Nanopore biosensors for detection of proteins and nucleic acids

• US 10,006,905 B2
• Filed: 03/25/2014
• Issued: 06/26/2018
• Est. Priority Date: 03/25/2013
• Status: Active Grant

• Current Assignee: Katholieke Universiteit Leuven
• Original Assignee: Katholieke Universiteit Leuven
• Inventors: Maglia, Giovanni, Soskine, Mikhael
• Primary Examiner(s): Li, Ruixiang
• Application Number:
  US14/779,895
• Publication Number:
  US 20160053300A1
• Time in Patent Office: 1,554 Days
• CPC Class Codes: C07K 14/255 Salmonella (G)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 33/48721 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 Class Codes: G01N 33/54306 Solid-phase reaction mechanisms

Abstract

Described herein are nanopore biosensors based on a modified cytolysin protein. The nanopore biosensors accommodate macromolecules including proteins and nucleic acids, and may additionally comprise ligands with selective binding properties.

Claims

1. A modified ClyA pore comprising a plurality of subunits, wherein each subunit comprises a polypeptide represented by an amino acid sequence of at least 80% identical to SEQ ID NO:

1 and wherein (a) exactly one Cys residue is substituted with Ala;

(b) exactly one Cys residue is substituted with Ser and exactly one Cys residue is substituted with Ala;

or (c) exactly one Cys residue is substituted with Ser and one or more of L99, E103, F166, and K294 are substituted with other amino acid residues, wherein the positions of the amino acid substitutions are numbered with reference to an amino acid sequence as set forth in SEQ ID NO;

1 starting from the third amino acid residue.

2. The modified ClyA pore of claim 1, wherein the Cys residue substituted with Ala is C87 and/or the Cys residue substituted with Ser is C285.

3. The modified ClyA pore of claim 1, wherein each subunit comprises at least one additional amino acid substitution selected from L99Q, E103G, F166Y, and K294R.

4. The modified ClyA pore of claim 1, wherein each subunit comprises a polypeptide represented by the amino acid sequence of SEQ ID NO:

2.

5. The modified ClyA pore of claim 1, wherein each subunit comprises an additional amino acid substitution of L203V.

6. The modified ClyA pore of claim 1, wherein each subunit comprises a polypeptide represented by the amino acid sequence of SEQ ID NO:

3.

7. The modified ClyA pore of claim 1, wherein the modified ClyA pore comprises at least 12 subunits.

8. The modified ClyA pore of claim 7, wherein the modified ClyA pore comprises 12 subunits.

9. The modified ClyA pore of claim 7, wherein the modified ClyA pore comprises 13 subunits.

10. The modified ClyA pore of claim 1, wherein the modified ClyA pore has a cis diameter of at least 3.5 nm.

11. The modified ClyA pore of claim 1, wherein the modified ClyA pore has a trans diameter of at least 6 nm.

12. The modified ClyA pore of claim 1, wherein the modified ClyA pore remains open when the voltage across the modified ClyA pore ranges from −

60 +90 to −

150 mV.

13. The modified ClyA pore of claim 1, wherein the modified ClyA pore translocates a DNA or protein analyte.

14. The modified ClyA pore of claim 13, wherein the DNA is single stranded DNA (ssDNA) or double stranded DNA (dsDNA).

15. The modified ClyA pore of claim 13, wherein the protein analyte is a protein with a molecular weight in the range of 15-70 kDa.

16. The modified ClyA pore of claim 1, wherein the amino acid sequence of SEQ ID NO:

1 is further modified to have at its C-terminus a Gly-Ser-Ser linker followed by a hexahistidine tag.

17. The modified ClyA pore of claim 16, wherein the first histidine of the hexahistidine tag is replaced with a tyrosine.

18. The modified ClyA pore of claim 1, which is in combination with a ligand that confers selective binding properties to an analyte.

19. A method for translocating DNA through a modified ClyA pore that is capable of translocating DNA, comprising the steps of providing the modified ClyA pore of claim 1, applying a voltage of at least +50 mV across the modified ClyA pore, adding a sample containing the DNA to the cis opening of the modified ClyA pore, and measuring the current flowing through the pore.

20. The method of claim 19, wherein a current blockade indicates translocation of the DNA.

21. The method of claim 19, wherein the modified ClyA pore is used under conditions of high ionic strength.

22. A device for translocating DNA, comprising:

a fluid-filled compartment separated by a membrane into a first chamber and a second chamber;

electrodes capable of applying potential across the membrane;

one or more modified ClyA pores of claim 1 inserted in the membrane;

a solution of high ionic strength in one chamber, wherein DNA translocates through the one or more modified ClyA pores from the first chamber to the second chamber.