Ultrafast sequencing of biological polymers using a labeled nanopore

• US 9,279,153 B2
• Filed: 05/22/2014
• Issued: 03/08/2016
• Est. Priority Date: 09/30/2009
• Status: Expired due to Fees

• Current Assignee: Quantapore, Inc.
• Original Assignee: Quantapore, Inc.
• Inventors: Huber, Martin
• Primary Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US14/285,474
• Publication Number:
  US 20140255935A1
• Time in Patent Office: 656 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/119 the label being proteinic
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• 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

Methods and systems for sequencing a biological molecule or polymer, e.g., a nucleic acid, are provided. One or more donor labels, which are attached to a pore or nanopore, may be illuminated or otherwise excited. A polymer having a monomer labeled with one or more acceptor labels, may be translocated through the pore. Either before, after or while the labeled monomer of the polymer passes through, exits or enters the pore, energy may be transferred from the excited donor label to the acceptor label of the monomer. As a result of the energy transfer, the acceptor label emits energy, and the emitted energy is detected in order to identify the labeled monomer of the translocated polymer and to thereby sequence the polymer.

Claims

1. A system for sequencing a polymer comprising:

a nanopore substrate assembly having a donor labeled nanopore which spans a substrate, the nanopore substrate assembly having a negatively charged electrode on a cis side and a positively charged electrode on a trans side;

a polymer comprising monomers each having an acceptor label, wherein the polymer is negatively charged and is loaded on the cis side of the nanopore substrate assembly, so that the labeled monomers of the polymer are translocated in single file through the nanopore from the cis side to the trans side;

an energy source for exciting the nanopore donor label, wherein the nanopore donor label is configured to emit energy at a first wavelength in response to such excitation such that energy is transferred from the nanopore donor label to a monomer acceptor label whenever such monomer acceptor label exits the nanopore within an energy transfer distance of the nanopore donor label, each acceptor label being configured to emit energy at a second wavelength which is different from the first wavelength after such energy transfer;

and

an optical detector for detecting the energy emitted from the monomer acceptor label as a result of the transfer of energy from the nanopore donor label to the monomer acceptor label, wherein the emitted energy is associated with a single monomer and facilitates the sequencing of the polymer.

2. The system of claim 1, wherein the donor label comprises one or more quantum dots and the acceptor label comprises one or more fluorophores.

3. The system of claim 1, wherein a plurality of nanopores are incorporated into the substrate in parallel, such that a plurality of polymers may be sequenced simultaneously.

4. The system of claim 1, wherein a plurality of nanopores are incorporated into a rotatable disc, such that the nanopores may be rotatably interchanged.

5. The method of claim 1, wherein the polymer is a nucleic acid and the monomer is a nucleotide.

6. The system of claim 1 wherein said enemy transfer distance is a Fö

rster radius.

7. The system of claim 1 wherein said nanopore donor label is on said trans side of said nanopore substrate assembly.

8. A system for sequencing a polymer comprising:

a nanopore substrate assembly having a donor labeled nanopore which spans a substrate, the nanopore substrate assembly having a negatively charged side and a positively charged side;

an aqueous buffer solution surrounding the nanopore substrate assembly;

a negative electrical conductor positioned on the negatively charged side of the nanopore substrate assembly;

a positive electrical conductor positioned on the positively charged side of the nanopore substrate assembly;

a polymer comprising a monomer having an acceptor label, wherein the polymer is loaded into a port positioned on the negatively charged side of the nanopore substrate assembly;

an energy source for exciting the nanopore donor label, wherein the nanopore donor label is configured to emit energy at a first wavelength;

wherein the system is configured so that when an electric field is applied applied by the conductors to force the labeled polymer to translocate through the nanopore from the negatively charged side to the positively charged side in a single file, energy is transferred from the nanopore donor label to the monomer acceptor label after the labeled monomer passes through and exits the nanopore and comes in proximity to the nanopore donor label, wherein the energy transfer results in the monomer acceptor label emitting energy at a second wavelength which is different from the first wavelength; and

an optical detector for detecting the energy emitted from the monomer acceptor label as a result of the transfer of energy from the nanopore donor label to the monomer acceptor label, wherein the emitted energy is associated with a single monomer and facilitates the sequencing of the polymer.

9. A method for sequencing a polymer comprising:

exciting a first label attached to a nanopore wherein the first label emits energy at a first wavelength;

translocating a polymer through the nanopore, wherein the polymer comprises monomers each labeled with a second label;

transferring energy from the excited first label to the second label of each monomer after the labeled monomer passes through and exits the nanopore, wherein the energy transfer results in the second label emitting energy at a second wavelength which is different from the first wavelength;

detecting the energy emitted by the second label as a result of the energy transfer, wherein the energy emitted by the second label is associated with a single monomer; and

deducing the polymer sequence based on the detection of the emitted energy from the second labels and identification of the monomers.

10. The method of claim 9, wherein the labeled polymer is translocated through the nanopore by applying an electric field across the nanopore.

11. The method of claim 9, wherein the first label is a Quantum dot.

12. The method of claim 9, wherein the second label is a fluorophore.

13. The method of claim 9, wherein a plurality of first labels exhibiting different spectral emission maxima are attached to the nanopore and a plurality of second labels having distinct absorption/emission spectra each associated with a specific monomer are attached to the polymer, and wherein each first label and corresponding second label undergo a FRET (Fö

rster Resonance Energy Transfer) interaction such that each second labeled monomer of the polymer emits a specific wavelength that is detected and used to sequence the polymer.

14. The method of claim 9, wherein detecting the energy emitted by the second label comprises optical detection of a specific wavelength emitted by the second label and associated with a specific nucleotide.

15. The method of claim 9, wherein the polymer is a nucleic acid and the monomer is a nucleotide and wherein said step of detecting generates time dependent signals associated with monomers.

16. The method of claim 15, wherein one of four nucleotide bases is labeled and the time dependent signal arising from a nucleotide label emission is converted into a nucleic acid sequence corresponding to positions of the labeled nucleotide in the nucleic acid sequence.

17. The method of claim 16, wherein the method is repeated for each of the four nucleotides in separate samples and four partial sequences are deduced and then aligned to assemble an entire nucleic acid sequence.