Method of preparation of nanopore and uses thereof

• US 11,795,191 B2
• Filed: 04/01/2019
• Issued: 10/24/2023
• Est. Priority Date: 04/09/2012
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University), THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF COMMERCE
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Ju, Jingyue, Kumar, Shiv, Tao, Chuanjuan, Chien, Minchen, Russo, James J., Kasianowicz, John J., Robertson, Joseph W. F.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US16/371,646
• Publication Number:
  US 20190309008A1
• Time in Patent Office: 1,667 Days
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/9125 with a definite EC number (2.7.7.-)
• 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

Abstract

This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to composition, method, and system for sequencing a nucleic acid using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule.

Claims

1. A method for nucleic acid sequencing, the method comprising providing an array of individually addressable sites, each site having a nanopore coupled to a nucleic acid polymerase, wherein the polymerase is attached to said nanopore within 0.7 nm of the entrance to said nanopore, and, at a given site of said array, polymerizing tagged nucleotides with the polymerase, wherein each tagged nucleotide comprises a tag capable of being cleaved in a nucleotide polymerization event and detected with the aid of the nanopore, wherein the tag is released and detected by the nanopore at said given site, wherein the nanopore is inserted into solvent-free planar lipid bilayer membranes, wherein the nucleotide sequence of a single-stranded DNA is determined, and comprises:

a) contacting the single-stranded DNA, wherein the single-stranded DNA is in an electrolyte solution and wherein the single-stranded DNA has a primer hybridized to a portion thereof, with;

(I) a nanopore of said array of individually addressable sites,

wherein the nanopore is an individually addressable nanopore in a membrane,

and

(II) at least four of said tagged nucleotides,

wherein each tagged nucleotide is a deoxyribonucleotide polyphosphate (dNPP) analogue,

under conditions permitting the polymerase to catalyze incorporation of one of the dNPP analogues into the primer if it is complementary to the nucleotide residue of the single-stranded DNA which is immediately 5′

to a nucleotide residue of the single-stranded DNA hybridized to the 3′

terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein each of the four dNPP analogues has the structure;

2. The method of claim 1, wherein the tag comprises an oligonucleotide, wherein the oligonucleotide comprises nucleotides having natural bases and at least one abasic site.

3. The method of claim 2, wherein the oligonucleotide comprises nucleotides having natural bases and

at least one nucleotide having a modified base.

4. The method of claim 1, wherein the tag is detectable by its charge, shape, size, or any combination thereof.

5. The method of claim 1, wherein the tag comprises an additional identifiable moiety.

6. The method of claim 5, wherein the additional identifiable moiety is a coumarin-based dye.

7. The method of claim 1, wherein the tag further comprises one or more of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a chemiluminescent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, an aliphatic acid, an aromatic acid, an alcohol, a thiol group, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, an azido group, or a combination thereof.

8. The method of claim 1, wherein the oligonucleotide of the tag has a length of approximately 30 bases.

9. The method of claim 1, wherein n is four.

10. The method of claim 1, wherein the oligonucleotide of the tag comprises a 5′

-OH, and the 5′

-OH of said oligonucleotide is attached to the terminal phosphate of the tagged nucleotide analogue.

11. The method of claim 1, wherein the oligonucleotide of the tag comprises a 3′

-OH, and the 3′

-OH of said oligonucleotide is attached to the terminal phosphate of the tagged nucleotide analogue.

12. The method of claim 1, wherein alkaline phosphatase molecules are covalently attached adjacent to the polymerase at the rim of each nanopore.

13. The method of claim 1, wherein the tag has a charge which is reverse in sign relative to the charge on the rest of the tagged nucleotide.

14. The method of claim 13, wherein the magnitude of the charge on the tag is the same as the magnitude of the charge on the remainder of the tagged nucleotide.

15. The method of claim 1, wherein the nanopore is an alpha hemolysin nanopore.

16. The method of claim 1, wherein the DNA polymerase is φ

29 DNA Polymerase.

17. The method of claim 1, wherein each dNPP analogue has a tag comprising an oligonucleotide having a length different from each other dNPP analogue.

18. The method of claim 1, wherein the polymerase catalyzes incorporation of the dNPP at a rate slower than the rate at which the tag is released and detected by the nanopore.

19. A method for sequencing a nucleic acid template, the method comprising providing an array of individually addressable sites, each site having a nanopore coupled to a nucleic acid polymerase, wherein the polymerase is attached to said nanopore within 0.7 nm of the entrance to said nanopore, and, at a given site of said array, polymerizing tagged nucleotides with the polymerase, wherein each tagged nucleotide comprises a tag capable of being cleaved in a nucleotide polymerization event and detected with the aid of the nanopore, wherein the tag is released and detected by the nanopore at said given site, wherein the nanopore is inserted into solvent-free planar lipid bilayer membranes, wherein alkaline phosphatase molecules are covalently attached adjacent to the polymerase at the rim of each nanopore, wherein the template is added along with a primer to form a loop, thereby determine the nucleic acid sequence.

20. The method of claim 19, wherein the nucleotide sequence of a single-stranded DNA is determined, and comprises:

a) contacting the single-stranded DNA, wherein the single-stranded DNA is in an electrolyte solution and wherein the single-stranded DNA has a primer hybridized to a portion thereof, with;

(III) a nanopore of said array of individually addressable sites,

wherein the nanopore is an individually addressable nanopore in a membrane, and

(IV) at least four of said tagged nucleotides,

wherein each tagged nucleotide is a deoxyribonucleotide polyphosphate (dNPP) analogue, under conditions permitting the polymerase to catalyze incorporation of one of the dNPP analogues into the primer if it is complementary to the nucleotide residue of the single-stranded DNA which is immediately 5′

to a nucleotide residue of the single-stranded DNA hybridized to the 3′

terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein each of the four dNPP analogues has the structure;