Nucleotide analogues
The present invention provides methods, compositions, mixtures and kits utilizing deoxynucleoside triphosphates comprising a 3′-O position capped by a group comprising methylenedisulfide as a cleavable protecting group and a detectable label reversibly connected to the nucleobase of said deoxynucleoside. Such compounds provide new possibilities for future sequencing technologies, including but not limited to Sequencing by Synthesis.
1. A deoxynucleoside triphosphate having the structure:
wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups; B is a nucleobase; A is an attachment group selected from the group consisting of exocyclic amine, propargyl amine, and propargyl hydroxyl;
C is a cleavable site core selected from the group consisting of:
wherein R 1 and R 2 are independently selected alkyl groups; L 1 and L 2 are connecting groups; and Label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes.
2. The deoxynucleoside triphosphate according to claim 1 , wherein said nucleobase is non-natural.
3. The deoxynucleoside triphosphate according to claim 2 , wherein the non-natural nucleobase is selected from the group consisting of 7-deaza guanine, 7-deaza adenine, 2-amino,7-deaza adenine, and 2-amino adenine.
4. The deoxynucleoside triphosphate according to claim 1 , wherein said cleavable protecting group is of the formula —CH 2 —SS—R, wherein R is selected from the group consisting of alkyl and substituted alkyl groups.
5. The deoxynucleoside triphosphate according to claim 1 , wherein said detectable label is a fluorescent label.
6. A labeled deoxynucleoside triphosphate according to the following structure:
wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups; B is a nucleobase, A is an attachment group selected from the group consisting of propargyl, exocyclic amine, propargyl amine, and propargyl hydroxyl; C is a cleavable site core selected from the group consisting of:
wherein R 1 and R 2 are independently selected alkyl groups;
and L 1 and L 2 are connecting groups, wherein L 1 is selected from the group consisting of —CONH(CH 2 ) x —, —CO—O(CH 2 ) x —, —CONH—(OCH 2 CH 2 O) x —, —CO—O(CH 2 CH 2 O) x —, and —CO(CH 2 ) x —, wherein x is 0-10; and wherein L 2 is selected from the group consisting of —CO—, —CONH—, —NHCONH—, —O—, —S—, —C═N, and —N═N—, alkyl, aryl, branched alkyl, branched aryl and combinations thereof and, wherein the label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes.
7. The labeled deoxynucleoside triphosphate according to claim 6 , wherein L 2 is not —S—.
8. A deoxynucleoside triphosphate having the structure:
wherein D is a cleavable protecting group selected from the group consisting of an disulfide alkyl, disulfide substituted alkyl groups, disulfide allyl, and disulfide substituted allyl groups; B is a nucleobase;
Linker comprises a cleavable oxymethylenedisulfide-containing site core, wherein said cleavable site core is selected from the group consisting of:
wherein R 1 and R 2 are independently selected alkyl groups; and Label is a detectable label selected from the group consisting of fluorophore dyes, energy transfer dyes, mass-tags, biotin, and haptenes.
9. The deoxynucleoside triphosphate according to claim 1 , wherein L 1 is selected from the group consisting of—CONH(CH 2 ) x —, —CO—O(CH 2 ) x —, —CONH—(OCH 2 CH 2 O) x —, —CO—O(CH 2 CH 2 O) x —, and —CO(CH 2 ) x —, wherein x is 0-10.
10. The deoxynucleoside triphosphate according to claim 1 , wherein L 2 is selected from the group consisting of
—NH—, —(CH 2 ) x —NH—, —C(Me) 2 (CH 2 ) x NH—, —CH(Me)(CH 2 ) x NH—, —C(Me) 2 (CH 2 ) x CO—, —CH(Me)(CH 2 ) x CO—, —(CH 2 ) x OCONH(CH 2 ) y O(CH 2 ) z NH—, —(CH 2 ) x CONH(CH 2 CH 2 O) y (CH 2 ) z NH—, —(CH 2 ) x OCONH(CH 2 CH 2 O) y (CH 2 ) z NH—, and —CONH(CH 2 ) x —, —CO(CH 2 ) x —, wherein x, y, and z are each independently selected from is 0-10.