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 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-C 1-6 -alkyl phosphine substituted with a plurality of hydroxyl, carboxyl, carboxylate, amino, or sulfonate groups.   
     
     
         4 . The composition of  claim 3 , wherein the aqueous solution comprises a plurality of different fluorescent labels. 
     
     
         5 . The composition of  claim 4 , wherein the fluorescent labels comprise fluorescein, rhodamine, alexa, bodipy, acridine, coumarin, pyrene, benzanthracene cyanine, or a mixture thereof. 
     
     
         6 . The composition of  claim 5 , wherein the fluorescent labels comprise Cy3 or Cy5. 
     
     
         7 . The composition of  claim 3 , wherein the aqueous solution comprises about 95% by volume of water. 
     
     
         8 . The composition of  claim 7 , wherein the aqueous solution comprises greater than 98% by volume of water. 
     
     
         9 . The composition of  claim 3 , wherein the water-soluble tri-C 1-6 -alkyl phosphine has a plurality of hydroxyl groups. 
     
     
         10 . The composition of  claim 3 , wherein the water-soluble tri-C 1-6 -alkyl phosphine has the structure of: 
       
         
           
           
               
               
           
         
       
       or a salt thereof. 
     
     
         11 . The composition of  claim 10 , wherein the salt of the water-soluble tri-C 1-6 -alkyl phosphine is a trisodium salt. 
     
     
         12 . The composition of  claim 3 , wherein the planar solid support comprises a glass surface, a silicon surface, a ceramic surface, or a plastic surface. 
     
     
         13 . The composition of  claim 12 , wherein the planar solid support comprises fabricated arrays of oligonucleotides. 
     
     
         14 . The composition of  claim 3 , wherein the aqueous mixture has a temperature of about 50° C. or higher. 
     
     
         15 . The composition of  claim 14 , wherein the aqueous mixture has a temperature of about 50° C. to about 80° C. 
     
     
         16 . The composition of  claim 3 , wherein the label in aqueous mixture comprises a multi-component label. 
     
     
         17 . The composition of  claim 14 , wherein the multi-component label comprises a fluorescent antibody. 
     
     
         18 . An oligonucleotide comprising a nucleotide residue, wherein said nucleotide residue comprises the structure: 
       
         
           
           
               
               
           
         
         wherein Z is —CH 2 N 3 ; 
         B is a nucleobase selected from the group consisting of a purine, a pyrimidine and a deazapurine; and 
       
       
         
           
           
               
               
           
         
       
       comprises 
       
         
           
           
               
               
           
         
       
     
     
         19 . 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. 
       
     
     
         20 . The oligonucleotide of  claim 18 , wherein the oligonucleotide is hybridized to a target polynucleotide. 
     
     
         21 . The oligonucleotide of  claim 20 , wherein the target polynucleotide is immobilized to a solid support. 
     
     
         22 . The oligonucleotide of  claim 21 , wherein the solid support comprises an array of immobilized target polynucleotides.