Nucleotide analogs

Abstract

The invention provides for nucleotide analogs and methods of using the same, e.g., for sequencing nucleic acids.

We claim: 
     
         1 . A method for sequencing a nucleic acid, the method comprising the steps of:
 exposing a nucleic acid duplex comprising a template portion and a primer portion to a nucleotide analog comprising an inhibitor that is charged or capable of becoming charged, and a polymerase, under conditions that permit template-dependent incorporation of the analog into the primer;   detecting incorporation of the analog;   removing or neutralizing the inhibitor; and   repeating the exposing, detecting, and removing steps at least once, thereby to determine the sequence of the template.   
     
     
         2 . The method of  claim 1 , wherein the template portion and/or the primer portion is directly or indirectly anchored to a support. 
     
     
         3 . The method of  claim 1 , wherein the detecting step comprises detecting individual analogs. 
     
     
         4 . The method of  claim 1  further comprising the step of removing unincorporated analog. 
     
     
         5 . The method of  claim 1 , wherein the inhibitor is selected from the group consisting of one or more carboxylic acid, one or more phosphate, one or more amino acid, one or more peptide, one or more sulfate, one or more caproic acid, and any combination thereof. 
     
     
         6 . The method of  claim 5 , wherein the amino acid is a negatively charged amino acid. 
     
     
         7 . The method of  claim 6 , wherein the amino acid is selected from the group consisting of aspartic acid, glutamic acid, histidine, lysine, and arginine. 
     
     
         8 . The method of  claim 5 , wherein the peptide is from about 2 to about 10 amino acids in length. 
     
     
         9 . The method of  claim 1 , wherein the inhibitor comprises multiple charged groups. 
     
     
         10 . The method of  claim 1 , wherein the inhibitor is negatively charged. 
     
     
         11 . The method of  claim 1 , wherein the inhibitor is positively charged. 
     
     
         12 . The method of  claim 1 , wherein the inhibitor does not cause steric inhibition of the polymerase. 
     
     
         13 . A nucleotide analog, comprising
 a nucleoside triphosphate;   an inhibitor comprising (a) one or more multiply charged groups or groups capable of becoming multiply charged, or (b) two or more singly charged groups or two or more groups capable of becoming singly charged;   a detectable label; and   a linker connecting the inhibitor and the label to the nucleoside triphosphate.   
     
     
         14 . The analog of  claim 13 , wherein the linker is cleavable. 
     
     
         15 . The analog of  claim 14 , wherein after the linker is cleaved, the residual analog has the structure of: 
       
         
           
           
               
               
           
         
       
       wherein
 B 1  is selected from the group consisting of purine bases, pyrimidine bases, and derivatives of purine and pyrimidine bases; 
 R′ is independently selected from the group consisting of —OH, —O—P(O)(OH) 2 , —O—C(O)—R x , —NHR y , and an —O-blocking agent, wherein R x  and R y  are alkyl groups; 
 R″ is independently selected from the group consisting of H and —OH; 
 R 7  is a phosphodiester or a phosphoryl group; and 
 z is an integer from about 1 to about 5. 
 
     
     
         16 . The analog of  claim 13 , wherein the charged groups consist of between about 2 to about 10 charged groups. 
     
     
         17 . The analog of  claim 13 , wherein the charged groups are selected from any combination of one or more carboxylic acid, one or more phosphate, one or more amino acid, one or more peptide, one or more sulfate, and one or more caproic acid. 
     
     
         18 . The analog of  claim 13 , wherein the label is optically detectable. 
     
     
         19 . The analog of  claim 18 , wherein the label is a fluorescent label. 
     
     
         20 . The analog of  claim 13 , wherein the inhibitor is not a steric inhibitor of a polymerase enzyme. 
     
     
         21 . The analog of  claim 13 , wherein the nucleoside triphosphate is selected from ATP, GTP, CTP, TTP, UTP, dATP, dGTP, dCTP, dTTP, dUTP, or an analog of any of the foregoing. 
     
     
         22 . A nucleotide analog of the following Formula II: 
       
         
           
           
               
               
           
         
       
       wherein
 NTP is a nucleoside or nucleotide triphosphate or an analog of either capable of incorporating onto the 3′ end of a polynucleotide strand hybridized to a template presenting the complement of the NTP; 
 L is a detectable label that facilitates the identification of the nucleotide analog; 
 Inhibitor comprises (a) one or more multiply charged groups or groups capable of becoming multiply charged, or (b) two or more singly charged groups or two or more groups capable of becoming singly charged; 
 R 1  and R 2  are independently a bond or a group, wherein at least one of R 1  and R 2  comprises a cleavable bond, which upon cleavage results in de-association of NTP from both L and Inhibitor; 
 R 3  is a bond or group linking R 2  to the Inhibitor moiety; and 
 R 4  is a bond or group linking R 2  to a L. 
 
     
     
         23 . The nucleotide analog of  claim 22 , wherein the Inhibitor does not comprise a nucleotide or nucleoside or analogs thereof. 
     
     
         24 . The nucleotide analog of  claim 22 , wherein the Inhibitor comprises a negatively charged group or a group capable of becoming negatively charged. 
     
     
         25 . The nucleotide analog of  claim 22 , wherein the Inhibitor comprises a positively charged group or a group capable of becoming positively charged. 
     
     
         26 . The nucleotide analog of  claim 22 , wherein the Inhibitor comprises two or more charged groups. 
     
     
         27 . The nucleotide analog of  claim 22 , wherein the Inhibitor comprises a charged group selected from the group consisting of —COOH, —PO 4 , —SO 4 , —SO 3 , —SO 2 , —NR w R v , where R w  and R v  independently is H, an alkyl or aryl group. 
     
     
         28 . The nucleotide analog of  claim 22 , wherein the Inhibitor comprises 
       
         
           
           
               
               
           
         
       
       wherein R 8  and R 9  independently is a H or an alkyl group; each of x and y is an integer from 0 to about 10. 
     
     
         29 . The nucleotide analog of  claim 28 , wherein R 8  and R 9  are H atoms and x=1 and y=2. 
     
     
         30 . The nucleotide analog of  claim 22 , wherein the Inhibitor does not comprise a —PO 4  group. 
     
     
         31 . The nucleotide analog of  claim 22 , wherein the Inhibitor does not comprise an aryl group. 
     
     
         32 . The nucleotide analog of  claim 22 , wherein the Inhibitor comprises an amino acid group or an amino acid analog group. 
     
     
         33 . The nucleotide analog of  claim 32 , wherein the Inhibitor comprises a peptide of 2 to 20 units of amino acids or analogs. 
     
     
         34 . The nucleotide analog of  claim 32 , wherein the Inhibitor comprises a group selected from the group consisting of Glu, Asp, Arg, His, Thr, Trp, Gln, Tyr and Lys. 
     
     
         35 . The nucleotide analog of  claim 22 , wherein R 3  comprises 
       
         
           
           
               
               
           
         
       
       wherein R 5  is a H or an alkyl group; p is an integer from 0 to about 10. 
     
     
         36 . The nucleotide analog of  claim 35 , wherein p is 5 or 6. 
     
     
         37 . The nucleotide analog of  claim 22 , wherein R 1  comprises a C—C triple bond. 
     
     
         38 . The nucleotide analog of  claim 22 , wherein R 1  comprises a S—S bond. 
     
     
         39 . The nucleotide analog of  claim 22 , wherein R 1  comprises a C—C triple bond and a S—S bond. 
     
     
         40 . The nucleotide analog of  claim 22 , wherein R 1  comprises 
       
         
           
           
               
               
           
         
       
       wherein R 6  is a H or an alkyl group; q and r independently is an integer from about 1 to about 10. 
     
     
         41 . The nucleotide analog of  claim 40 , wherein q is 1 or 2 and r is 1, 2 or 3. 
     
     
         42 . The nucleotide analog of  claim 22 , wherein NTP is selected from dATP, dGTP, dCTP, dTTP, dUTP, ATP, GTP, CTP, TTP, UTP or an analog thereof. 
     
     
         43 . The nucleotide analog of  claim 22 , wherein the L is an optically-detectable moiety. 
     
     
         44 . The nucleotide analog of  claim 43 , wherein the optically-detectable moiety comprises a fluorophore. 
     
     
         45 . The nucleotide analog of  claim 44 , wherein the fluorophore is Cy5 or ATTO 647N. 
     
     
         46 . The nucleotide analog of  claim 22 , wherein the group of the Inhibitor that is charged or capable of becoming charged is from about 5 to about 60 bonds away from the NTP. 
     
     
         47 . The nucleotide analog of  claim 36 , wherein p is 5 and the detectable label comprises ATTO 647N. 
     
     
         48 . The nucleotide analog of  claim 22 , wherein R 3  comprises 
       
         
           
           
               
               
           
         
       
       wherein k is an integer from about 1 to about 5. 
     
     
         49 . The nucleotide analog of  claim 48 , wherein the Inhibitor comprises a —COOH group. 
     
     
         50 . The nucleotide analog of  claim 49 , wherein the Inhibitor comprises two or more —COOH groups. 
     
     
         51 . The nucleotide analog of  claim 22 , wherein R 3  comprises 
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2  are independently H or alkyl groups, and may together form 3, 4, 5, or 6-member rings, and j is an integer from about 1 to about 5. 
     
     
         52 . The nucleotide analog of  claim 22 , wherein R 1  comprises 
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3 , and R 4  are independently H or alkyl groups, and two or more of which may together form one or more 3, 4, 5, or 6-member rings, and j is an integer from about 1 to about 3.