# US 2020/0399692 A1 · Modified nucleotides

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- Number: US 2020/0399692 A1
- Document ID: US20200399692A1
- Status: Published application
- Kind: A1
- Application number: 16/877,442
- Filed: 2020-05-18
- Published: 2020-12-24
- Earliest claimed: 2002-08-23
- Assignees: Illumina Cambridge Limited
- Applicants: Illumina Cambridge Limited
- Inventors: John MILTON; Xiaolin WU; Mark Edward Brennan SMITH; Joseph BRENNAN; Colin Lloyd BARNES; Xiaohai LIU; Silke RUEDIGER
- Web page: https://patentgrep.com/patent/US20200399692A1

## CPC

- C12Q 1/6869: Methods for sequencing
- C07H 1/00: Processes for the preparation of sugar derivatives
- C07H 19/00: Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides ; Anhydro-derivatives thereof
- C07H 19/06: Pyrimidine radicals
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/14: Pyrrolo-pyrimidine radicals
- C07H 19/16: Purine radicals
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12Q 2525/186: incorporating a non-extendable or blocking moiety
- C12Q 2535/113: Cycle sequencing
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure —O—Z wherein Z is any of —C(R′)2-O—R″, —C(R′)2-N(R″)2, —C(R′)2-N(H)R″, —C(R′)2-S—R″ and —C(R′)2-F, wherein each R″ is or is part of a removable protecting group; each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R′)2 represents an alkylidene group of formula ═C(R′″)2 wherein each R′″ may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R″ is exchanged for H or, where Z is —C(R′)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′OH; with the proviso that where Z is —C(R′)2-S—R″, both R′ groups are not H.

## Claims

### Claim 1

(canceled)

### Claim 2

(canceled)

### Claim 3

A composition comprising:
a planar solid support;
a plurality of different target polynucleotides immobilized at distinct regions on the solid support, wherein the distinct regions comprise multiple copies of one of the plurality of different target polynucleotides;
a plurality of different complementary oligonucleotides hybridized to the different target polynucleotides; and
an aqueous solution contacting the solid support, wherein the aqueous solution comprises:
at least 75% by volume water as a continuous phase of the aqueous solution;
at least one fluorescent label; and
a water-soluble tri-C1-6-alkyl phosphine substituted with a plurality of hydroxyl, carboxyl, carboxylate, amino, or sulfonate groups.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the aqueous solution comprises a plurality of different fluorescent labels.

### Claim 5 (depends on 4)

The composition of claim 4, wherein the fluorescent labels comprise fluorescein, rhodamine, alexa, bodipy, acridine, coumarin, pyrene, benzanthracene cyanine, or a mixture thereof.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the fluorescent labels comprise Cy3 or Cy5.

### Claim 7 (depends on 3)

The composition of claim 3, wherein the aqueous solution comprises about 95% by volume of water.

### Claim 8 (depends on 7)

The composition of claim 7, wherein the aqueous solution comprises greater than 98% by volume of water.

### Claim 9 (depends on 3)

The composition of claim 3, wherein the water-soluble tri-C1-6-alkyl phosphine has a plurality of hydroxyl groups.

### Claim 10 (depends on 3)

The composition of claim 3, wherein the water-soluble tri-C1-6-alkyl phosphine has the structure of:
or a salt thereof.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the salt of the water-soluble tri-C1-6-alkyl phosphine is a trisodium salt.

### Claim 12 (depends on 3)

The composition of claim 3, wherein the planar solid support comprises a glass surface, a silicon surface, a ceramic surface, or a plastic surface.

### Claim 13 (depends on 12)

The composition of claim 12, wherein the planar solid support comprises fabricated arrays of oligonucleotides.

### Claim 14 (depends on 3)

The composition of claim 3, wherein the aqueous mixture has a temperature of about 50° C. or higher.

### Claim 15 (depends on 14)

The composition of claim 14, wherein the aqueous mixture has a temperature of about 50° C. to about 80° C.

### Claim 16 (depends on 3)

The composition of claim 3, wherein the label in aqueous mixture comprises a multi-component label.

### Claim 17 (depends on 14)

The composition of claim 14, wherein the multi-component label comprises a fluorescent antibody.

### Claim 18

An oligonucleotide comprising a nucleotide residue, wherein said nucleotide residue comprises the structure:
wherein Z is —CH2N3;
B is a nucleobase selected from the group consisting of a purine, a pyrimidine and a deazapurine; and
comprises

### Claim 19 (depends on 18)

The oligonucleotide of claim 18, wherein the nucleobase is selected from the group consisting of
wherein the asterisk * indicates the attachment point to the sugar moiety of the nucleotide residue.

### Claim 20 (depends on 18)

The oligonucleotide of claim 18, wherein the oligonucleotide is hybridized to a target polynucleotide.

### Claim 21 (depends on 20)

The oligonucleotide of claim 20, wherein the target polynucleotide is immobilized to a solid support.

### Claim 22 (depends on 21)

The oligonucleotide of claim 21, wherein the solid support comprises an array of immobilized target polynucleotides.