Selective modification of polymer subunits to improve nanopore-based analysis

Patent metadata

Field	Value
Publication number	US-2024360502-A1
Application number	US-202418769519-A
Country	US
Kind code	A1
Filing date	Jul 11, 2024
Priority date	Aug 30, 2013
Publication date	Oct 31, 2024
Grant date	—

Abstract

The present disclosure provides method and systems for improving nanopore-based analyses of polymers. The disclosure provides methods for selectively modifying one or more monomeric subunit(s) of a kind a pre-analyte polymer that results polymer analyte with a modified subunit. The polymer analyte produces a detectable signal in a nanopore-based system. The detectable signal, and/or its deviation from a reference signal, indicates the location of the modified subunit in the polymer analyte and, thus, permits the identification of the subunit at that location in the original pre-analyte polymer.

First claim

1 . A method for detecting the presence of a target nucleic acid subunit of a kind in a pre-analyte polymer, the method comprising:
(a) selectively modifying a guanine or cytosine nucleobase subunit of a nucleic acid pre-analyte polymer to produce an abasic site, wherein selectively modifying the guanine or cytosine nucleobase subunit comprises contacting the target polymer subunit of a kind with an agent to produce a modified subunit and converting the modified subunit to an abasic site, thereby producing the nucleic acid polymer analyte; (b) translocating the nucleic acid polymer analyte comprising the abasic site from a first conductive liquid medium to a second conductive liquid medium through a nanopore, wherein the nanopore provides liquid communication between the first conductive liquid medium and the second conductive liquid medium; (c) measuring an ion current between the first conductive liquid medium and the second conductive liquid medium as the nucleic acid polymer analyte passes through the nanopore; and (d) detecting the abasic site based on the measured ion current to detect the presence of the target nucleic acid subunit of a kind in the nucleic acid pre-analyte polymer.

2 . The method of claim 1 , wherein selectively modifying the cytosine residue comprises selectively converting the cytosine residue into a uracil residue to produce the modified subunit.

3 . The method of claim 2 , wherein converting the cytosine residue into the uracil residue comprises contacting the pre-analyte polymer with an agent selected from bisulfite, cytosine deaminase, NO, N 2 O 3 , and Echinomycin.

4 . The method of claim 2 , wherein converting the uracil residue into an abasic site comprises contacting the pre-analyte polymer with a nucleic acid error correction enzyme.

5 . The method of claim 4 , wherein the nucleic acid error correction enzyme is uracil deglycosylase (UNG).

6 . The method of claim 1 , wherein selectively modifying the cytosine residue comprises selectively methylating the cytosine residue to produce the modified subunit.

7 . The method of claim 6 , wherein selectively methylating the cytosine residue comprises contacting the pre-analyte polymer with a methyltransferase enzyme or incorporating a methyl-cytosine analog into the pre-analyte polymer sequence in place of the cytosine residue using a polymerase enzyme.

8 . The method of claim 6 , wherein converting the methylated cytosine residue into an abasic site comprises contacting the pre-analyte polymer with a nucleic acid error correction enzyme.

9 . The method of claim 1 , wherein selectively modifying the guanine residue comprises selectively methylating the guanine residue to produce the modified subunit.

10 . The method of claim 9 , wherein selectively methylating the guanine residue comprises incorporating a methyl-guanine analog into the pre-analyte polymer sequence in place of the guanine residue using a polymerase enzyme or contacting the pre-analyte polymer with a methyltransferase enzyme.

11 . The method of claim 10 , wherein converting the methylated guanine residue into an abasic site comprises contacting the pre-analyte polymer with a nucleic acid error correction enzyme.

12 . The method of claim 1 , wherein the nucleic acid is DNA or RNA, and wherein the pre-analyte polymer further comprises a peptide nucleic acid (PNA).

13 . The method of claim 1 , wherein the pre-analyte polymer is DNA or RNA.

14 . The method of claim 1 , wherein step (d) comprises:
(i) comparing the measured ion current to an ion current corresponding to a reference polymer comprising the target nucleobase without modification; and (ii) detecting the presence of a difference in the ion currents compared in step (i) wherein the presence of a difference in ion currents indicates the presence of the abasic site in the polymer analyte or detecting the absence of a difference in the ion currents compared in step (i), wherein the absence of a difference in ion currents indicates the absence of the abasic site in the polymer analyte.

15 . The method of claim 14 , wherein the reference polymer comprises the same nucleic acid sequence as the pre-analyte polymer.

16 . The method of claim 14 , further comprising translocating the reference polymer from the first conductive liquid medium to the second conductive liquid medium through the nanopore and measuring an ion current to provide the ion current corresponding to the reference polymer before sub-step (i).

17 . The method of claim 1 , comprising determining the identity of the target polymer subunit at a position in the pre-analyte polymer sequence that corresponds to the position of the modified nucleobase subunit in the polymer analyte.

18 . The method of claim 1 , wherein the protein nanopore is alpha-hemolysin, Mycobacterium smegmatis porin A (MspA), or a homolog thereof.

19 . A method for analyzing a nucleic acid analyte comprising:
(a) incorporating a modified guanine or cytosine into the nucleic acid analyte; (b) contacting the nucleic acid analyte with an error correcting enzyme capable of removing the modified guanine or cytosine to provide an abasic site in the nucleic acid analyte; (c) translocating the nucleic acid analyte comprising the abasic site from a first conductive liquid medium to a second conductive liquid medium through a nanopore, wherein the nanopore provides liquid communication between the first conductive liquid medium and the second conductive liquid medium; (d) measuring an ion current between the first conductive liquid medium and the second conductive liquid medium as the nucleic acid polymer analyte passes through the nanopore; and (e) detecting the abasic site based on the measured ion current.

20 . A nanopore-based system for detecting the presence of a target polymer subunit of a kind in a pre-analyte polymer, comprising:
a nanopore; a first conductive medium; a second conductive medium; a pre-analyte polymer comprising a target polymer subunit of a kind; at least one agent for selectively modifying the target polymer subunit of a kind in the pre-analyte polymer; and at least one agent to convert the selectively modified target polymer subunit of a kind to an abasic site to produce a polymer analyte, wherein the nanopore provides liquid communication between the first conductive liquid medium and the second conductive liquid medium, wherein the abasic site produces a distinct current blockade signal in the nanopore-based system upon translocation of the polymer analyte from the first conductive liquid medium to the second conductive liquid medium through the nanopore, and wherein detecting the distinct current blockade signal by measuring an ion current between the first conductive liquid medium and the second conductive liquid medium as the polymer analyte passes through the nanopore detects the presence of the target polymer subunit of a kind in the pre-analyte polymer.

Assignees

• Univ Washington Through Its Center For Commercialization
• Illumina Inc

Inventors

• Gundlach Jens H
• Laszlo Andrew
• Derrington Ian
• Mandell Jeffrey G

Classifications

• G01N33/48721

  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 title

• B01D57/02

  by electrophoresis (treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title