Modified nucleotides

• US 7,771,973 B2
• Filed: 06/01/2009
• Issued: 08/10/2010
• Est. Priority Date: 12/23/2002
• Status: Expired due to Term

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Smith, Mark, Wu, Xiaolin, Ruediger, Silke, Brennan, Joseph, Milton, John, Liu, Xiaohai, Barnes, Colin
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/455,397
• Publication Number:
  US 20090325172A1
• Time in Patent Office: 435 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/26.6
• US Class Current: 435/91.1
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: 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
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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

1. A method for determining the sequence of a target single-stranded polynucleotide, comprising monitoring the sequential incorporation of complementary nucleotides, wherein at least one incorporation is of a nucleotide 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 allyl, —

C(R′

)₂—

N₃, —

C(R^IV)₂—

O—

R″

, —

C(R′

)₂—

N(R″

)₂, —

C(R′

)₂—

N(H)R″

, or —

C(R^IV)₂—

S—

R″

,

wherein —

C(R^IV)₂—

O—

R″

is of the formula —

CR⁴(R⁵)—

O—

CR⁴(R⁵)—

OR⁶ or of the formula —

CR⁴(R⁵)—

O—

CR⁴(R⁵)—

SR⁶; and

wherein —

C(R^IV)₂—

S—

R″

is of the formula —

CR⁴(R⁵)—

S—

CR⁴(R⁵)—

OR⁶ or of the formula —

CR⁴(R⁵)—

S—

CR⁴(R⁵)—

SR⁶;

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;

each R⁴ and R⁵ is independently a hydrogen atom or an alkyl group; and

R⁶ is alkyl, cycloalkyl, alkenyl, cycloalkenyl or benzyl,

and wherein the blocking group is removed prior to introduction of the next complementary nucleotide.

2. The method of claim 1 wherein the nucleotide comprises a base linked to a detectable label.

4. The method of claim 1 wherein said incorporating step is accomplished by a Thermococcus sp.

5. The method of claim 4 wherein the Thermococcus sp is 9°

N or a single mutant or double mutant thereof.

6. The method of claim 5 wherein the double mutant is -Y409V A485L.

13. The method of claim 1 wherein Z is an azidomethyl group.

14. The method of claim 1 wherein Z is an allyl group and the blocking group removal is performed in aqueous solution with a transition metal complex comprising a transition metal and one or more phosphine ligands.

15. The method of claim 14 wherein said transition metal is selected from the group comprising platinum, palladium, rhodium, ruthenium, osmium and iridium.

16. The method of claim 14 wherein the phosphine ligands comprise derivatised triaryl phosphine ligands or derivatised trialkyl phosphine ligands.

17. The method of claim 16 wherein the phosphine ligands are derivatised with one or more functionalities selected from the group comprising amino, hydroxyl, carboxyl and sulfonate groups.

3. A method for determining the sequence of a target single-stranded polynucleotide, comprising:

(a) providing a plurality of different nucleotides wherein each of said plurality of different nucleotides has 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 allyl, —

C(R′

)₂—

N₃, —

C(R^IV)₂—

O—

R″

, —

C(R′

)₂—

N(R″

)₂, —

C(R′

)₂—

N(H)R″

, or —

C(R^IV)₂—

S—

R″

,

wherein —

C(R^IV)₂—

O—

R″

is of the formula —

CR⁴(R⁵)—

O—

CR⁴(R⁵)—

OR⁶ or of the formula —

CR⁴(R⁵)—

O—

CR⁴(R⁵)—

SR⁶; and

wherein —

C(R^IV)₂—

S—

R″

is of the formula —

CR⁴(R⁵)—

S—

CR⁴(R⁵)—

OR⁶ or of the formula —

CR⁴(R⁵)—

S—

CR⁴(R⁵)—

SR⁶;

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;

each R⁴ and R⁵ is independently a hydrogen atom or an alkyl group; and

R⁶ is alkyl, cycloalkyl, alkenyl, cycloalkenyl or benzyl,

and wherein each type of nucleotide comprises a base linked to a detectable label and the detectable label linked to each type of nucleotide can be distinguished upon detection from the detectable label used for other types of nucleotides;

(b) incorporating the nucleotide into the complement of the target single-stranded polynucleotide;

(c) detecting the label of the nucleotide of (b), thereby determining the type of nucleotide incorporated;

(d) removing the label of the nucleotide of (b) and the blocking group; and

(e) optionally repeating steps (b)-(d) one or more times;

thereby determining the sequence of a target single-stranded polynucleotide.

7. The method of claim 3 wherein the label of the nucleotide and the blocking group are removed in a single chemical treatment step.

8. The method according to claim 3, wherein each of the nucleotides are brought into contact with the target sequentially, with removal of non-incorporated nucleotides prior to addition of the next nucleotide, and wherein detection and removal of the label and the blocking group is carried out either after addition of each nucleotide, or after addition of all four nucleotides.

9. The method according to claim 3, wherein each of the nucleotides are brought into contact with the target together simultaneously, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label and the blocking group.

10. The method according to claim 3, comprising a first step and a second step, wherein in the first step, a first composition comprising two of the four nucleotides is brought into contact with the target and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label, and wherein in the second step, a second composition comprising the two nucleotides not included in the first composition is brought into contact with the target, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label and blocking group, and wherein the first and second steps are optionally repeated one or more times.

11. The method according to claim 3, comprising a first step and a second step, wherein in the first step, a composition comprising one of the four nucleotides is brought into contact with the target, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label and blocking group and wherein in the second step, a second composition comprising the three nucleotides not included in the first composition is brought into contact with the target, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label and blocking group and wherein the first step and the second step are optionally repeated one or more times.

12. The method according to claim 3, comprising a first step and a second step, wherein in the first step, a first composition comprising three of the four nucleotides is brought into contact with the target, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label and blocking group and wherein in the second step, a composition comprising the nucleotide not included in the first composition is brought into contact with the target, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label and blocking group and wherein the first step and the second step are optionally repeated one or more times.