Macromolecule blocks for nanopore sequencing

Patent metadata

Field	Value
Publication number	US-2025109433-A1
Application number	US-202418887792-A
Country	US
Kind code	A1
Filing date	Sep 17, 2024
Priority date	Sep 22, 2023
Publication date	Apr 3, 2025
Grant date	—

Abstract

In one aspect, the disclosed technology relates to nanopore sequencing with a polynucleotide comprising a plurality of nucleotides, wherein each nucleotide comprises a macromolecular block. In some embodiments, the macromolecular blocks are configured for slowing or halting the polynucleotide translocation through a nanopore. In some embodiments the macromolecular blocks have linear and branched structures.

First claim

What is claimed is:

1 . A modified nucleotide comprising a modification covalently attached to its nucleobase or its sugar, wherein the modification comprises a macromolecule block selected from the group consisting of an oligonucleotide block, a polyethylene glycol block, a peptide block, and a fluorinated block.

2 . The modified nucleotide of claim 1 , wherein the macromolecule block is the oligonucleotide block, and the oligonucleotide block comprises natural DNA/RNA nucleotides, modified nucleic acids, or a combination thereof.

3 . The modified nucleotide of claim 2 , wherein the oligonucleotide block comprises a linear structure, a branched structure, a hairpin type structure, or a cyclic structure.

4 . The modified nucleotide of claim 2 , wherein the oligonucleotide block is selected from the group consisting of:

wherein each M is a natural nucleotide or a modified nucleic acid;
n is an integer selected from 1 to 70;
p is an integer selected from 1 to 30;
q is an integer selected from 2 to 10; and
* is an end group selected from the group consisting of

wherein r is 2, 3, 4, or 5, s is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and “B” is Uracil, Cytosine, Guanine, or Adenine.

5 . The modified nucleotide of claim 1 , wherein the macromolecule block is the polyethylene glycol block, and the polyethylene block comprises a linear or a branched structure.

6 . The modified nucleotide of claim 5 , wherein the polyethylene glycol block is selected from the group consisting of:

wherein x is an integer selected from 1 to 10;
y is an integer selected from 1 to 30; and
* is an end group selected from the group consisting of

7 . The modified nucleotide of claim 1 , wherein the macromolecule block is the peptide block.

8 . The modified nucleotide of claim 7 , wherein the peptide block is selected from the group consisting of:

wherein each R is independently a side chain residue of an amino acid selected from the group consisting of

wherein R′ is —OH, —CO 2 − , —OSO 3 − , or —OPO 3 2− ;
R″ is H, acyl, acryl, alloc, benzoyl, BOC, Fmoc, formyl, or Cbz;
z is an integer selected from 3 to 6; and
n is an integer selected from 1 to 5.

9 . The modified nucleotide of claim 1 , wherein the macromolecule block is the fluorinated block.

10 . The modified nucleotide of claim 9 , wherein the fluorinated block comprises a perfluoroalkyl block, fluoro-peptide block, or perfluoroalkylated oligo block.

11 . The modified nucleotide of claim 9 , wherein the modification is selected from the group consisting of:

wherein:
h is an integer selected from 1 to 5;
i is an integer selected from 1 to 5;
j is an integer selected from 1 to 6;
n is an integer selected from 1 to 5;
z is an integer selected from 1 to 6;
R″ is H, acyl, acryl, alloc, benzoyl, BOC, Fmoc, formyl, or Cbz; and
X is C, O, PO 2 , PO 3 , or NR.

12 . The modified nucleotide of claim 1 , wherein the modification further comprises a covalent coupling between the macromolecule block and the modified nucleotide, wherein the covalent coupling comprises a moiety selected from the group consisting of amine-NHS ester, amine-imidoester, amine-pentofluorophenyl ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-alkyne, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, and azide-norbornene.

13 . The modified nucleotide of claim 1 , wherein the modification further comprise one or more covalently linked moiety selected from the group consisting of dyes, synthetic polymers, and small molecules.

14 . An oligonucleotide comprising one or more modified nucleotides according to claim 1 .

15 . A method for determining a sequence of a target polynucleotide in a nanopore-based sequencing system, the method comprising:
providing a daughter strand of the target polynucleotide comprising one or more modified nucleotides according to claim 1 , and reading the modified nucleotides by applying a reading voltage across a read head to identify a first reporter element in a constriction of a nanopore based on a first electrical response in the system, wherein one or more nucleotides translocate through a nanopore.

16 . The method of claim 15 , wherein the macromolecule block is configured to slow, pause, or halt the translocation.

17 . A method for determining a sequence of a target polynucleotide in a nanopore-based sequencing system, the method comprising:
providing the target polynucleotide and a plurality of the macromolecule blocks according to claim 1 in an electrolyte for the nanopore-based sequencing system; applying a voltage bias to cause the target polynucleotide to translocate through a constriction of a nanopore, wherein one or more of the plurality of the macromolecule blocks are non-covalently associated with the target polynucleotide; and detecting and identifying one or more nucleotides as the nucleotides pass through the constriction based on an electrical response in the system.

18 . The method of claim 17 , wherein the macromolecule blocks are associated with the target polynucleotide via hydrogen bonding, Van-der-Waals interaction, ionic interaction, or hydrophobic interaction.

19 . The method of claim 17 , wherein the macromolecule blocks are configured to slow, pause, or halt the translocation.

Assignees

• Illumina Inc

Inventors

• Bohra Hassan Zakiruddin
• Teo Yin Nah
• Neelakandan Ramesh
• Garcia Erin
• Salam Abdul Sadeer Abd
• Lukamto Daniel Hartoyo
• Ghosh Soumadwip
• Yang Xiangyuan
• Mandell Jeffrey G

Classifications

• C12Q1/6853

  using modified primers or templates · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C07H21/02

  with ribosyl as saccharide radical · CPC title

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• C07H19/00

  Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides ; Anhydro-derivatives thereof · CPC title