Alpha-hemolysin variants

Patent metadata

Field	Value
Publication number	US-10752948-B2
Application number	US-201916238874-A
Country	US
Kind code	B2
Filing date	Jan 3, 2019
Priority date	Sep 24, 2015
Publication date	Aug 25, 2020
Grant date	Aug 25, 2020

Abstract

Described herein are engineered alpha-hemolysin subunits having mutated oligomerization domains for assembling into heptameric nanopores in lipid bilayers.

First claim

What is claimed is:

1. A hetero-oligomeric α-hemolysin (αHL) heptamer, comprising at least one preceding and at least one following subunit, each subunit comprising at least one αHL monomer and/or at least one polypeptide comprising concatenated αHL monomers,
wherein the at least one αHL monomer and/or the at least one polypeptide comprising concatenated αHL monomers comprises a mutation corresponding to H35L of SEQ ID NO:3; and
wherein the heptamer comprises exactly 7 αHL monomers, each αHL monomer comprising a first oligomerization domain and a second oligomerization domain, the first oligomerization domain of each αHL monomer being linked to the second oligomerization domain of a preceding αHL monomer and the second oligomerization domain of each αHL monomer being linked to the first oligomerization domain of a following αHL monomer.

2. The hetero-oligomeric αHL heptamer of claim 1 , wherein at least one αHL monomer further comprises a time-to-thread (TTT) substitution at a position corresponding to one or both of position 12 and position 17 of SEQ ID NO: 3.

3. An isolated polypeptide comprising one or more alpha-hemolysin monomers, wherein at least one of the alpha-hemolysin monomers comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3 and an H35L substitution.

4. The isolated polypeptide of claim 3 , wherein one or more of the alpha-hemolysin monomers further comprise a time-to-thread (TTT) substitution at a position corresponding to position 12 and/or position 17 of SEQ ID NO: 3.

5. The isolated polypeptide of claim 4 , wherein the TTT substitution is selected from the group consisting of T12K, T12R, N17K, and N17R.

6. The isolated polypeptide of claim 3 having one alpha-hemolysin monomer.

7. The isolated polypeptide of claim 3 having at least two alpha-hemolysin monomers, wherein each monomer of the polypeptide is separated from each adjacent monomer in the polypeptide by a flexible linker.

8. The isolated polypeptide of claim 7 having from two to seven alpha-hemolysin monomers.

9. The isolated polypeptide of claim 3 , wherein at least one of the one or more alpha-hemolysin monomers further comprises an attachment component.

10. The isolated polypeptide of claim 9 , wherein the attachment component comprises a SpyTag or SpyCatcher peptide.

11. The isolated polypeptide of claim 9 , wherein the attachment component joins the alpha-hemolysin monomer to a DNA polymerase, an RNA polymerase, or a reverse transcriptase.

12. A heptameric pore complex comprising seven alpha-hemolysin monomers, wherein at least one of the alpha-hemolysin monomers comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3 and an H35L substitution.

13. The heptameric pore complex of claim 12 , further comprising a polymerase linked to one of the alpha-hemolysin monomers.

14. The heptameric pore complex of claim 13 , wherein the polymerase is covalently linked to the alpha-hemolysin monomer.

15. The heptameric pore complex of claim 12 , wherein one or more of the alpha-hemolysin monomers further comprises a time-to-thread (TTT) substitution at a position corresponding to one or both of position 12 and position 17 of SEQ ID NO: 3.

16. The heptameric pore complex of claim 15 , wherein the TTT substitution or substitutions is/are selected from the group consisting of T12K, T12R, N17K, and N17R.

17. The heptameric pore complex of claim 12 , wherein each alpha-hemolysin monomer of the heptameric pore complex is disposed within a separate polypeptide from the other alpha-hemolysin monomers of the heptameric pore complex.

18. The heptameric pore complex of claim 12 , wherein from two to seven of the alpha-hemolysin monomers of the heptameric pore complex are disposed within a single polypeptide.

19. The isolated polypeptide of claim 12 , wherein at least one of the one or more alpha-hemolysin monomers further comprises an attachment component.

20. The isolated polypeptide of claim 19 , wherein the attachment component comprises a SpyTag or SpyCatcher peptide.

21. The isolated polypeptide of claim 19 , wherein the attachment component joins the alpha-hemolysin monomer to a DNA polymerase, an RNA polymerase, or a reverse transcriptase.

22. A method of making a heptameric pore complex, the method comprising heating a plurality of polypeptides of claim 3 in the presence of a lipid at a temperature greater than 25° C. for a sufficient period of time for the polypeptides to self-aggregate into alpha-hemolysin heptamers.

23. The method of claim 22 , wherein the temperature at which the polypeptides are heated is 30° C. or higher.

24. The method of claim 23 , wherein the temperature at which the polypeptides are heated is from 30° C. to 50° C.

25. A chip for nucleic acid sequencing, said chip comprising a heptameric pore complex of claim 12 disposed in a membrane adjacent to or in proximity to an electrode.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Craig Timothy Kellogg
• Cech Cynthia Ann
• Dorwart Michael
• Jensen Liv Elisabeth
• Porter Marshall Winston
• Tzitzilonis Christos
• Yang Alexander Hyun-Min

Classifications

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title

• C07K14/31

  from Staphylococcus (G) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2565/607

  being a sensor, e.g. electrode · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title