US-12624389-B2 - Methods for sequencing biopolymers

Abstract

The present disclosure provides devices, systems, and methods related to sequencing a biopolymer. In particular, the present disclosure relates to methods for sequencing a polynucleotide using a bioelectronic device that includes protein assemblies used as coupling molecules in bioelectronic circuits. The present disclosure also provides multimeric protein assemblies with various combinations of live and dead subunits arranged to maximize conduction.

Inventors

• Stuart Lindsay
• Eathen Ryan
• Bintian ZHANG
• Xu Wang

Assignees

• ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY

Dates

Publication Date

20260512

Application Date

20220603

Claims (16)

1. 1 . A bioelectronic device comprising: a first electrode; a second electrode separated from the first electrode by a gap; a protein; and a linker comprising: an assembly of monomers comprising a first ligand-binding monomer configured to bind to one or more ligands and a first non-ligand-binding monomer configured to not bind to the one or more ligands, and a tag comprising a first glutamate moiety or a first aspartate moiety or both; wherein the protein is attached to the first electrode and to the second electrode via the linker.
2. 2 . The bioelectronic device of claim 1 , further comprising a second ligand-binding monomer.
3. 3 . The bioelectronic device of claim 1 , further comprising a second non-ligand-binding monomer and a third non-ligand binding monomer.
4. 4 . The bioelectronic device of claim 1 , further comprising a second non-ligand-binding monomer.
5. 5 . The bioelectronic device of claim 1 , wherein the assembly of monomer comprises a first side and a second side opposite a first side and the first ligand-binding monomer is disposed on the first side, the bioelectronic device further comprising a second ligand-binding monomer disposed the second side.
6. 6 . The bioelectronic device of claim 1 , wherein the assembly of monomer comprises a first side, the bioelectric device further comprising a second ligand-binding monomer disposed on a first side of the assembly of monomers and the first ligand-binding monomer is disposed the same first side.
7. 7 . The bioelectronic device of claim 1 , wherein the linker further comprises a peptide or a polypeptide.
8. 8 . The bioelectronic device of claim 7 , wherein the linker further comprises streptavidin.
9. 9 . The bioelectronic device of claim 1 , wherein the tag further comprises a second glutamate moiety.
10. 10 . The bioelectronic device of claim 1 , wherein the tag further comprises a hexaglutamate moiety.
11. 11 . The bioelectronic device of claim 1 , wherein the first ligand-binding monomer includes a C-terminal end and the tag is coupled to a C-terminal end.
12. 12 . The bioelectronic device of claim 1 , wherein the one or more ligands includes biotin.
13. 13 . The bioelectronic device of claim 1 , wherein the protein comprises biotin covalently attached to the protein.
14. 14 . The bioelectronic device of claim 1 , wherein the first electrode or the second electrode or both comprises gold, palladium, platinum, silver, copper, or a combination thereof.
15. 15 . The bioelectronic device of claim 1 , wherein the protein is selected from the group consisting of a polymerase, a nuclease, a proteasome, a glycopeptidase, a glycosidase, a kinase, and an endonuclease.
16. 16 . The bioelectronic device of claim 1 , wherein the protein includes an inactive region to which the linker is attached.

Description

RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/197,901 filed Jun. 7, 2021, which is incorporated herein by reference in its entirety for all purposes. GOVERNMENT SUPPORT This invention was made with government support under R01 HG011079 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD The present disclosure provides devices, systems, and methods related to sequencing a biopolymer. In particular, the present disclosure relates to methods for sequencing a polynucleotide using a bioelectronic device that includes protein assemblies used as coupling molecules in bioelectronic circuits. The present disclosure also provides multimeric protein assemblies with various combinations of live and dead subunits arranged to maximize conduction. BACKGROUND Proteins connected to electrodes via well-defined chemical contacts are remarkably good electrical conductors. Their contact resistance is high, probably owing to the tunnel barrier presented by an outer shell of hydrated residues, but once carriers are injected, the long decay length (on the order of 10 nm) gives rise to improved conductance relative to conventional molecular wires, for lengths in excess of 5 nm. The specific self-assembly of proteins allows quite complex circuits to be built. For example, because polymerase Φ29 interacts with metals unpredictably owing to seven surface cysteines, streptavidin bridges were used to connect doubly-biotinylated Φ29 to biotinylated electrodes, allowing enzyme fluctuations to be recorded electrically. Streptavidin is a tetrameric protein with an extremely high affinity for biotin, and it is an important component of nano-scale protein assemblies. The chemical nature of the electrical contact has a strong effect on the conductance of streptavidin. Streptavidin molecules connected to electrodes via surface thiols (by means of lysine modification) have a significantly lower conductance than molecules attached to electrodes by means of the non-covalent biotin-streptavidin interaction, despite an eight-atom saturated chain linker between the biotin and the electrode. However, the distribution of measured conductances for streptavidin molecules (and complexes connected via streptavidin) has multiple peaks, indicative of multiple modes of connection, made possible by the tetravalent nature of streptavidin. This ambiguity in connection geometries is not easily resolved. SUMMARY Embodiments of the present disclosure include a bioelectronic device comprising a first electrode and a second electrode separated by a gap. In accordance with these embodiments, the bioelectronic device further comprises a protein attached to the first and second electrodes via a linker, and the linker includes an assembly of monomers comprising at least one ligand-binding monomer and at least one non-ligand-binding monomer. In some embodiments, the assembly of monomers are configured to maximize electronic conductance. In some embodiments, the at least one ligand-binding monomer comprises two ligand-binding monomers. In some embodiments, the at least one ligand-binding monomer comprises three ligand-binding monomers. In some embodiments, the at least one non-ligand-binding monomer comprises two non-ligand-binding monomers. In some embodiments, the assembly of monomers comprises two ligand-binding monomers arranged in trans. In some embodiments, the assembly of monomers comprises two ligand-binding monomers arranged in cis. In some embodiments, the assembly of monomers comprises three ligand-binding monomers and exhibits a higher conductance compared to an assembly of monomers comprising two ligand-binding monomers. In some embodiments, the linker comprises a peptide or polypeptide. In some embodiments, the linker comprises streptavidin. In some embodiments, the linker further comprises a tag. In some embodiments, the tag comprises a negative charge. In some embodiments, the tag comprises at least one of a glutamate moiety and/or an aspartate moiety. In some embodiments, the tag comprises at least two glutamate moieties. In some embodiments, the tag comprises a hexaglutamate moiety. In some embodiments, the tag is coupled to the C-terminal end of each of the at least one ligand-binding monomers in the assembly. In some embodiments, the ligand is biotin. In some embodiments, the protein is biotinylated. In some embodiments, the first and/or the second electrode comprises gold, palladium, platinum, silver, copper, or any alloys thereof. In some embodiments, the protein is selected from the group consisting of a polymerase, a nuclease, a proteasome, a glycopeptidase, a glycosidase, a kinase and an endonuclease. In some embodiments, the linker is attached to an inactive region of the protein. Embodiments of the present disclosure also include a method of modulating electronic conductance through a protein using any of the devices descri