# US 10,480,025 B2 · Labelled nucleotides

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- Number: US 10,480,025 B2
- Document ID: US10480025B2
- Status: Granted
- Kind: B2
- Application number: 15/202,456
- Filed: 2016-07-05
- Published: 2017-01-05
- Granted: 2019-11-19
- Earliest claimed: 2001-12-04
- Assignees: Illumina Cambridge Limited
- Applicants: Illumina Cambridge Limited
- Inventors: Shankar Balasubramanian; Colin Lloyd Barnes; Xiaohai Liu; John Milton; Xiaolin Wu; Harold Swerdlow
- Web page: https://patentgrep.com/patent/US10480025B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- 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
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/6823: Release of bound markers
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 2525/186: incorporating a non-extendable or blocking moiety
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Nucleosides and nucleotides are disclosed that are linked to detectable labels via a cleavable linker group.

## Claims

### Claim 1

A nucleotide or nucleoside molecule having a ribose or deoxyribose sugar moiety and a base linked to a detectable label via a cleavable linker, wherein the sugar moiety comprises a protecting group attached via a 3′ oxygen atom, and wherein said protecting group comprises an azido group that can be modified or removed to expose a 3′ OH group.

### Claim 2 (depends on 1)

The molecule of claim 1, wherein the base is a purine, or a pyrimidine.

### Claim 3 (depends on 1)

The molecule of claim 1, wherein the base is a deazapurine.

### Claim 4 (depends on 1)

The molecule of claim 1, which is a deoxyribonucleotide triphosphate.

### Claim 5 (depends on 1)

The molecule of claim 1, wherein the detectable label is a fluorophore.

### Claim 6 (depends on 1)

The molecule of claim 1, wherein the cleavable linker is acid labile, photolabile or comprises a disulphide linkage.

### Claim 7 (depends on 1)

The molecule of claim 1, wherein the cleavable linker and the protecting group are cleavable under identical conditions.

### Claim 8 (depends on 1)

The molecule of claim 1, wherein the protecting group comprises azidomethyl (CH2N3).

### Claim 9

A kit comprising a plurality of nucleotides, each having a ribose or deoxyribose sugar moiety and a base linked to a detectable label via a cleavable linker, wherein the sugar moiety comprises a protecting group attached via the 3′ oxygen atom, and said protecting group comprises an azido group that can be modified or removed to expose a 3′ OH group, and wherein the detectable label linked to each nucleotide can be distinguished upon detection from the detectable labels used for structurally different nucleotides.

### Claim 10 (depends on 9)

The kit of claim 9, wherein the cleavable linker and the protecting group are cleavable under identical conditions.

### Claim 11 (depends on 9)

The kit of claim 9, wherein the protecting group comprises azidomethyl (CH2N3).

### Claim 12 (depends on 9)

The kit of claim 9, further comprising an enzyme and buffers appropriate for the action of the enzyme.

### Claim 13 (depends on 12)

The kit of claim 12, wherein the enzyme is a terminal transferase, a polymerase or a reverse transcriptase.

### Claim 14 (depends on 4)

The molecule of claim 4, wherein the detectable label is a fluorophore.

### Claim 15 (depends on 14)

The molecule of claim 14, wherein the protecting group comprises azidomethyl (CH2N3).

### Claim 16 (depends on 15)

The molecule of claim 15, wherein said protecting group consists of azidomethyl bound via the carbon atom of the azidomethyl group to the 3′ oxygen atom, and wherein the azidomethyl protecting group can be removed to expose a 3′ OH group.

### Claim 17 (depends on 16)

The molecule of claim 16, wherein the cleavable linker and the protecting group are cleavable under identical conditions.

### Claim 18 (depends on 17)

The molecule of claim 17, wherein the base is a deazapurine.

### Claim 19 (depends on 18)

The molecule of claim 18, wherein the deazapurine is a 7-deazaadenine, and wherein said cleavable linker is bound to the 7-deazaadenine at the 7-position.

### Claim 20 (depends on 18)

The molecule of claim 18, wherein the deazapurine is a 7-deazaguanine, and wherein said cleavable linker is bound to the 7-deazaguanine at the 7-position.

### Claim 21 (depends on 17)

The molecule of claim 17, wherein the base is a cytosine, and wherein said cleavable linker is bound to the cytosine at the 5-position.

### Claim 22 (depends on 17)

The molecule of claim 17, wherein the base is a uracil, and wherein said cleavable linker is bound to the uracil at the 5-position.

### Claim 23 (depends on 19)

The molecule of claim 19, wherein the cleavable linker comprises a disulfide moiety.

### Claim 24 (depends on 20)

The molecule of claim 20, wherein the cleavable linker comprises a disulfide moiety.

### Claim 25 (depends on 21)

The molecule of claim 21, wherein the cleavable linker comprises a disulfide moiety.

### Claim 26 (depends on 22)

The molecule of claim 22, wherein the cleavable linker comprises a disulfide moiety.

### Claim 27 (depends on 19)

The molecule of claim 19, wherein the cleavable linker comprises an azido moiety.

### Claim 28 (depends on 20)

The molecule of claim 20, wherein the cleavable linker comprises an azido moiety.

### Claim 29 (depends on 21)

The molecule of claim 21, wherein the cleavable linker comprises an azido moiety.

### Claim 30 (depends on 22)

The molecule of claim 22, wherein the cleavable linker comprises an azido moiety.

### Claim 31

A nucleotide molecule, wherein:
the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
the nucleotide molecule comprises a 7-deazaadenine base,
the 7-deazaadenine base of the nucleotide molecule is substituted with a linker at the 7-deaza position,
the linker is bound to a fluorophore,
the linker comprises a cleavable moiety located between the 7-deazaadenine base and the fluorophore,
the cleavable moiety comprises a disulfide moiety or an azido moiety,
the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the 7-deazaadenine base, and
upon cleavage of the azidomethyl moiety, a 3-OH group is formed and the formation of the 3-OH group allows addition of a nucleotide to the 3′ oxygen atom.

### Claim 32 (depends on 31)

The nucleotide molecule of claim 31, wherein the cleavable moiety comprises a disulfide moiety.

### Claim 33 (depends on 31)

The nucleotide molecule of claim 31, wherein the cleavable moiety comprises an azido moiety.

### Claim 34

A 7-substituted 7-deaza-2′-deoxy-adenosine triphosphate nucleotide molecule, wherein:
the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
the 7-deazaadenine base of the molecule is linked to a fluorophore via a cleavable linker at the 7-deaza position of the base,
the linker comprises a cleavable moiety located between the 7-deazaadenine base and the fluorophore,
the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the 7-deazaadenine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.

### Claim 35

A nucleotide molecule having the structure:
wherein:
X is triphosphate,
R1 is —OCH2N3,
R2 is hydrogen,
Linker is a cleavable linker comprising a cleavable moiety,
Label is a fluorophore, and
upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.

### Claim 36 (depends on 35)

The nucleotide molecule of claim 35, wherein the cleavable moiety and the —CH2N3 of R1 are cleavable in a chemical reaction under identical conditions.

### Claim 37 (depends on 36)

The nucleotide molecule of claim 36, wherein the cleavable moiety comprises a disulfide moiety.

### Claim 38 (depends on 36)

The nucleotide molecule of claim 36, wherein the cleavable moiety comprises an azido moiety.

### Claim 39

A nucleotide molecule, wherein:
the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
the nucleotide molecule comprises a 7-deazaguanine base,
the 7-deazaguanine base of the nucleotide molecule is substituted with a linker at the 7-deaza position,
the linker is bound to a fluorophore,
the linker comprises a cleavable moiety located between the 7-deazaguanine base and the fluorophore,
the cleavable moiety comprises a disulfide moiety or an azido moiety,
the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the 7-deazaguanine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′ oxygen atom.

### Claim 40 (depends on 39)

The nucleotide molecule of claim 39, wherein the cleavable moiety comprises a disulfide moiety.

### Claim 41 (depends on 39)

The nucleotide molecule of claim 39, wherein the cleavable moiety comprises an azido moiety.

### Claim 42

A 7-substituted 7-deaza-2′-deoxy-guanosine triphosphate nucleotide molecule, wherein:
the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
the 7-deazaguanine base of the molecule is linked to a fluorophore via a cleavable linker at the 7-deaza position of the base,
the linker comprises a cleavable moiety located between the 7-deazaguanine base and the fluorophore,
the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the 7-deazaguanine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.

### Claim 43

A nucleotide molecule having the structure:
wherein:
X is triphosphate,
R1 is —OCH2N3,
R2 is hydrogen,
Linker is a cleavable linker comprising a cleavable moiety,
Label is a fluorophore, and
upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.

### Claim 44 (depends on 43)

The nucleotide molecule of claim 43, wherein the cleavable moiety and CH2N3 of the —CH2N3 of R1 are cleavable in a chemical reaction under identical conditions.

### Claim 45 (depends on 44)

The nucleotide molecule of claim 44, wherein the cleavable moiety comprises a disulfide moiety.

### Claim 46 (depends on 44)

The nucleotide molecule of claim 44, wherein the cleavable moiety comprises an azido moiety.

### Claim 47

A nucleotide molecule, wherein:
the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
the nucleotide molecule comprises a cytosine base,
the cytosine base of the nucleotide molecule is substituted with a linker at the 5 position of the base,
the linker is bound to a fluorophore,
the linker comprises a cleavable moiety located between the cytosine base and the fluorophore,
the cleavable moiety comprises a disulfide moiety or an azido moiety,
the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the cytosine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′ oxygen atom.

### Claim 48 (depends on 47)

The nucleotide molecule of claim 47, wherein the cleavable moiety comprises a disulfide moiety.

### Claim 49 (depends on 47)

The nucleotide molecule of claim 47, wherein the cleavable moiety comprises an azido moiety.

### Claim 50

A 5-substituted 2′-deoxy-cytidine triphosphate nucleotide molecule, wherein:
the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
the cytosine base of the molecule is linked to a fluorophore via a cleavable linker at the 5 position of the base,
the linker comprises a cleavable moiety located between the cytosine base and the fluorophore,
the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the cytosine base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.

### Claim 51

A nucleotide molecule having the structure:
wherein:
X is triphosphate,
R1 is —OCH2N3,
R2 is hydrogen,
Linker is a cleavable linker comprising a cleavable moiety,
Label is a fluorophore, and
upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.

### Claim 52 (depends on 51)

The nucleotide molecule of claim 51, wherein the cleavable moiety and the —CH2N3 of R1 are cleavable in a chemical reaction under identical conditions.

### Claim 53 (depends on 52)

The nucleotide molecule of claim 52, wherein the cleavable moiety comprises a disulfide moiety.

### Claim 54 (depends on 52)

The nucleotide molecule of claim 52, wherein the cleavable moiety comprises an azido moiety.

### Claim 55

A nucleotide molecule, wherein:
the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
the nucleotide molecule comprises a uracil base,
the uracil base of the nucleotide molecule is substituted with a linker at the 5 position of the base,
the linker is bound to a fluorophore,
the linker comprises a cleavable moiety located between the uracil base and the fluorophore,
the cleavable moiety comprises a disulfide moiety or an azido moiety,
the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the uracil base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′ oxygen atom.

### Claim 56 (depends on 55)

The nucleotide molecule of claim 55, wherein the cleavable moiety comprises a disulfide moiety.

### Claim 57 (depends on 55)

The nucleotide molecule of claim 55, wherein the cleavable moiety comprises an azido moiety.

### Claim 58

A 5-substituted 2′-deoxy-uridine triphosphate nucleotide molecule, wherein:
the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
the uracil base of the molecule is linked to a fluorophore via a cleavable linker at the 5 position of the base,
the linker comprises a cleavable moiety located between the uracil base and the fluorophore,
the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the uracil base, and
upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.

### Claim 59

A nucleotide molecule having the structure:
wherein:
X is triphosphate,
R1 is —OCH2N3,
R2 is hydrogen,
Linker is a cleavable linker comprising a cleavable moiety,
Label is a fluorophore, and
upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.

### Claim 60 (depends on 59)

The nucleotide molecule of claim 59, wherein the cleavable moiety and the —CH2N3 of R1 are cleavable in a chemical reaction under identical conditions.

### Claim 61 (depends on 60)

The nucleotide molecule of claim 60, wherein the cleavable moiety comprises a disulfide moiety.

### Claim 62 (depends on 60)

The nucleotide molecule of claim 60, wherein the cleavable moiety comprises an azido moiety.