UNNATURAL POLYMERASE SUBSTRATES THAT CAN SUSTAIN ENZYMATIC SYNTHESIS OF DOUBLE STRANDED NUCLEIC ACIDS FROM A NUCLEIC ACID TEMPLATE AND METHODS OF USE

• US 20090269759A1
• Filed: 01/16/2009
• Published: 10/29/2009
• Est. Priority Date: 04/29/2008
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Menchen, Steven M. JR., Khan, Shoeb I., Lam, Joe, Ma, Zhaochun, Chen, Jer-Kang, Kenney, Paul, Huang, Boli, Rosenblum, Barnett
• Granted Patent: US 8,058,414 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety

Abstract

Nucleotide analogs that can sustain the enzymatic synthesis of double-stranded nucleic acid from a nucleic template are described. The nucleotide analogs include: (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine, uracil and their analogs; (ii) a label attached to the base or analog of the base via a cleavable linker; (iii) a deoxyribose; and (iv) one or more phosphate groups. The linker and/or the label inhibits template directed polymerase incorporation of a further nucleotide substrate onto an extended primer strand. In addition, cleavage of the linker leaves a residue attached to the base which is not present in the natural nucleotide and which does not inhibit extension of the primer strand. The nucleotide analogs can therefore be used as reversible terminators in sequencing by synthesis methods without blocking the 3′ hydroxyl group. Methods of sequencing DNA using the substrates are also described.

Claims

1. A compound represented by the following formula (I):

2. The compound of claim 1 where R₁ is an organic substituent selected from the group consisting of neutral forms of substituted or un-substituted alkyls, substituted or un-substituted aromatics, substituted or un-substituted heterocycles, substituted or un-substituted aromatic heterocycles, or anionic or cationic forms thereof.

3. The compound of claim 2, wherein cleavage of the chemically labile group L₂ leaves a linker residue moiety attached to the base having a terminal group F.

4. The compound of claim 3, wherein the terminal group F is not an amino group.

5. The compound of claim 3, wherein the terminal group F is an hydroxyl group.

6. The compound of claim 1, wherein l is 2 or 3.

7. The compound of claim 1, wherein the compound has a structure represented by the following formula (II):

8. The compound of claim 1, wherein L₂ comprises a chemically labile group selected from the group consisting of:

—

C(O)—

S—

;

—

C(O)O—

;

—

CH—

(SCH₃)—

Ph— and

[RO]₂P(O)S—

;

wherein Ph is a substituted phenyl group or a substituted aromatic ring; and

wherein R₁ is an organic substituent selected from the group consisting of neutral forms of substituted or un-substituted alkyls, substituted or un-substituted aromatics, substituted or un-substituted heterocycles, substituted or un-substituted aromatic heterocycles, or anionic or cationic forms thereof.

9. The compound of claim 2, wherein L₁ forms a permanent linkage between the base and the heteroatom F, and L₂ forms a transient linkage between the heteroatom F and R₂.

10. The compound of claim 9, wherein the heteroatom is O, S, or N.

11. The compound of claim 9, wherein L₁ comprises a moiety represented by the formula:

12. The compound of claim 9, wherein L₁ comprises a moiety represented by the formula:

13. The compound of claim 9, wherein L₂-R₂ comprises a moiety represented by the formula:

14. The compound of claim 1, wherein the compound has a structure represented by the following formula (VI):

15. The compound of claim 1, wherein the chemically labile group L₂ is activated by Ag⁺ to cleave F-L₂.

16. The compound of claim 1, wherein L₂ comprises a moiety selected from the group consisting of:

17. The compound of claim 1, wherein L₂ comprises a moiety of the following formula:

25. The compound of claim 1, wherein R₁ is a group that suppresses the template directed polymerase incorporation rate of the nucleoside triphosphate onto the 3′

end of an extending oligonucleotide prior to cleavage of L₂-R₂ from the previous incorporation event.

26. The compound of claim 25, wherein R₁ is a reporter or quencher.

27. The compound of claim 1, wherein the compound has a structure represented by the following formula (VI):

28. The compound of claim 27, wherein R₁

is an organic substituent selected from the group consisting of neutral forms of substituted or un-substituted alkyls, substituted or un-substituted aromatics, substituted or un-substituted heterocycles, substituted or un-substituted aromatic heterocycles, or anionic or cationic forms thereof.

29. The compound of claim 7, wherein R₁ is not H.

30. The compound of claim 29, wherein R₁ is an organic substituent selected from the group consisting of neutral forms of substituted or un-substituted alkyls, substituted or un-substituted aromatics, substituted or un-substituted heterocycles, substituted or un-substituted aromatic heterocycles, or anionic or cationic forms thereof.

18. A method of sequencing a nucleic acid comprising:

(a) hybridizing a primer to a target polynucleotide to form a primer-target duplex, wherein the target polynucleotide is attached to a solid support at the 3′

or 5′

end;

(b) contacting the primer-target duplex with a polymerase and one or more nucleotide analogs to incorporate a nucleotide analog onto the 3′

end of the primer thereby forming an extended primer strand, wherein the incorporated nucleotide analog terminates the polymerase reaction and wherein each of the one or more nucleotide analogs comprises;

(i) a base selected from the group consisting of adenine, guanine, cytosine, thymine, uracil and their analogs;

(ii) a label attached to the base or analog thereof via a cleavable linker;

(iii) a deoxyribose; and

(iv) one or more phosphate groups, wherein the label is unique for the base and wherein the combination of a polyphosphate terminal substituent, the linker and/or the label inhibits the template directed polymerase incorporation of a further nucleotide analog onto the extended primer strand;

(c) washing the surface of the solid support to remove unincorporated nucleotide analogs;

(d) detecting the unique label attached to the just-incorporated nucleotide analog to thereby identify the just-incorporated nucleotide analog;

(e) cleaving the cleavable linker between the just incorporated nucleotide analog and the unique label thereby allowing the incorporation of a further nucleotide analog onto the extended primer strand;

(f) washing the surface of the solid support to remove cleaved compound fragments; and

(e) repeating steps (b), (c), (d), (e) and (f).

19. The method of claim 18, wherein each of the one or more nucleotide analogs has a structure represented by the following formula (I):

20. The method of claim 19, wherein R₁ is an organic substituent selected from the group consisting of neutral forms of substituted or un-substituted alkyls, substituted or un-substituted aromatics, substituted or un-substituted heterocycles, substituted or un-substituted aromatic heterocycles, or anionic or cationic forms thereof, L₃ is H, l is 2, and m is 5.

21. The method of claim 19, wherein the one or more nucleotide analogs comprise:

a first nucleotide analog wherein B is adenine having a first label;

a second nucleotide analog wherein B is guanine having a second label;

a third nucleotide analog wherein B is cytosine having a third label; and

a fourth nucleotide analog wherein B is thymine having a fourth label.

22. The method of claim 21, wherein the first, second, third and fourth labels are each fluorescent labels.

23. The method of claim 19, wherein R₁ is a group that suppresses the template directed polymerase incorporation rate of the nucleoside triphosphate onto the 3′

end of an extending oligonucleotide prior to cleavage of L₂-R₂ from the previous incorporation event.

24. The method of claim 23, wherein R₁ is a reporter or quencher.

31. The method of claim 18, wherein each of the one or more nucleotide analogs has a structure represented by the following formula (II):

32. The method of claim 31, wherein R₁ is a substituted or un-substituted hydrocarbon group.

33. The method of claim 32, wherein R₁ is a substituted or un-substituted methyl, propargyl or benzyl group.

34. The method of claim 31, wherein each of the one or more nucleotide analogs has a structure represented by the following formula (VI):