US2016168636A1PendingUtilityA1

Nucleotide analogs

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Assignee: FLUIDIGM CORPPriority: May 18, 2007Filed: Oct 14, 2015Published: Jun 16, 2016
Est. expiryMay 18, 2027(~0.9 yrs left)· nominal 20-yr term from priority
Inventors:Suhaib SiddiqiJ. William EfcavitchJudith MitchellSubramanian MarappanJayson BowersMirna Jaroza
C12Q 1/6876C07H 19/16A61K 49/0052C07H 19/06A61K 49/0041C12Q 2563/107C12Q 1/6869
49
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Claims

Abstract

The invention generally relates to nucleotide analogs and methods of their use in sequencing-by-synthesis reactions. In certain embodiments, the invention provides a nucleotide analog including a detectable label attached to a nitrogenous base portion of a nucleotide analog by a cleavable linker, in which contact of the analog with at least one activating agent results in cleavage of the label and elimination of the linker, thereby producing a natural nucleotide, a 9-deaza-G, 9-deaza-A, or ψ-uridine.

Claims

exact text as granted — not AI-modified
1 . A nucleotide analog comprising a detectable label attached to the nitrogenous base portion of the nucleotide by a cleavable linker,
 wherein contact of the analog with a reducing agent results in release of the label and a first portion of the linker, thereby producing a natural nucleotide, a deaza-guanine, a deaza-adenine, or ψ-uridine with a residual portion of the linker attached thereto, characterized in that the residual portion of the linker is neutral in charge.   
     
     
         2 . The analog according to  claim 1 , wherein the reducing agent is selected from dithiothreitol (DTIT), β-mercaptoethanol, dithiocrythretol (DIE), GSH, cysteine, cysteamine, tris(3-hydroxypropyl)phosphine (THPP), glutathione, and salts of sulfurous acid. 
     
     
         3 . The analog according to  claim 1 , wherein the reducing agent is tricarboxyethyl phosphine (TCEP). 
     
     
         4 . The analog according to  claim 1 , wherein the linker is attached to C7 of a purine. 
     
     
         5 . The analog according to  claim 1 , wherein the linker is attached to C5 of deaza-guanine or deaza-adenine. 
     
     
         6 . The analog according to  claim 1 , wherein the detectable label is a fluorescent label. 
     
     
         7 . The analog according to  claim 1 , wherein the linker does not contain an amide bond 
     
     
         8 . The analog according to  claim 1 , wherein the linker comprises a disulfide bond. 
     
     
         9 . The analog according to  claim 1 , wherein the residual portion of the linker is an alkyne. 
     
     
         10 . The analog according to  claim 1 , wherein the residual portion of the linker terminates with a hydroxyl group. 
     
     
         11 . The analog according to  claim 1 , wherein the residual portion of the linker is —C≡C—CH 2 OH. 
     
     
         12 . The analog according to  claim 1 , wherein the residual portion of the linker terminates in a thiol group. 
     
     
         13 . The analog according to  claim 1 , wherein after cleavage of the linker by contact with the reducing agent, part of the first portion of the linker temporarily remains attached to the analog, which thereafter undergoes a cyclization reaction and is released from the analog, leaving the residual portion. 
     
     
         14 . The analog according to  claim 13 , wherein the part of the first portion that is released from the analog by the cyclization reaction is a thioester. 
     
     
         15 . The analog according to  claim 1 , comprising a chemical structure selected from: 
       
         
           
           
               
               
           
         
         wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, and D is a group that comprises a detectable label. 
       
     
     
         16 . The analog according to  claim 15 , wherein L comprises: 
       
         
           
           
               
               
           
         
       
     
     
         17 . The analog according to  claim 1 , comprising a chemical structure selected from: 
       
         
           
           
               
               
           
         
         wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, and D is a group that comprises a detectable label. 
       
     
     
         18 . The analog according to  claim 1 , wherein contact of the analog with a reducing agent results in release of the label and the first portion of the linker, thereby producing a pyrimidine with the residual portion of the linker attached thereto. 
     
     
         19 . The analog according to  claim 1 , comprising an inhibitor that is configured so that the analog can be added to an oligonucleotide by a polymerase, whereupon it inhibits the polymerase from adding a further nucleotide or nucleotide analog to the oligonucleotide. 
     
     
         20 . The analog according to  claim 19 , configured so that the inhibitor is released from the analog along with the detectable label upon cleavage of the cleavable linker. 
     
     
         21 . A nucleotide analog configured to reversibly label an oligonucleotide and inhibit polymerization, the nucleotide analog comprising;
 (1) a nitrogenous base portion;   (2) a detectable label;   (3) a separate polymerase inhibitor; and   (4) a cleavable linker joining both the detectable label and the polymerase inhibitor to the nitrogenous base portion;
 wherein once the analog is added to an oligonucleotide by a polymerase, 
 (a) the inhibitor inhibits the polymerase from adding a further nucleotide; and 
 (b) contacting the analog with a reducing agent results in release of the label, the inhibitor, and a first portion of the linker to produce a nucleotide or nucleotide analog with a residual portion of the linker attached thereto, such that the polymerase can add a further nucleotide or nucleotide analog to the oligonucleotide. 
   
     
     
         22 . The analog according to  claim 21 , wherein the linker is attached to C7 of a purine or to C5 of deaza-guanine or deaza-adenine. 
     
     
         23 . The analog according to  claim 21 , comprising a chemical structure selected from: 
       
         
           
           
               
               
           
         
         wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, D is a group that comprises a detectable label, and E is a group that inhibits polymerase activity. 
       
     
     
         24 . The analog according to  claim 21 , comprising a chemical structure selected from: 
       
         
           
           
               
               
           
         
         wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, D is a group that comprises a detectable label, and E is a group that inhibits polymerase activity. 
       
     
     
         25 . The analog according to  claim 1 , incorporated into an oligonucleotide. 
     
     
         26 . The analog according to  claim 21 , incorporated into an oligonucleotide. 
     
     
         27 . A method of releasing a label from an analog according to  claim 1 , comprising contacting the analog with a reducing agent, thereby producing a natural nucleotide or a deaza-nucleotide with the residual portion of the linker attached thereto. 
     
     
         28 . The method of  claim 27 , wherein the residual portion comprises a thiol group. 
     
     
         29 . The method of  claim 28 , further comprising capping the thiol group. 
     
     
         30 . The method of  claim 29 , whereby the thiol group is capped with an ethyl group, a methyl group, acetamide, acetate, ethyl acetate, or butanone. 
     
     
         31 . A method of sequencing a nucleic acid, comprising iteratively carrying out the steps of adding a nucleotide analog according to  claim 1  in a template-dependent manner to a primer that is duplexed with the nucleic acid, detecting incorporation of the nucleotide analog into the duplex, and then contacting the duplex with a reducing agent, thereby producing a natural nucleotide or a deaza-nucleotide from the added nucleotide analog with the residual portion of the linker attached thereto. 
     
     
         32 . The method of  claim 31 , wherein the nucleotide analog comprises an inhibitor,
 wherein the inhibitor is configured so that the analog can be added to an oligonucleotide by a polymerase, whereupon it inhibits the polymerase from adding a further nucleotide or nucleotide analog to the oligonucleotide, and   wherein the inhibitor is further configured to be released from the analog along with the detectable label upon cleavage of the cleavable linker.

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