Modified nucleotides
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.
1. A polynucleotide molecule of formula PN—OZ, wherein Z is selected from
-allyl, —CR′ 2 O-allyl, —CR′ 2 NR″(allyl), —CR′ 2 NH(allyl), —CR′ 2 N(allyl) 2 or -CR′ 2 -—S-allyl, PN is a 3′-terminal nucleotide of said polynucleotide; each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, amido group or two R′ taken together represent an alkylidene group ═C(R′″), wherein R′″ is selected from hydrogen, halogen, and alkyl; R″ is a removable protecting group; and said polynucleotide further comprises a detectable label linked by a linker.
2. A molecule according to claim 1 wherein said linker is cleavable.
3. A molecule according to claim 1 wherein said detectable label is a fluorophore.
4. A molecule according to claim 1 wherein said linker is acid labile, photolabile or contains a disulfide linkage.
5. A molecule according to claim 1 , wherein said linker is a phosphine-cleavable azide-containing linker.
6. A molecule according to claim 1 , wherein said formula is PN—O-allyl.
7. A molecule according to claim 1 , wherein the formula is PN—OCR′ 2 NR″(allyl), PN—OCR′ 2 NH(allyl), or PN—OCR′ 2 N(allyl) 2 .
8. A molecule according to claim 1 , wherein the formula is PN—OCR′ 2 —S-allyl.
9. A molecule according to claim 1 , wherein said linker is non-cleavable.
10. A molecule according to claim 1 , wherein said formula is PN—OCR′ 2 -allyl.
11. A molecule according to claim 1 , wherein each incidence of R′ is hydrogen.
12. A molecule according to any one of claims 6 , 7 , 8 , 10 or 11 , wherein said detectable label is linked to the 3′-terminal nucleotide.
13. A molecule according to any one of claims 6 , 7 , 8 , 10 , or 11 , wherein said molecule is hybridized to a target single-stranded polynucleotide.
14. A molecule according to claim 12 wherein said detectable label is a fluorophore.
15. A molecule according to claim 1 wherein said detectable label is a fluorophore.
16. A molecule according to claim 1 , wherein at least one R′ is aryl.
17. A molecule according to claim 1 , wherein at least one R′ comprises said detectable label.