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US-20260125749-A1 - HAIRPIN LOOP METHOD FOR DOUBLE STRAND POLYNUCLEOTIDE SEQUENCING USING TRANSMEMBRANE PORES

US20260125749A1US 20260125749 A1US20260125749 A1US 20260125749A1US-20260125749-A1

Abstract

The invention relates to a new method of sequencing a double stranded target polynucleotide. The two strands of the double stranded target polynucleotide are linked by a bridging moiety. The two strands of the target polynucleotide are separated using a polynucleotide binding protein and the target polynucleotide is sequenced using a transmembrane pore.

Inventors

  • Clive Gavin Brown
  • James Anthony Clarke
  • Graham James Hall
  • Gavin Harper
  • Andrew John Heron
  • James White

Assignees

  • OXFORD NANOPORE TECHNOLOGIES PLC

Dates

Publication Date
20260507
Application Date
20241106

Claims (18)

  1. 1 .- 35 . (canceled)
  2. 36 . A method for nanopore sensing, the method comprising: (i) transiently coupling a nucleic acid construct to a membrane comprising a nanopore, wherein the nucleic acid construct comprises a target polynucleotide, a leader that facilitates passing the nucleic acid construct through the nanopore, and a marker; (ii) applying a voltage across the membrane sufficient to cause the nucleic acid construct to move through the nanopore; and (iii) sensing the nucleic acid construct as it moves through the nanopore.
  3. 37 . The method of claim 36 , wherein the membrane is an amphiphilic layer, a lipid bilayer or a block copolymer.
  4. 38 . The method of claim 37 , wherein the amphiphilic layer is a 1,2-diphytanoyl-glycero-3-phosphocholine lipid bilayer.
  5. 39 . The method of claim 37 , wherein the block copolymer is a triblock copolymer having outer hydrophilic layers and an inner hydrophobic core.
  6. 40 . The method of claim 36 , wherein nucleic acid construct is coupled to the membrane via a tether.
  7. 41 . The method of claim 40 , wherein the teacher is a lipid, a fatty acid, a sterol, a carbon nanotube, or an amino acid.
  8. 42 . The method of claim 41 , wherein the tether is a cholesterol TEG tether.
  9. 43 . The method of claim 36 , wherein the nanopore is a protein nanopore.
  10. 44 . The method of claim 43 , wherein the protein nanopore is α-hemolysin (α-HL) nanopore or a MspA nanopore.
  11. 45 . The method of claim 36 , wherein the marker is positioned within the leader.
  12. 46 . The method of claim 36 , wherein the marker is positioned between the leader and the target polynucleotide.
  13. 47 . The method of claim 36 , wherein the marker results in a distinctive current when they interact with the pore.
  14. 48 . The method of claim 36 , wherein the marker indicative of the source of the target polynucleotide.
  15. 49 . The method of claim 36 , wherein the marker comprises an abasic region, nucleotide sequence, unnatural nucleotide, fluorophore, or cholesterol.
  16. 50 . The method of claim 36 , wherein the sensing comprises reading nucleotides of the nucleic acid construct as the nucleotides move through the nanopore.
  17. 51 . The method of claim 50 , wherein the nucleotides are read from single channel currents obtained from electrodes connected to a buffered solution on cis and trans sides of the membrane comprising the nanopore.
  18. 52 . A method of sequencing a double stranded target polynucleotide, comprising: (a) providing a construct comprising the target polynucleotide, wherein the two strands of the target polynucleotide are linked at or near one end of the target polynucleotide by a bridging moiety; (b) separating the two strands of the target polynucleotide to provide a single stranded polynucleotide comprising one strand of the target polynucleotide linked to the other strand of the target polynucleotide by the bridging moiety; (c) moving the single stranded polynucleotide through a transmembrane pore such that a proportion of the nucleotides in the single stranded polynucleotide interact with the pore; and (d) measuring the current passing through the pore during each interaction and thereby determining the sequence of the target polynucleotide, wherein the separating in step (b) comprises contacting the construct with a polynucleotide binding protein which separates the two strands of the target polynucleotide.

Description

FIELD OF THE INVENTION The invention relates to a new method of sequencing a double stranded target polynucleotide. The two strands of the target polynucleotide are linked by a bridging moiety. The two strands of the target polynucleotide are separated by a polynucleotide binding protein. Sequencing of the target polynucleotide is carried out using a transmembrane pore. BACKGROUND OF THE INVENTION There is currently a need for rapid and cheap nucleic acid (e.g. DNA or RNA) sequencing technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of nucleic acid and require a high quantity of specialist fluorescent chemicals for signal detection. Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology. When a potential is applied across a nanopore, there is a drop in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nanopore detection of the nucleotide gives a current blockade of known signature and duration. The concentration of a nucleotide can then be determined by the number of blockade events (where an event is the translocation of an analyte through the nanopore) per unit time to a single pore. In the “Strand Sequencing” method, a single polynucleotide strand is passed through the pore and the nucleotides are directly identified. Strand Sequencing can involve the use of a nucleotide handling enzyme, such as Phi29 DNA polymerase, to control the movement of the polynucleotide through the pore. Nanopore sequencing, using enzymes to control the translocation of dsDNA through the nanopore, has in the past focused on only reading one strand of a dsDNA construct. When the enzyme is used as polymerase, the portion to be sequenced is single stranded. This is fed through the nanopore and the addition of dNTPs at a primer/template junction on top of the strand pulls the single stranded portion through the nanopore in a controlled fashion. The majority of the published literature uses this approach to control strand movement (Lieberman et al. (2010) “Processive Replication of Single DNA Molecules in a Nanopore Catalyzed by phi29 DNA Polymerase” J. Am. Chem. Soc. 132 (50): 17961-17972). In the polymerase mode, the complementary strand cannot be sequenced. When the enzyme is used as a double stranded exonuclease as published (Lieberman et al. (2010) supra), the unzipping of the complementary strand is accompanied by the digestion of this strand. It is therefore not possible to sequence the complementary strand with this approach. The complementary strand cannot therefore be captured and sequenced by the nanopore. Hence, only half of the DNA information in dsDNA is sequenced. In more detail, when both polymerase and exonuclease activity are inhibited (by running without tri-phosphates bases and with excess of EDTA), enzymes such as Phi29 DNA polymerase have been shown to unzip dsDNA when pulled through a nanopore by a strong applied field (FIG. 1) (Lieberman et al. (2010) supra). This has been termed unzipping mode. Unzipping mode implies that it is the unzipping of dsDNA above or through the enzyme, and importantly, it is the requisite force required to disrupt the interactions of both strands with the enzyme and to overcome the hydrogen bonds between the hybridised strands. In the past the second complementary strand was considered to be essential for efficient enzyme binding. In addition, it was thought that the requisite force required to unzip the strand above or in the enzyme was a dominant braking effect slowing DNA through the pore. Herein we describe how enzymes such as Phi29 DNA polymerase can act as a molecular brake for ssDNA, enabling sufficient controlled movement through a nanopore for sequencing around the hairpin turns of specially designed dsDNA constructs to sequence both the sense and anti-sense strands of dsDNA (FIG. 2). Unzipping mode has in the past predominantly been performed using templates where the distal part of the analyte is blunt ended (FIG. 1). Small hairpins have occasionally been used, but were only included to simplify DNA design. Previous work has not considered the use of hairpins on long dsDNA to provide the ability to read both strands. This is because the unzipping movement model has not considered Phi29 DNA polymerase or related enzymes capable of controlling the movement of the DNA when entering ssDNA regions (i.e. when moving around the hairpin and along the anti-sense strand-FIG. 2). SUMMARY OF THE INVENTION The inventors have surprisingly demonstrated that both strands of a double stranded target polynucleotide can be sequenced by a nanopore when the two strands are linked by a bridging moiety and then separated. Furthermore