Alpha-hemolysin variants and uses thereof

Patent metadata

Field	Value
Publication number	US-12378604-B2
Application number	US-202117185021-A
Country	US
Kind code	B2
Filing date	Feb 25, 2021
Priority date	Apr 21, 2016
Publication date	Aug 5, 2025
Grant date	Aug 5, 2025

Abstract

Described herein are variants of alpha-hemolysin having at least one mutation, such as a mutation to a positive charge. In certain examples, the mutation is selected from V149K, E287R, H35G, T109K, P151K, K147N, E111N, M113A, or combinations thereof in the mature, wild-type alpha-hemolysin amino acid sequence. The α-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having a decreased time-to-thread. The decreased time-to-thread, for example, increases DNA sequencing efficiency and accuracy.

First claim

What is claimed is:

1. An alpha-hemolysin variant comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14, wherein said amino acid sequence comprises (i) an H35 substitution of SEQ ID NO: 14 and (ii) a positively charged amino acid substituted for one or more positions of SEQ ID NO: 14 selected from the group consisting of T109, V149, P151, and E287 of SEQ ID NO: 14.

2. The alpha-hemolysin variant of claim 1 , wherein the amino acid sequence comprises at least 98% sequence identity to the sequence set forth as SEQ ID NO: 14.

3. The alpha-hemolysin variant of claim 1 , wherein the variant further comprises a substitution at H144A of SEQ ID NO: 14.

4. The alpha-hemolysin variant according to claim 1 , wherein the variant further comprises two or more glycine residues at residues 127 through 131.

5. The alpha-hemolysin variant of claim 1 , wherein the variant further comprises one or more H35G, E111N, M113A, H144A, or K147N substitutions of SEQ ID NO: 14.

6. The alpha-hemolysin variant of claim 1 , wherein the variant further comprises a polymerase attached thereto.

7. The alpha-hemolysin variant of claim 6 , wherein the polymerase is a DNA polymerase.

8. The alpha-hemolysin variant of claim 7 , wherein the DNA polymerase is covalently attached to the alpha-hemolysin variant.

9. The alpha-hemolysin variant of claim 8 , wherein the DNA polymerase is covalently attached to the alpha-hemolysin variant via an isopeptide bond.

10. The alpha-hemolysin variant of claim 9 , wherein the DNA polymerase is covalently attached to the alpha-hemolysin variant via a Spy Tag and SpyCatcher binding pair to form a SpyTag/SpyCatcher linkage.

11. The alpha-hemolysin variant of claim 1 , wherein the at least 95% sequence identity to SEQ ID NO: 14 is a sequence identity according to CLUSTAL-W program in MacVector version 13.0.7, operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

12. A heptameric nanopore assembly comprising seven alpha-hemolysin subunits arranged in a nanopore, wherein at least one of the seven alpha-hemolysin subunits comprises the alpha-hemolysin variant of claim 1 .

13. The heptameric nanopore assembly of claim 12 , wherein a DNA polymerase is covalently attached to one of the seven alpha-hemolysin subunits.

14. The heptameric nanopore assembly of claim 13 , wherein the polymerase is covalently attached to one of the seven alpha-hemolysin subunits via an isopeptide bond.

15. The heptameric nanopore assembly of claim 13 , wherein the nanopore has a decreased time to thread (TTT) relative to a heptameric nanopore assembly consisting of native alpha-hemolysins.

16. The heptameric nanopore assembly of claim 15 , wherein the nanopore has at least a 10% decreased TTT relative to the heptameric nanopore assembly consisting of the native alpha-hemolysins.

17. The heptameric nanopore assembly of claim 15 , wherein the nanopore has at least a 20% decreased TTT relative to the heptameric nanopore assembly consisting of the native alpha-hemolysins.

18. The heptameric nanopore assembly of claim 15 , wherein the nanopore has at least a 50% decreased TTT relative to the heptameric nanopore assembly consisting of the native alpha-hemolysins.

19. The heptameric nanopore assembly of claim 15 , wherein the nanopore has at least an 80% decreased TTT relative to the heptameric nanopore assembly consisting of the native alpha-hemolysins.

20. The heptameric nanopore assembly claim 12 , wherein the amino acid sequence comprises at least 98% sequence identity to the sequence set forth as SEQ ID NO: 14.

21. The heptameric nanopore assembly claim 12 , wherein the alpha-hemolysin variant further comprises a substitution at H144A of SEQ ID NO: 14.

22. The heptameric nanopore assembly claim 12 , wherein the alpha-hemolysin variant further comprises two or more glycine residues at residues 127 through 131.

23. The heptameric nanopore assembly claim 12 , wherein the alpha-hemolysin variant further comprises one or more H35G, E111N, M113A, H144A, or K147N substitutions of SEQ ID NO: 14.

24. The heptameric nanopore assembly claim 12 , wherein the at least 95% sequence identity to SEQ ID NO: 14 is a sequence identity according to CLUSTAL-W program in MacVector version 13.0.7, operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Craig Timothy K
• Ambroso Mark R
• Harris Corissa J
• Dipietro Matthew M
• Zou Yaozhong
• Porter Marshall W

Classifications

• G01N2333/31

  from Staphylococcus (G) · CPC title

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• C07K14/31

  from Staphylococcus (G) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• G01N33/48721

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title