Nanopore-based polymer analysis with mutually-quenching fluorescent labels

• US 9,885,079 B2
• Filed: 10/08/2015
• Issued: 02/06/2018
• Est. Priority Date: 10/10/2014
• Status: Active Grant

• Current Assignee: Quantapore, Inc.
• Original Assignee: Quantapore, Inc.
• Inventors: Huber, Martin
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US14/878,292
• Publication Number:
  US 20160122812A1
• Time in Patent Office: 852 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

The invention is directed to a method for determining a monomer sequence of a polymer that is translocated through a nanopore. Monomers of the polymer are labeled with fluorescent labels such that in free solution fluorescent labels of adjacent monomers substantially quench each other and wherein the nanopore constrains fluorescent labels within its bore into a constrained state wherein no detectable fluorescent signal can be generated. By exciting the fluorescent label of each monomer as it exits the nanopore and transitions from a constrained state to a quenched state with an adjacent fluorescent label, a fluorescent signal can be generated by the exiting fluorescent label that allows its monomer to be identified, thereby permitting a monomer sequence to be determined from a sequence of fluorescent signals as the polymer translocates through the nanopore.

Claims

1. A method of determining a nucleotide sequence of at least one polynucleotide, the method comprising the steps of:

translocating at least one single stranded polynucleotide through a nanopore, wherein different kinds of nucleotides of the single stranded polynucleotide are labeled with different fluorescent labels from a mutually quenching set and wherein the nanopore has a bore dimensioned to force the fluorescent labels within the nanopore into a constrained state wherein substantially no detectable signal is generated and wherein each fluorescent label of the mutually quenching set (i) quenches fluorescence of every other fluorescent label of the same set whenever inch labels are attached to adjacent nucleotides of a polynucleotide in free solution, and (ii) generates a distinct fluorescent signal in a non-quenched state;

exciting the fluorescent label of each nucleotide upon exiting the nanopore and during its transition from a constrained state to formation of a quenched state with an adjacent nucleotide;

measuring a fluorescent signal generated by the exiting fluorescent label to identify the nucleotide to which the fluorescent label is attached; and

determining a nucleotide sequence of the polynucleotide from a sequence of fluorescent signals.

2. The method of claim 1 wherein nucleotides of said polynucleotide are labeled with second members of a FRET pair, each second member producing a FRET signal indicative of the nucleotide to which it is attached, and wherein nucleotides of said polynucleotide pass in sequence by a first member of the FRET pair positioned adjacent to said nanopore so that each second member upon exiting said nanopore passes within a FRET distance of the first member of the FRET pair;

and

wherein said step of exciting includes exposing the first member to a light beam of a first wavelength so that FRET occurs between the first and second members of the FRET pair within the FRET distance to generate a FRET signal of a second wavelength indicative of the nucleotide exiting said nanopore.

3. The method of claim 2 wherein said nanopore is disposed in a solid phase membrane and wherein said first member of said FRET pair is attached to the solid phase membrane adjacent to said nanopore.

4. The method of claim 2 wherein said nanopore is a protein nanopore and wherein said first member of said FRET pair is attached to the protein nanopore.

5. The method of claim 2 wherein said first member of said FRET pair is a donor and said second member of said FRET pair is an acceptor.

6. The method of claim 5 wherein said donor is a quantum dot.

7. The method of claim 5 wherein said acceptor is a fluorescent organic dye.

8. The method of claim 5 wherein said step of exposing includes exposing said donor to an evanescent wave from a total internally reflected excitation.

9. The method of claim 1 wherein said nanopore is in a nanopore array comprising a plurality of substantially identical nanopores.

10. The method of claim 9 wherein said nanopore array comprises a solid phase membrane separating a first chamber from a second chamber, the solid phase membrane comprising a planar array of apertures each having said protein nanopore immobilized therein.

17. The method of claim 1 wherein said nanopore is a protein nanopore.

18. The method of claim 17 wherein said protein nanopore is a hemolysin or a variant thereof.

19. The method of claim 1 wherein said single stranded polynucleotides are labeled by polymerase extension.

20. The method of claim 1 wherein one or more of said fluorescein labels are selected from among rhodamine dyes, fluorescein dyes and cyanine dyes.

11. A method of determining a nucleotide sequence of at least one polynucleotide, the method comprising the steps of:

translocating at least one single stranded polynucleotide through a nanopore, wherein substantially all different kinds of nucleotides of the single stranded polynucleotide are labeled with adducts and/or different fluorescent labels from a mutually quenching set and wherein the nanopore has a bore dimensioned to force the fluorescent labels within the nanopore into a constrained state so that substantially no detectable signal is generated and wherein the adducts and fluorescent labels are selected so that translocation speed through the nanopore is reduced to permit detection of individual nucleotides and wherein each adduct is an organic molecule having a molecular weight in the range of that of an organic dye and wherein each fluorescent label of the mutually quenching set (i) quenches fluorescence of every other fluorescent label of the same set whenever such labels are attached to adjacent nucleotides of a polynucleotide in free solution, and (ii) generates a distinct fluorescent signal in a non-quenched stat;

exciting the fluorescent label of each nucleotide upon exiting the nanopore and during its transition from a constrained state to formation of a quenched state with an adjacent nucleotide;

measuring a fluorescent signal generated by the exiting fluorescent label to identify the nucleotide to which the fluorescent label is attached; and

determining a nucleotide sequence of the polynucleotide from a sequence of fluorescent signals.

12. The method of claim 11 wherein said adducts and said fluorescent labels are selected so that said translocation speed is in the range of 1 to 1000 nucleotides per second.

13. The method of claim 12 wherein said nanopore is a protein nanopore.

14. The method of claim 13 wherein said nanopore is a hemolysin or a mutant thereof.

15. The method of claim 12 wherein said single stranded polynucleotides are labeled by polymerase extension.

16. The method of claim 12 wherein one or more of said adducts and/or fluorescent labels are selected from among rhodamine dyes, fluorescein dyes and cyanine dyes.