Nanopore protein conjugates and uses thereof

Patent metadata

Field	Value
Publication number	US-11150233-B2
Application number	US-201816146053-A
Country	US
Kind code	B2
Filing date	Sep 28, 2018
Priority date	Mar 31, 2016
Publication date	Oct 19, 2021
Grant date	Oct 19, 2021

Abstract

Described herein are nanopore protein conjugates that can be used in DNA sequencing reactions. The nanopore protein conjugates includes a nanopore protein monomer that is joined to a DNA binding domain. The nanopore protein monomer is available to oligomerize with other nanopore protein monomers, while the DNA binding domain is available to bind to a template DNA strand. In certain examples, the nanopore protein monomer is an alpha-hemolysin monomer or variant thereof and the DNA binding domain is an Sso7d protein or variant thereof, such as an Sso7d-like protein. Also provided are nanopore protein assemblies incorporating the nanopore protein conjugates, along with methods of using the nanopore protein assemblies in sequencing reactions.

First claim

We claim:

1. A nanopore protein conjugate comprising a nanopore protein monomer and a DNA binding domain, wherein the DNA binding domain comprises an Sso7d domain.

2. The nanopore protein conjugate of claim 1 , wherein the nanopore protein monomer comprises an α-hemolysin (α-HL) domain.

3. The nanopore protein conjugate of claim 2 , wherein the α-HL domain comprises an amino acid sequence at least 75% identical to the amino acid sequence set forth as SEQ ID NO: 3 and wherein the Sso7d domain comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2.

4. The nanopore protein conjugate of claim 3 , wherein the α-HL domain comprises a substitution at a position corresponding to position 1, 2, 3, 4, 9, 12, 17, 35, 47, 106, 128, 129, 130, 131, 144, 149, or 287 of SEQ ID NO: 3 and wherein the substitution comprises one or more positive charges.

5. The nanopore protein conjugate of claim 4 , wherein the α-HL domain further comprises an H144A substitution.

6. The nanopore protein conjugate of claim 4 , wherein the α-HL domain comprises a substitution at a position corresponding to residues T12 and N17.

7. The nanopore protein conjugate of claim 4 , wherein the α-HL domain comprises a substitution selected from one or more of T12K, T12R, N17K, N17R, or combinations thereof.

8. The nanopore protein conjugate of claim 4 , wherein the substitution is selected from the group consisting of T12R, N17K, and N17R.

9. The nanopore protein conjugate of claim 1 , wherein the nanopore protein conjugate comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5.

10. A heptameric nanopore assembly comprising at least one of the nanopore protein conjugates according to claim 1 .

11. The heptameric nanopore assembly of claim 10 , further comprising a DNA polymerase that is joined to a nanopore monomer of the heptameric nanopore assembly.

12. The heptameric nanopore assembly of claim 11 , wherein the DNA polymerase is joined to the nanopore monomer of the heptameric nanopore assembly via a SpyTag/SpyCatcher linkage.

13. A method for detecting a target molecule, comprising:
providing a chip comprising a nanopore assembly according to claim 10 , wherein the nanopore is disposed within a membrane;
positioning a sensing electrode adjacent or in proximity to the membrane;
contacting the nanopore with a nucleic acid molecule, wherein the nucleic acid molecule is associated with a reporter molecule and comprises an address region and a probe region, wherein the reporter molecule is associated with the nucleic acid molecule at the probe region, and wherein the reporter molecule is coupled to a target molecule;
sequencing the address region while the nucleic acid molecule is in contact with the nanopore to determine a nucleic acid sequence of said address region; and
identifying, with the aid of a computer processor, the target molecule based upon a nucleic acid sequence of the sequenced address region.

14. The nanopore protein conjugate of claim 1 , wherein the nanopore protein conjugate comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5.

15. The nanopore protein conjugate of claim 1 , wherein the nanopore protein conjugate comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 5.

16. The nanopore protein conjugate of claim 1 , wherein the nanopore protein conjugate comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 5.

17. The nanopore protein conjugate of claim 1 , wherein the nanopore protein conjugate comprises SEQ ID NO: 5.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Craig Timothy
• Harris Corissa
• Dipetro Matt
• Ambroso Mark
• Zou Yaozhong
• Porter Marshall
• Delkaninia Issa
• Noble Alexxa
• Fairman James
• Wu Wiseley
• Bajaj Kapil
• Bellesia Giovanni
• Shin Seong-Ho

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• G01N33/48721 Primary

  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

• G01N2333/31

  from Staphylococcus (G) · CPC title

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title

• G01N27/44791

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