Cleavable cyclic loop nucleotides for nanopore sequencing

Patent metadata

Field	Value
Publication number	US-2023357307-A1
Application number	US-202318311876-A
Country	US
Kind code	A1
Filing date	May 3, 2023
Priority date	May 4, 2022
Publication date	Nov 9, 2023
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 linker construct between two positions of the nucleotide, wherein the linker construct optionally comprises a reporter moiety corresponding to the identity of the nucleotide, and wherein the linker construct is a part of the cleavable cyclic loop nucleotide comprising a cleavable site. In some embodiments, the nucleotides further comprise arresting constructs for slowing or halting the polynucleotide translocation through a nanopore.

First claim

1 . A compound having one of the following structures:

wherein
X is —O—, —CH 2 —, —NH—,

X′ is ═N—SO 2 —, ═NH—CO—, or

Y is —O—, —S—, —NH—, or —Se—;
L 1 is a first linking group;
L 2 is a second linking group; and
SP is a spacer.

2 . The compound of claim 1 , wherein SP comprises one or more of the following moieties:
(1) alkyl chains having 5 to 50 carbons, (2) oligonucleotides, modified oligonucleotides or polyphosphates having 1 to 100 repeating units, (3) polypeptides having 1 to 100 repeating units, (4) hydrophilic polymers having 1 to 100 repeating units, and (5) hydrophobic polymers having 1 to 100 repeating units.

3 . The compound of claim 2 , wherein the hydrophilic polymers are selected from the group consisting of polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, and polyethyleneimine.

4 . The compound of claim 2 , wherein the hydrophobic polymers are selected from the group consisting of polylactic acid, polymethylmethacrylate, and polystyrene.

5 . The compound of claim 1 , wherein each of L 1 and L 2 independently comprises a conjugating moiety selected from the group consisting of amine-NHS ester, amine-imidoester, amine-pentafluorophenyl 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.

6 . The compound of claim 5 , wherein each of L 1 and L 2 independently further comprises a first linker between the conjugating moiety and X, and a second linker between the conjugating moiety and SP.

7 . The compound of claim 6 , wherein the first linker and the second linker are independently selected from the group consisting of polynucleotide having 1 to 100 repeating units, polypeptide having 1 to 100 repeating units, alkyl chains having 5 to 50 carbons, hydrophilic polymers having 1 to 100 repeating units comprising polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, or polyethyleneimine, hydrophobic polymers having 1 to 100 repeating units comprising polylactic acid, polymethylmethacrylate, or polystyrene, and combinations thereof.

8 . The compound of claim 1 , wherein SP or the Base further comprises an arresting construct.

9 . (canceled)

10 . The compound of claim 8 , wherein the arresting construct is a linear, a branched or a cyclic polymer.

11 . The compound of claim 10 , wherein the arresting construct comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide/polynucleotide, a peptide/polypeptide, or combinations thereof.

12 . An oligonucleotide comprising one of the following structures:

wherein
X is —O—, —CH 2 —, —NH—,

X′ is ═N—SO 2 —, ═NH—CO—, or

Y is —O—, —S—, —NH—, or —Se—
one of R 1 , R 2 , and R 3 is allyl, while the others are H;
L 1 is a first linking group;
L 2 is a second linking group; and
SP is a spacer.

13 . The oligonucleotide of claim 12 , wherein structure (VII) can further be represented by the following structures:

14 . The oligonucleotide of claim 12 , wherein structure (VIII) can further be represented by the following structures:

15 . The oligonucleotide of claim 12 , wherein structure (X) can further be represented by the following structures:

16 . An oligonucleotide comprising one of the following structures:

wherein
Y is —O—, —S—, —NH—, or —Se—; and
one of Y 1 , Y 2 , and Y 3 is —S— or —Se—, and the others are —O— or —NH—.

17 . The oligonucleotide of claim 12 , wherein SP comprises one or more of the following moieties:
(1) alkyl chains having 5 to 50 carbons, (2) oligonucleotides or modified oligonucleotides or phosphoramidite analogs having 1 to 100 repeating units, (3) polypeptides having 1 to 100 repeating units, (4) hydrophilic polymers having 1 to 100 repeating units selected from the group consisting of polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, and polyethyleneimine, and (5) hydrophobic polymers having 1 to 100 repeating units selected from the group consisting of polylactic acid, polymethylmethacrylate, and polystyrene.

18 . The oligonucleotide of claim 12 , wherein
each of L 1 and L 2 independently comprises a conjugating moiety selected from the group consisting of amine-NHS ester, amine-imidoester, amine-pentafluorophenyl 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.

19 . The oligonucleotide of claim 18 , wherein each of L 1 and L 2 independently further comprises a first linker between the conjugating moiety and X, and a second linker between the conjugating moiety and SP.

20 . The oligonucleotide of claim 19 , wherein the first linker and the second linker are independently selected from the group consisting of an oligonucleotide or modified oligonucleotide or phosphoramidite analogs having 1 to 100 repeating units, polypeptide having 1 to 100 repeating units, alkyl chains having 5 to 50 carbons, hydrophilic polymers having 1 to 100 repeating units comprising polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, or polyethyleneimine, hydrophobic polymers having 1 to 100 repeating units comprising polylactic acid, polymethylmethacrylate, or polystyrene, and

Assignees

• Illumina Inc
• Illumina Singapore Pte Ltd

Inventors

• Salam Abdul Sadeer Abd
• Yang Xiangyuan
• Bohra Hassan Zakiruddin
• Teo Yin Nah
• Lee Min Yen
• Neelakandan Ramesh
• Mandell Jeffrey G
• Garcia Erin
• Soh Sharyuen
• Lukamto Daniel Hartoyo

Classifications

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12Q2525/186

  incorporating a non-extendable or blocking moiety · CPC title

• C12Q2525/117

  incorporating modified base · CPC title

• C12Q2525/113

  incorporating modified backbone · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title