoded by the nucleic acid sequence, or a removal of the narrowest constriction of the tunnel loop of the protein encoded by the nucleic acid sequence;
- (iv) a modification to produce a modification of a tunnel loop residue of a protein encoded by the nucleic acid sequence, the modification resulting in an alteration of an electrostatic charge property of a tunnel loop of the protein encoded by the nucleic acid sequence;
- (v) a modification which alters an external charge of a protein encoded by the nucleic acid sequence;
- (vi) a modification which promotes binding of a protein encoded by the nucleic acid sequence to a solid-state matrix;
- (vii) a modification which extends an N-terminus or a C-terminus of a protein encoded by the nucleic acid sequence or an N-terminus or a C-terminus of a cleaved portion of the protein encoded by the nucleic acid sequence;
- and
- (viii) a deletion of an amino acid residue of at least one of a C-terminal region and an N-terminal region of a protein encoded by the nucleic acid sequence.
7 more elements
A nucleic acid molecule comprising a modification of a nucleic acid sequence of SEQ ID NO:
- 2, the nucleic acid molecule comprising one of:
- SEQ ID NO:
- 28;
- SEQ ID NO:
- 12;
- SEQ ID NO:
- 38;
- SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 22, SEQ ID NO:
- 24, SEQ ID NO:
- 26, SEQ ID NO:
- 20, SEQ ID NO:
- 10, SEQ ID NO:
- 14, SEQ ID NO:
- 30, SEQ ID NO:
- 32, SEQ ID NO:
- 36, SEQ ID NO:
- 34, SEQ ID NO:
- 4, SEQ ID NO:
- 6, and SEQ ID NO:
- 8.
20 more elements
A nucleic acid molecule comprising a modification of a nucleic acid sequence of SEQ ID NO:
- 2, the nucleic acid molecule comprising one of:
- SEQ ID NO:
- 28;
- SEQ ID NO:
- 38;
- SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 24, SEQ ID NO:
- 26, SEQ ID NO:
- 20, SEQ ID NO:
- 10, SEQ ID NO:
- 14, SEQ ID NO:
- 30, SEQ ID NO:
- 32, SEQ ID NO:
- 36, SEQ ID NO:
- 34, SEQ ID NO:
- 4, SEQ ID NO:
- 6, and SEQ ID NO:
- 8.
17 more elements
A vector comprising a modification of a nucleic acid sequence of SEQ ID NO:
- 2, the modification comprising one or more of:
- (i) a modification in a portion of the nucleic acid sequence that encodes an upper internal surface residue or a lower internal surface residue of a protein encoded by the nucleic acid sequence, the modification producing an alteration of an electrostatic surface potential of an upper internal surface or a lower internal surface of the protein encoded by the nucleic acid sequence;
- (ii) a modification comprising an insertion of a cysteine residue into a protein encoded by the nucleic acid sequence or a replacement of an amino acid residue of a protein encoded by the nucleic acid sequence with a cysteine residue;
- (iii) a modification to produce a modification of a tunnel loop residue of a protein encoded by the nucleic acid sequence, the modification of the tunnel loop residue comprising an expansion of a narrowest constriction of a tunnel loop of the protein encoded by the nucleic acid sequence, a restriction of the narrowest constriction of the tunnel loop of the protein encoded by the nucleic acid sequence, or a removal of the narrowest constriction of the tunnel loop of the protein encoded by the nucleic acid sequence;
- (iv) a modification to produce a modification of a tunnel loop residue of a protein encoded by the nucleic acid sequence, the modification resulting in an alteration of an electrostatic charge property of a tunnel loop of the protein encoded by the nucleic acid sequence;
- (v) a modification which alters an external charge of a protein encoded by the nucleic acid sequence;
- (vi) a modification which promotes binding of a protein encoded by the nucleic acid sequence to a solid-state matrix;
- (vii) a modification which extends an N-terminus or a C-terminus of a protein encoded by the nucleic acid sequence or an N-terminus or a C-terminus of a cleaved portion of the protein encoded by the nucleic acid sequence;
- and
- (viii) a deletion of an amino acid residue of at least one of a C-terminal region and an N-terminal region of a protein encoded by the nucleic acid sequence.
7 more elements
A vector comprising a modification of a nucleic acid sequence of SEQ ID NO:
- 2, the modification comprising one or more of:
- SEQ ID NO:
- 28;
- SEQ ID NO:
- 12;
- SEQ ID NO:
- 38;
- SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 22, SEQ ID NO:
- 24, SEQ ID NO:
- 26, SEQ ID NO:
- 20, SEQ ID NO:
- 10, SEQ ID NO:
- 14, SEQ ID NO:
- 30, SEQ ID NO:
- 32, SEQ ID NO:
- 36, SEQ ID NO:
- 34, SEQ ID NO:
- 4, SEQ ID NO:
- 6, and SEQ ID NO:
- 8.
20 more elements
A vector comprising a modification of a nucleic acid sequence of SEQ ID NO:
- 2, the modification comprising one or more of:
- SEQ ID NO:
- 28;
- SEQ ID NO:
- 38;
- SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 24, SEQ ID NO:
- 26, SEQ ID NO:
- 20, SEQ ID NO:
- 10, SEQ ID NO:
- 14, SEQ ID NO:
- 30, SEQ ID NO:
- 32, SEQ ID NO:
- 36, SEQ ID NO:
- 34, SEQ ID NO:
- 4, SEQ ID NO:
- 6, and SEQ ID NO:
- 8.
17 more elements
Dependencies are inferred from the claim text.