Design and synthesis of novel disulfide linker based nucleotides as reversible terminators for dna sequencing by synthesis

A method for sequencing a nucleic acid comprising:

• a) contacting a nucleic acid having a primer hybridized to a portion thereof, with a polymerase and a first type of nucleotide analogue under conditions permitting the nucleotide polymerase to catalyze incorporation of the nucleotide analogue into the primer if the nucleotide analogue is complementary to a nucleotide residue of the nucleic acid that is immediately 5′ to a nucleotide residue of the nucleic acid hybridized to the 3′ terminal nucleotide residue of the primer, so as to form a nucleic acid extension product, wherein the nucleotide analogue has the structure:
• wherein,
• B is a nucleobase;
• L3 is a cleavable linker having the structure:
• wherein L2 is a linker;
• R6 is a polymerase-compatible cleavable dithio moiety that when bound to the 3′-O of the nucleotide analogues prevents a polymerase from catalyzing a polymerase reaction with the 3′-O of the nucleotide analogue, wherein R6 has the structure:
• Wherein R1A and R8Bare independently hydrogen, —CH3, —CX3, —CHX2, —CH2X, —CN, -Ph, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 2 to 6 membered heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3 to 6 membered heterocycloalkyl, substituted or unsubstituted phenyl, or substituted or unsubstituted 5 to 6 membered heteroaryl, wherein X is a halogen;
• wherein R9, R10, and R11 are each independently hydrogen, —CX3, —CHX2, —CH2X, —OCH3, —SCH3, —NHCH3, —CN, -Ph, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 2 to 6 membered heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3 to 6 membered heterocycloalkyl, substituted or unsubstituted phenyl, or substituted or unsubstituted 5 to 6 membered heteroary, wherein X is a halogen;
• R5 is an anchor moiety, wherein the identity of the anchor moiety is predetermined and correlated to the identity of the nucleobase;
• b) removing any nucleotide analogue not incorporated into the primer in step a);
• c) contacting the nucleic acid in step a) with at least one compound having the formula R12-L4-R13, wherein R12 is a complementary anchor moiety binder that rapidly reacts with the anchor moiety, thereby forming a conjugate with the anchor moiety, L4 is a covalent or a non-covalent linker, and R13 is a detectable label;
• d) detecting the presence of the detectable label so as to thereby determine whether the nucleotide analogue of step a) was incorporated so as to thereby determine the identity of the complementary nucleotide residue in the template DNA, and
• wherein if the base of the nucleotide analogue a) is not complementary to the nucleotide residue of the nucleic acid which is immediately 5′ to the nucleotide residue of the single-stranded DNA hybridized to the 3′ terminal nucleotide residue of the primer, then iteratively repeating steps a) through c) with a second, third, and then fourth type of nucleotide analogue, wherein each different type of nucleotide analogue has a different base from each other type of nucleotide analogue, until the nucleotide analogue has a base that is complementary.
• e) contacting the nucleic acid with a cleaving agent, so as to (i) cleave the cleavable linker attached to the nucleobase, and (ii) cleave the cleavable dithio moiety, thereby resulting in a 3′-OH on the growing DNA strand;
• and
• f) iteratively performing steps a) through e) for each nucleotide residue of the nucleic acid to be sequenced so as to thereby determine the sequence of the nucleic acid.

The compound of 2, wherein

• L1 is L1A-L1B-L1C-L1D-L1E;
• and
• L1A, L1B, L1C, L1D and L1E are independently a bond, substituted or unsubstituted C1-C8 alkylene, substituted or unsubstituted 2 to 8 membered heteroalkylene, substituted or unsubstituted C3-C8 cycloalkylene, substituted or unsubstituted 3 to 8 membered heterocycloalkylene, substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene;
• wherein at least one of L1A, L1B, L1C, L1D and L1E is not a bond.

The compound of 3, wherein

• L1 is L1A-L1B-L1C-L1D-L1E;
• and
• L1A, L1B, L1C, L1D and L1E are independently a bond, substituted or unsubstituted C1-C8 alkylene, substituted or unsubstituted 2 to 8 membered heteroalkylene, substituted or unsubstituted C3-C8 cycloalkylene, substituted or unsubstituted 3 to 8 membered heterocycloalkylene, substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene;
• wherein at least one of L1A, L1B, L1C, L1D and L1E is not a bond.