2-Nitrobenzyl-Modified Ribonucleotides

Abstract

This disclosure provides novel reversibly terminated ribonucleotides which can be used as a reagent for DNA sequencing reactions. Methods of sequencing nucleic acids using the disclosed nucleotides are also provided.

1 . A ribonucleoside having a formula SM-BASE wherein SM is a ribose, BASE is a pyrimidine or purine, and wherein said ribose comprises a reversible chain terminating moiety at a 2′ position in said ribose. 
     
     
         2 . The method of  claim 1  wherein said base is selected from the group consisting of adenine, guanine, cytosine and uracil. 
     
     
         3 . The ribonucleoside of  claim 1  which is a ribonucleotide selected from the group consisting of ribonucleoside 5′-phosphate, a ribonucleoside 5′-diphosphate or a ribonucleoside 5′-triphosphate. 
     
     
         4 . The ribonucleoside of  claim 1  which is a ribonucleotide selected from the group consisting of adenosine 5′-monophosphate, adenosine 5′-diphosphate, adenosine 5′-triphosphate, guanosine 5′-monophosphate, guanosine 5′-diphosphate, guanosine 5′-triphosphate, uridine 5′-monophosphate, uridine 5′-diphosphate, uridine 5′-triphosphate, cytidine 5′-monophosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate. 
     
     
         5 . The ribonucleoside of  claim 1  wherein said reversible chain terminating moiety is connected to said ribose by a reversible linkage. 
     
     
         6 . The ribonucleoside of  claim 5  wherein said reversible linkage is a bond that is cleavable by electromagnetic radiation, chemical treatment, or a combination thereof. 
     
     
         7 . The ribonucleoside of  claim 6  wherein said electromagnetic radiation is light. 
     
     
         8 . The ribonucleoside of  claim 1  wherein the reversible chain terminating moiety is a 2-Nitrobenzyl group. 
     
     
         9 . The ribonucleoside of  claim 1  wherein reversible chain terminating moiety is a Desyl group. 
     
     
         10 . The ribonucleoside of  claim 1  wherein reversible chain terminating moiety is a p-hydroxyphenacyl caging group. 
     
     
         11 . The ribonucleoside of  claim 1  wherein said ribonucleotide is labeled with a detectable label. 
     
     
         12 . The ribonucleoside of  claim 11  wherein said detectable label is a moiety is selected from the group consisting of green fluorescent protein, blue fluorescent protein, red fluorescent protein, beta-galactosidase, chloramphenicol acetyltransferase, beta-glucoronidase, luciferases, b-lactamase, digoxygenin, fluorescent dye molecule, fluorescein, cy3, cy5, alkaline phosphatase and horse radish peroxidase. 
     
     
         13 . The ribonucleoside of  claim 11  wherein said detectable label may be removed by photobleaching. 
     
     
         14 . The ribonucleoside of  claim 11  wherein said detectable label is connected to the ribonucleotide by a reversible linkage. 
     
     
         15 . The ribonucleoside of  claim 14  wherein said reversible linkage is a bond that is cleavable by electromagnetic radiation, chemical treatment, or a combination thereof. 
     
     
         16 . A method of producing a reversibly terminating ribonucleoside comprising the steps of:
 (a) providing a ribonucleoside having a formula SM-BASE wherein SM is a ribose, BASE is a pyrimidine or purine;   (b) attaching a reversible chain terminating moiety at a 2′ position of said ribose.   
     
     
         17 . The method of  claim 16  further comprising the step of attaching a detectable moiety to said ribonucleoside before or after step (b). 
     
     
         18 . The method of  claim 16  wherein said reversible chain terminating moiety is selected from the group consisting of a 2-Nitrobenzyl group, a Desyl group and a p-hydroxyphenacyl caging group. 
     
     
         19 . A method of sequencing a target nucleic acid comprising the steps of
 (a) elongating a primer which is complexed with a target nucleic acid to form an incorporated ribonucleotide with a RNA polymerase and at least one first species of ribonucleotide having the formula PM-SM-BASE wherein PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, wherein said ribose comprises a chain terminating moiety connected by a reversible linkage at a 2′ position of said ribose;   (b) detecting said incorporated ribonucleotide to determine a sequence of said target nucleic acid.   
     
     
         20 . The method of  claim 19  further comprising the step of:
 (c) removing said chain terminating moiety of said incorporated ribonucleotide by breaking said reversible linkage; 
 (d) repeating steps (a), (b) and (c) with at least one second species of ribonucleotide having the formula PM-SM-BASE wherein PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, wherein said ribose comprises a chain terminating moiety connected by a reversible linkage at a 2′ position of said ribose. 
 
     
     
         21 . The method of  claim 19  wherein said complex between said primer and said target nucleic acid is formed by hybridization or synthesis by RNA polymerase. 
     
     
         22 . The method of  claim 19  wherein said RNA polymerase is a phage-encoded RNA polymerase selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase and SP6 RNA polymerase. 
     
     
         23 . A method of sequencing a target nucleic acid comprising the steps of:
 (a) elongating a primer which is complexed with a target nucleic acid with a RNA polymerase and ribonucleotides ATP, GTP, UTP, and CTP to form an incorporated nucleotide, wherein said ribonucleotides having the formula PM-SM-BASE wherein PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, wherein said ribose comprises a chain terminating moiety connected by a reversible linkage at a 2′ position of said ribose and wherein each of said ribonucleotide is reversibly labeled with a detectable label;   (b) detecting said incorporated nucleotide by detecting said detectable label to determine a sequence of said target nucleic acid.   
     
     
         24 . The method of  claim 23  further comprising the step of:
 (c) removing said chain terminating moiety and said detectable label from said incorporated ribonucleotide; 
 (d) repeating steps (a), (b) and (c) until a desired amount of nucleic acid sequence is determined. 
 
     
     
         25 . The method of  claim 23  wherein said complex between said primer and said target nucleic acid is formed by hybridization or synthesis by RNA polymerase. 
     
     
         26 . The method of  claim 23  or  24  wherein said RNA polymerase is a phage-encoded RNA polymerase selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase and SP6 RNA polymerase. 
     
     
         27 . The method of  claim 23  wherein said RNA polymerase comprises a steric gate deletion which can incorporate a 2′ modified ribonucleotide into an elongating nucleic acid. 
     
     
         28 . The method of  claim 23  wherein said RNA polymerase comprises additional amino acid substitutions in a nucleotide binding and catalytic pocket which can incorporate a 2′ modified ribonucleotide into an elongating nucleic acid.