Modified nucleotides

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.

1 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . A modified nucleoside triphosphate molecule comprising a base and a deoxyribose sugar moiety, wherein the 3′ carbon atom of the sugar moiety has covalently attached thereto a group of the structure:
   —O—Z
 
 wherein Z is a removable protecting group comprising an azido group. 
 
     
     
         4 . The molecule of  claim 3 , wherein the removable protecting group is azidomethyl. 
     
     
         5 . The molecule of  claim 4 , wherein the base is a purine, a pyrimidine or a deazapurine. 
     
     
         6 . The molecule of  claim 4 , wherein the base is adenine, 7-deazaadenine, guanine, or 7-deazaguanine. 
     
     
         7 . The molecule of  claim 4 , wherein the base is cytosine, thymine or uracil. 
     
     
         8 . The molecule of  claim 4 , wherein the base is linked to a detectable label via a cleavable linker. 
     
     
         9 . A kit comprising a plurality of different modified nucleoside triphosphate molecules, each comprising a base and a deoxyribose sugar moiety wherein the 3′ carbon atom of the sugar moiety has covalently attached thereto a group of the structure:
   —O—Z
 
 wherein Z is a removable protecting group comprising an azido group. 
 
     
     
         10 . The kit of  claim 9 , wherein the removable protecting group is azidomethyl. 
     
     
         11 . The kit of  claim 10 , wherein each base is a purine, a pyrimidine or a deazapurine. 
     
     
         12 . The kit of  claim 10 , wherein the kit comprises at least four different modified nucleoside triphosphate molecules. 
     
     
         13 . The kit of  claim 12 , wherein the kit comprises a set of modified dATP, dTTP, dCTP and dGTP or a set of modified dATP, dUTP, dCTP and dGTP. 
     
     
         14 . The kit of  claim 10 , further comprising a polymerase capable of incorporating the modified nucleoside triphosphate molecules into a polynucleotide strand, wherein the polymerase is 9° N or a mutant thereof comprising Y409V and A485L substitutions. 
     
     
         15 . A method of controlling the incorporation of nucleotides into a polynucleotide strand, comprising incorporating into a polynucleotide strand a modified nucleoside triphosphate molecule comprising a base and a deoxyribose sugar moiety wherein the 3′ carbon atom of the sugar moiety has covalently attached thereto a group of the structure:
   —O—Z
 
 wherein Z is a removable protecting group comprising an azido group; 
 wherein the incorporation of the modified nucleoside triphosphate molecule prevents incorporation of a subsequent nucleotide into the polynucleotide strand; and 
 wherein the incorporating step is performed using a polymerase. 
 
     
     
         16 . The method of  claim 15 , wherein the removable protecting group is azidomethyl. 
     
     
         17 . The method of  claim 16 , wherein the base is a purine, a pyrimidine or a deazapurine. 
     
     
         18 . The method of  claim 16 , wherein at least four different modified nucleoside triphosphate molecules are brought into contact with the polynucleotide strand simultaneously. 
     
     
         19 . The method of  claim 18 , wherein the modified nucleoside triphosphate molecules comprise a set of modified dATP, dTTP, dCTP and dGTP or a set of modified dATP, dUTP, dCTP and dGTP. 
     
     
         20 . The method of  claim 16 , further comprising removing the removable protecting group from the sugar moiety to generate a 3′ hydroxyl group and incorporating a subsequent nucleotide into the polynucleotide strand. 
     
     
         21 . The method of  claim 20 , wherein the removable protecting group is removed using a water-soluble phosphine. 
     
     
         22 . The method of  claim 21 , wherein the water-soluble phosphine is a trialkyl phosphine derivatized with one or more functionalities selected from the group comprising amino, hydroxyl, carboxyl and sulfonate groups.