Synthesis of four-color 3′-O-allyl modified photocleavable fluorescent nucleotides and related methods

1. A method for determining the sequence of a single-stranded DNA template comprising the following steps:

(A) contacting the single-stranded DNA template with 9° N polymerase in the presence of (i) a primer and (ii) four nucleotide analogues under conditions such that the 9° N polymerase catalyzes DNA synthesis of a DNA extension product which has incorporated at its 3′ end, a nucleotide analogue complementary to, and base-paired with, a nucleotide residue which is not base-paired and is located at the 5′ end of the single-stranded DNA template to be sequenced,

wherein each of the four nucleotide analogues comprises: (a) a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil, (b) a deoxyribose, (c) a unique fluorophore cleavably attached to each base of the same type base, and (d) a removable chemical moiety bound to the 3′-oxygen of the deoxyribose which blocks further DNA synthesis when it is incorporated into the extension product; and

wherein each fluorescent nucleotide analogue when incorporated into the DNA extension product is characterized by a predetermined fluorescence emission wavelength different from the fluorescence emission wavelength of each of the other three fluorescent nucleotide analogues when incorporated into the DNA extension product;

(B) removing nucleotide analogues not incorporated into the DNA extension product;

(C) determining the identity of the fluorescent nucleotide analogue incorporated into the DNA extension product based upon its characteristic fluorescence emission wavelength;

(D) treating the nucleotide analogue incorporated into the DNA extension product so as to remove the chemical moiety bound to the 3′-oxygen of the deoxyribose and cleave the fluorophore from the base;

(E) repeating each of steps (A) to (D) to successively determine the identity of the nucleotide analogues incorporated at the 3′ end of each succeeding extension product so synthesized so as to thereby determine the sequence of the single-stranded DNA template.

2. The method of claim 1 , wherein the single-stranded DNA template is bound to a solid substrate.

3. The method of claim 1 , wherein the primer in (A) is a self-priming moiety.

4. The method of claim 1 , wherein in (A) the fluorophore is photocleavably attached to each base of the same type base.

5. The method of claim 4 , wherein all the fluorophores are photocleavably attached to all the bases.

6. The method of claim 1 , wherein in (A) the removable chemical moiety bound to the 3′-oxygen of the deoxyribose is —CH 2 —CH—CH 2 or —CH 2 —O—CH 3 .

7. The method of claim 1 , wherein in (A) the 9° N polymerase is 9° N polymerase (exo-) A485L/Y409V.

8. The method of claim 1 , wherein the treatment in (D) to remove the chemical moiety from the 3′-oxygen is performed in the presence of a palladium (Pd) catalyst.

9. The method of claim 8 , wherein the palladium catalyst is Na 2 PdCl 4 .

10. The method of claim 1 , wherein the treatment in (D) to remove the chemical moiety is performed at about pH 8.8.

11. The method of claim 4 , wherein the treatment in (D) to photocleave the fluorophore from the base comprises light irradiation.

12. The method of claim 1 , wherein the treatment in (D) results in removal of the chemical moiety from the 3′-oxygen and cleavage of the fluorophore from the base substantially concurrently.

13. The method of claim 1 , wherein the treatment in (D) first results in removal of the chemical moiety from the 3′-oxygen and then cleavage of the fluorophore from the base.

14. The method of claim 1 , wherein the treatment in (D) first results in cleavage of the fluorophore from the base and then removal of the chemical moiety from the 3′-oxygen.

15. A method for determining the sequence of a single-stranded RNA template comprising the following steps:

(A) contacting the single-stranded RNA template with an RNA polymerase in the presence of (i) a primer and (ii) four nucleotide analogues under conditions such that the RNA polymerase catalyzes RNA synthesis of an RNA extension product which has incorporated at its 3′ end, a nucleotide analogue complementary to, and base-paired with, a nucleotide residue which is not base-paired and is located at the 5′ end of the single-stranded RNA template to be sequenced,

wherein each of the four nucleotide analogues comprises: (a) a base selected from the group consisting of adenine, guanine, cytosine, and uracil, (b) a ribose, (c) a unique fluorophore cleavably attached to each base of the same type base, and (d) a removable chemical moiety bound to the 3′-oxygen of the ribose which blocks further RNA synthesis when it is incorporated into the extension product; and

wherein each fluorescent nucleotide analogue when incorporated into the RNA extension product is characterized by a predetermined fluorescence emission wavelength different from the fluorescence emission wavelength of each of the other three fluorescent nucleotide analogues when incorporated into the RNA extension product;

(B) removing nucleotide analogues not incorporated into the RNA extension product;

(C) determining the identity of the fluorescent nucleotide analogue incorporated into the RNA extension product based upon its characteristic fluorescence emission wavelength;

(D) treating the nucleotide analogue incorporated into the RNA extension product so as to remove the chemical moiety bound to the 3′-oxygen of the ribose and cleave the fluorophore from the base;

(E) repeating each of steps (A) to (D) to successively determine the identity of the nucleotide analogues incorporated at the 3′ end of each succeeding extension product so synthesized so as to thereby determine the sequence of the single-stranded RNA template.

16. The method of claim 15 , wherein the single-stranded RNA template is bound to a solid substrate.

17. The method of claim 15 , wherein the primer in (A) is a self-priming moiety.

18. The method of claim 15 , wherein in (A) the fluorophore is photocleavably attached to each base of the same type base.

19. The method of claim 18 , wherein all the fluorophores are photocleavably attached to all the bases.

20. The method of claim 15 , wherein in (A) the removable chemical moiety bound to the 3′-oxygen of the ribose is —CH 2 —CH═CH 2 or —CH 2 —O—CH 3 .

21. The method of claim 15 , wherein the treatment in (D) to remove the chemical moiety from the 3′-oxygen is performed in the presence of a palladium (Pd) catalyst.

22. The method of claim 21 , wherein the palladium catalyst is Na 2 PdCl 4 .

23. The method of claim 15 , wherein the treatment in (D) to remove the chemical moiety is performed at about pH 8.8.

24. The method of claim 18 , wherein the treatment in (D) to photocleave the fluorophore from the base comprises light irradiation.

25. The method of claim 15 , wherein the treatment in (D) results in removal of the chemical moiety from the 3′-oxygen and cleavage of the fluorophore from the base substantially concurrently.

26. The method of claim 15 , wherein the treatment in (D) first results in removal of the chemical moiety from the 3′-oxygen and then cleavage of the fluorophore from the base.

27. The method of claim 15 , wherein the treatment in (D) first results in cleavage of the fluorophore from the base and then removal of the chemical moiety from the 3′-oxygen.

28. The method of claim 6 , wherein if the removable chemical moiety bound to the 3′-oxygen of the deoxyribose is —CH 2 —CH═CH 2 , then the treatment in (D) to remove the chemical moiety is performed at about pH 8.8.