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US-20260125429-A1 - MEMBRANE TRANSLOCATION COMPOSITIONS AND METHODS

US20260125429A1US 20260125429 A1US20260125429 A1US 20260125429A1US-20260125429-A1

Abstract

Provided herein, inter alia, are compositions and methods relating to α-Hemolysin polypeptide variants for translocation across phospholipid membranes.

Inventors

  • Alexander HARJUNG
  • Neal DEVARAJ

Assignees

  • THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date
20260507
Application Date
20250805

Claims (20)

  1. 1 . An α-Hemolysin polypeptide comprising an amino acid insert sequence between amino acids corresponding to amino acid positions D128 and K131 of SEQ ID NO:3, wherein the amino acid insert sequence is 5 to 70 amino acids in length.
  2. 2 . The α-Hemolysin polypeptide of claim 1 , wherein the α-Hemolysin polypeptide without said amino acid insert sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3.
  3. 3 . The α-Hemolysin polypeptide of claim 1 , wherein the amino acid sequence between amino acids corresponding to amino acid positions D128 and K131 of SEQ ID NO:3 comprises an amino acid deletion.
  4. 4 . The α-Hemolysin polypeptide of claim 1 , wherein the α-Hemolysin polypeptide comprises a cysteine mutation corresponding to amino acid position 130 of SEQ ID NO:3.
  5. 5 . The α-Hemolysin polypeptide of claim 1 , wherein the α-Hemolysin polypeptide comprises a histidine to leucine mutation corresponding to amino acid position H48 of SEQ ID NO:3.
  6. 6 . The α-Hemolysin polypeptide of claim 1 , wherein the α-Hemolysin polypeptide comprises a deletion corresponding to amino acid positions D2-G23 of SEQ ID NO:3.
  7. 7 . The α-Hemolysin polypeptide of claim 1 , wherein the amino acid insert sequence is less than 41 amino acids in length.
  8. 8 . The α-Hemolysin polypeptide of claim 1 , wherein the amino acid insert sequence is less than 52 amino acids in length.
  9. 9 . The α-Hemolysin polypeptide of claim 1 , wherein the amino acid insert sequence comprises a cysteine.
  10. 10 . The α-Hemolysin polypeptide of claim 1 , wherein the amino acid insert sequence comprises a glycine linker.
  11. 11 . The α-Hemolysin polypeptide of claim 1 , additionally comprising a bioconjugate reactive moiety.
  12. 12 . The α-Hemolysin polypeptide of claim 11 , wherein the bioconjugate reactive moiety is attached to an amino acid of the amino acid insert sequence.
  13. 13 . The α-Hemolysin polypeptide of claim 1 , additionally comprising a cargo molecule.
  14. 14 . The α-Hemolysin polypeptide of claim 13 , wherein the cargo molecule is attached to the amino acid insert sequence.
  15. 15 . The α-Hemolysin polypeptide of claim 13 , wherein the cargo molecule is covalently attached to a cysteine in the amino acid insert sequence.
  16. 16 . The α-Hemolysin polypeptide of claim 13 , wherein the cargo molecule is covalently attached by a bioconjugate to a cysteine in the amino acid insert sequence.
  17. 17 . The α-Hemolysin polypeptide of claim 13 , wherein the cargo molecule is selected from the group consisting of a polynucleotide, a polypeptide, a lipid, a carbohydrate, and a small molecule.
  18. 18 . A phospholipid membrane comprising the α-Hemolysin polypeptide of claim 1 .
  19. 19 .- 26 . (canceled)
  20. 27 . A peptide of the formula: wherein PS1 is an amino acid sequence having at least 90% sequence identity to SEQ ID: 35; PS2 is an amino acid sequence having at least 90% sequence identity to SEQ ID: 36; L1 is a peptidyl linker having 1-70 amino acids; and AA1 and AA2 are independently hydrogen or amino acid sequences having 1-100 amino acids.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a continuation of International Application No. PCT/US2024/015069, filed Feb. 8, 2024, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/483,836, filed Feb. 8, 2023, each which are hereby incorporated by reference in its entirety. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under N00014-22-1-2800 awarded by the Office of Naval Research. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING The instant application contains a Sequence Listing submitted as an electronic .xml file named 2024-06-06 Sequence_Listing_ST26 048537-657001WO.xml created Jun. 6, 2024 and is 71,628 bytes in size. BACKGROUND Functionalization of the cell membrane with cell-surface proteins and receptors is a requirement for many cellular functions. It enables communication and interaction with the extracellular environment and plays a key role in various cellular processes such as cell-cell adhesion. Natural cells use transmembrane proteins to functionalize the cell membrane. They have therefore evolved different complex mechanisms for the insertion of proteins into cell membranes. Since these highly complex pathways are dependent on multiple proteins and cofactors, reconstituting them into artificial cells would be extremely challenging. For this reason, artificial systems are mostly limited to reconstituting membrane proteins through detergent-based methods, which are often low-yielding and non-trivial to successfully perform on complex transmembrane protein systems. Likewise, it is a common practice to functionalize preformed vesicles with proteins or peptides from the outside, by anchoring them to the membrane. However, none of these methods resemble the natural process in which cells express a protein in the cytoplasm and then insert the protein into the cell membrane. Pore forming toxins (PFTs) can self-insert into biological membranes, independent of any insertion machinery. α-hemolysin (αHL) is an example of a PFT. Pore formation occurs when αHL binds to the lipid membrane, first as a soluble monomer, and then subsequently forming a heptameric complex, which spontaneously translocates across the lipid membrane as a barrel-like structure. αHL has been extensively studied due to its biological role in bacterial infection and its utility in nanopore sequencing. Due to its self-insertion and pore-forming ability, αHL has also been used in artificial cell systems to make lipid membranes permeable to small molecules, which can promote internal biochemical reactions like transcription and translation. BRIEF SUMMARY Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect. In an aspect is provided an α-Hemolysin polypeptide comprising an amino acid insert sequence between amino acids corresponding to positions D128 and K131 of SEQ ID NO:3, wherein the amino acid insert sequence is 5 to 70 amino acids in length. In embodiments, the α-Hemolysin polypeptide without said amino acid insert sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In embodiments, the amino acid sequence between amino acids corresponding to D128 and K131 of SEQ ID NO:3 comprises an amino acid deletion. In embodiments, the α-Hemolysin polypeptide comprises a cysteine mutation corresponding to a position of amino acid 130 of SEQ ID NO:3. In embodiments, the α-Hemolysin polypeptide comprises a histidine to leucine mutation. In embodiments, the α-Hemolysin polypeptide comprises a deletion corresponding to amino acids D2-G23 of SEQ ID NO:3. In embodiments, the amino acid insert sequence is less than 41 amino acids in length. In embodiments, the amino acid insert sequence is less than 52 amino acids in length. In embodiments, the amino acid insert sequence comprises a cysteine. In embodiments, the amino acid insert sequence comprises a glycine linker. In embodiments, the α-Hemolysin additionally comprises a bioconjugate reactive moiety. In embodiments, the bioconjugate reactive moiety is attached to an amino acid of the amino acid insert sequence. In embodiments, the α-Hemolysin additionally comprises a cargo molecule. In embodiments, the cargo molecule is attached to the amino acid insert sequence. In embodiments, the cargo molecule is covalently attached to a cysteine in the amino acid insert sequence. In embodiments, the cargo molecule is covalently attached by a bioconjugate to a cysteine in the amino acid insert sequence. In embodiments, the cargo molecule is selected from the group of a polynucleotide, a polypeptide, a lipid, a carbohydrate, or a small molecule. In an aspect is provided a phospholipid membrane comprising an α-Hemolysi