REVERSIBLE TERMINATOR MOLECULES AND METHODS OF THEIR USE

• US 20140242579A1
• Filed: 07/26/2013
• Published: 08/28/2014
• Est. Priority Date: 02/28/2013
• Status: Active Grant

• Current Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Original Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Inventors: ZHOU, Wei, MCGALL, Glenn, JAIN, Moti, MEI, Rui
• Granted Patent: US 9,650,406 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

The present invention relates generally to methods of sequencing of polynucleotides and compounds, compositions and kits useful for sequencing of polynucleotides. The chemical compounds include nucleotide and nucleoside analogs which possess a blocking group covalently attached to the 3′ hydroxyl of the sugar moiety. The blocking group may optionally be additionally covalently attached to a linker and/or detectable label. The nucleotide analogs may be ribonucleotide or deoxyribonucleotide analogs. Methods include incorporation of the reversible terminator molecules into growing polynucleotide strands by polymerase enzymes, such as in single base sequencing methodologies utilizing sequential reversible termination techniques.

Claims

1. One or more reversible terminator compound selected from the group consisting of:

2. The compound according to claim 1, wherein B is selected from the group consisting of naturally occurring guanine, cytosine, adenine, uracil and thymine.

3. The compound according to claim 1, wherein R is chlorophenyl.

4. The compound according to claim 1, wherein R is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl and hexyl.

5. The compound according to claim 1, wherein linker L has the structure depicted in Scheme I:

6. The compound according to claim 5, wherein the linker is selected from one or more of the group consisting of:

—

(CH₂)₆—

NHC(O)(CH₂)_n—

NHC(O)—

,

—

CH₂(CH₂OCH₂)₃CH₂—

NHC(O)—

(CH₂)_n—

NHC(O)—

,

—

CH₂CH(CH₂CH₂OH)(OCH₂CH₂)_n—

NHC(O)—

(CH₂)_n—

NHC(O)—

,

7. The compound according to claim 5, wherein Q is any one or more of the following:

—

O—

, —

S—

S—

,

8. The compound according to claim 6, wherein X is a halogen.

9. The compound according to claim 1, wherein D1 and/or D2 are fluorescent detectable labels.

10. The compound according to claim 1, wherein D1 and D2 are acceptor-donor FRET pair labels, respectively, or fluorophore-quencher labels, respectively.

11. The compound according to claim 2, wherein each reversible terminator associated with each base B is individually attached to a different fluorescent label such that each base B is detectably distinguishable from any other different base B.

28. A method of sequencing a polynucleotide, which comprises:

performing a polymerization reaction in a reaction system comprising a target polynucleotide to be sequenced, one or more polynucleotide primers which hybridize with the target polynucleotide to be sequenced, a catalytic amount of a polymerase enzyme and the compound of claim 1.

12. A nucleoside comprising a sugar moiety, wherein the sugar moiety has covalently bound thereto at the 3′

hydroxyl group a phosphate moiety and a removable blocking group, wherein the blocking group is an alkyl or aryl group.

13. The nucleoside according to claim 12, wherein the blocking group comprises a substituted alkyl or aryl group.

14. The nucleoside according to claim 12, wherein the nucleoside has covalently attached to the 5′

hydroxyl group one or more phosphate groups.

15. The nucleoside according to claim 14, wherein the 5′

hydroxyl group has covalently attached thereto three or five phosphate groups.

16. The nucleoside according to claim 12, wherein the blocking group is removable by incubation with one or more enzymes.

17. The nucleoside according to claim 16, where the one or more enzymes are selected from the group consisting of:

phosphodiesterase and phospholipase.

18. The nucleoside according to claim 16, wherein the enzyme is endonuclease IV.

19. The nucleoside according to claim 12, wherein the blocking group further comprises a linker moiety and optionally a label.

20. The nucleoside according to claim 19, wherein the linker is enzymatically cleavable.

21. The nucleoside according to claim 19, wherein the linker is sufficiently long to allow cleavage of the linker by one or more enzymes.

22. The nucleoside according to claim 19, wherein the linker is selected from the group consisting of:

—

(CH₂)₆—

NHC(O)(CH₂)_n—

NHC(O)—

,

—

CH₂(CH₂OCH₂)₃CH₂—

NHC(O)—

(CH₂)_n—

NHC(O)—

,

—

CH₂CH(CH₂CH₂OH)(OCH₂CH₂)_n—

NHC(O)—

(CH₂)_n—

NHC(O)—

,

23. The nucleoside according to claim 19, wherein the label is a fluorescent dye.

24. The nucleoside according to claim 19, where the base is selected from the group consisting of naturally occurring:

C, A, T, G, and mixtures thereof.

25. The nucleoside according to claim 24, wherein each different nucleoside comprising a different base comprises a blocking group comprising a label, and wherein each label is different allowing differential detection of each of C, A, T, and G.

26. The nucleoside according to claim 12, wherein the phosphate moiety covalently attached to the 3′

hydroxyl group comprises at least one sulfur atom or BH₃ group substituted for one of the non-bonding phosphate oxygen atoms.

27. The nucleoside according to claim 12, wherein the 2′

group on the sugar moiety has covalently attached thereto a hydroxyl group.