Polypeptide nanopores synthetically functionalized with positively charged species, and methods of making and using the same

Patent metadata

Field	Value
Publication number	US-12578321-B2
Application number	US-202218552532-A
Country	US
Kind code	B2
Filing date	Mar 10, 2022
Priority date	Mar 31, 2021
Publication date	Mar 17, 2026
Grant date	Mar 17, 2026

Abstract

Polypeptide nanopores synthetically functionalized with positively charged species, and methods of making and using the same, are provided herein. In some examples, a polypeptide nanopore includes a first side, a second side, a channel extending through the first and second sides, and a mutated amino acid residue. The mutated amino acid residue may be synthetically functionalized with a positively charged species that inhibits translocation of cations through the channel.

First claim

What is claimed is:

1 . A polypeptide nanopore comprising a first side, a second side, a channel extending through the first and second sides, and a mutated amino acid residue which is synthetically functionalized with a positively charged species that inhibits translocation of cations through the channel, wherein the positively charged species has a net charge of +2, wherein the mutated amino acid residue comprises a natural amino acid residue or an unnatural amino acid residue, and wherein the unnatural amino acid residue comprises an alkyne, azide, or alkene.

2 . The polypeptide nanopore of claim 1 , wherein the polypeptide nanopore comprises MspA, Fragaceatoxin C, α-hemolysin, aerolysin, CsgG, or CsgG/CsgF.

3 . The polypeptide nanopore of claim 1 , wherein the polypeptide nanopore comprises multiple polypeptide subunits.

4 . The polypeptide nanopore of claim 1 , comprising a plurality of the positively charged species.

5 . A polypeptide nanopore comprising a first side, a second side, a channel extending through the first and second sides, and a mutated amino acid residue which is synthetically functionalized with a positively charged species that inhibits translocation of cations through the channel, wherein the polypeptide nanopore comprises MspA, and the mutated amino acid residue is located at residue 90 , 91 , or 93 .

6 . The polypeptide nanopore of claim 5 , wherein the positively charged species comprises a nonmetal cation.

7 . The polypeptide nanopore of claim 5 , wherein the nonmetal cation comprises NR 4 +, where each R group independently comprises hydrogen, a saturated alkyl group, an unsaturated alkyl group, an aromatic species, oxygen, nitrogen, silicon, sulfur, boron, phosphorous, a thiol, an ester derivation, an amide derivation, an amine derivation, a carbonyl derivation, a heterocycle, oligo (siloxane), oligo (ethylene oxide), an amino acid, a nucleobase, a reactive handle for further bioconjugation, a photoactive label, a photoactive dye, a redox-active label, or a redox-active dye, and at least one of the R groups is covalently linked to the mutated amino acid residue through at least one bond; or
wherein the nonmetal cation comprises C 5 H 4 R(NR) + , C 3 H 2 R(NH)(NR) + , C 8 H 7 R(NR 2 ) + , C(NR 2 ) 3 + , SR 3 + , PR 4 + , BR 2 + , C 3 R 3 + (cyclopropenium), C 3 R 3 (NR)S + (thiozonium), or C 3 R 3 (NR)O + (oxazonium), and where each R group independently comprises hydrogen, a saturated alkyl group, an unsaturated alkyl group, an aromatic species, oxygen, nitrogen, silicon, sulfur, boron, phosphorous, a thiol, an ester derivation, an amide derivation, an amine derivation, a carbonyl derivation, a heterocycle, oligo (siloxane), oligo (ethylene oxide), an amino acid, a nucleobase, a reactive handle for further bioconjugation, a photoactive label, a photoactive dye, a redox-active label, a redox-active dye, a nitrogenous aromatic and pi-conjugated species, apyridinium, an imidazolium, an indolium, a guanidinium, a carbazolium, a quinolinium, a functionalized derivative of a pyridinium, or a functionalized derivative of a purinium, and at least one of the R groups is covalently linked to the mutated amino acid residue through at least one bond.

8 . A polypeptide nanopore comprising a first side, a second side, a channel extending through the first and second sides, and a mutated amino acid residue which is synthetically functionalized with a positively charged species that inhibits translocation of cations through the channel, wherein the positively charged species comprises a cationic metal coordination complex.

9 . The polypeptide nanopore of claim 8 , wherein the cationic metal coordination complex comprises at least one metal ion complexed to one or more nonmetal ligands.

10 . The polypeptide nanopore of claim 9 , wherein the at least one metal ion comprises a transition metal cation or a noble metal cation.

11 . The polypeptide nanopore of claim 9 , wherein the at least one of the one or more nonmetal ligands is covalently bound to the mutated amino acid residue.

12 . A polypeptide nanopore comprising a first side, a second side, a channel extending through the first and second sides, and a mutated amino acid residue which is synthetically functionalized with a positively charged species that inhibits translocation of cations through the channel, wherein the positively charged species comprises an ionophore.

13 . The polypeptide nanopore of claim 12 , wherein the ionophore is covalently bound to the mutated amino acid residue.

14 . A polypeptide nanopore comprising a first side, a second side, a channel extending through the first and second sides, and a mutated amino acid residue which is synthetically functionalized with a positively charged species that inhibits translocation of cations through the channel, wherein the polypeptide nanopore comprises multiple polypeptide subunits, and wherein at least one of the multiple polypeptide subunits does not comprise the mutated amino acid residue.

15 . A polypeptide nanopore comprising a first side, a second side, a channel extending through the first and second sides, and a mutated amino acid residue which is synthetically functionalized with a positively charged species that inhibits translocation of cations through the channel, wherein the polypeptide nanopore comprises multiple polypeptide subunits, and wherein at least two of the polypeptide subunits are cross-linked to one another.

16 . The polypeptide nanopore of claim 15 , wherein the at least two of the polypeptide subunits are crosslinked through the positively charged species.

17 . The polypeptide nanopore of claim 15 , having an overall net charge of between about +2 and +18.

18 . A polypeptide nanopore comprising a first side, a second side, a channel extending through the first and second sides, and a mutated amino acid residue which is synthetically functionalized with a positively charged species that inhibits translocation of cations through the channel, wherein the positively charged species has a net charge of +2, wherein the polypeptide nanopore comprises multiple polypeptide subunits, and wherein each of the polypeptide subunits is coupled to a respective positively charged species.

Assignees

• Illumina Inc

Inventors

• Savagian Lisa
• Simpson Burton
• Park Sang
• Boyanov Boyan
• Mandell Jeffrey G
• Mcdonald Seth M

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• B82B3/0038

  Manufacturing processes for forming specific nanostructures not provided for in groups B82B3/0014 - B82B3/0033 · CPC title

• B82B1/008

  Nanostructures not provided for in groups B82B1/001 - B82B1/007 · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q2563/137

  Metal/ion, e.g. metal label · CPC title