Reversible terminator molecules and methods of their use

• US 9,650,406 B2
• Filed: 07/26/2013
• Issued: 05/16/2017
• 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, Jain, Moti, Mei, Rui, McGall, Glenn
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/952,377
• Publication Number:
  US 20140242579A1
• Time in Patent Office: 1,390 Days
• Field of Search: 536 41, 536 231, 536 266, 435 612
• 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. A reversible terminator compound selected from the group consisting of Formula:

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 that is optionally substituted.

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 K is a halogen.

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

7. The compound according to claim 1, wherein D1 and D2 are acceptor-donor FRET pair labels.

8. A composition, comprising two or more reversible terminators of claim 1, 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.

29. The compound according to claim 1, wherein R is the C₁-C₆ alkyl and the C₁-C₆ alkyl is optionally substituted by a substituent selected from the group consisting of —

OR′

, ═

O, ═

NR′

, ═

N—

OR′

, —

NR′

R″

, —

SR′

, -halogen, —

SiR′

R″

R′

″

, —

OC(O)R′

, —

C(O)R′

, —

CO₂R′

, —

CONR′

R″

, —

OC(O)NR′

R″

, —

NR″

C(O)R′

, —

NR′

—

C(O)NR″

R′

″

, —

NR″

C(O)₂R′

, —

NR—

C(NR′

R″

R′

″

)═

NR″

″

, —

NR′

—

C(NR″

R′

″

)═

NR″

″

, —

S(O)R′

, —

S(O)₂R′

, —

S(O)₂NR′

R″

, —

NRSO₂R′

, —

CN and —

NO₂;

wherein R′

, R″

, R′

″ and

R″

″

are each independently selected from hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkyl, alkoxy, thioalkoxy, and arylalkyl, and

wherein when R′ and

R″

are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.

30. The compound according to claim 1, wherein R is the aryl group, and the aryl group is optionally substituted by a substituent selected from the group consisting of halogen, —

OR′

, ═

O, ═

NR′

, ═

N—

OR′

, —

NR′

R″

, —

SR′

, -halogen, —

SiR′

R″

R′

″

, —

OC(O)R′

, —

C(O)R′

, —

CO₂R′

, —

CONR′

R″

, —

OC(O)NR′

R″

, —

NR″

C(O)R′

, —

NR′

—

C(O)NR″

R′

″

, —

NR″

C(O)₂R′

, —

NR′

, —

C(NR′

R″

R′

″

)═

NR′

″

, —

NR′

, —

C(NR″

R′

″

)═

NR″

″

, —

S(O)R′

, —

S(O)₂R′

, —

S(O)₂NR′

R″

, —

NRSO₂R′

, —

CN and —

NO₂, —

R′

, —

N₃, —

CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl;

wherein R′

, R″

, R′

″ and

R″

″

are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

9. A nucleotide comprising:

a sugar moiety covalently bound at a 3-oxygen to a phosphate moiety, wherein the atoms at the bonding positions are oxygen, and an atom or group at a non-bonding position of the phosphate moiety is selected from an oxygen atom, a sulfur atom or a BH₃ group; and

a removable blocking group bound to the phosphate moiety,

wherein the blocking group further comprises a linker group, and

wherein the linker group 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)_m—

NHC(O)—

(CH₂)_n—

NHC(O)—

,

10. The nucleotide according to claim 9, wherein the nucleotide has covalently attached to the 5′

hydroxyl group one or more phosphate groups.

11. The nucleotide according to claim 10, wherein the 5′

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

12. The nucleotide according to claim 9, wherein the blocking group is removable by incubation with one or more enzymes.

13. The nucleotide according to claim 12, where the one or more enzymes are selected from the group consisting of:

phosphodiesterase and phospholipase.

14. The nucleotide according to claim 12, wherein the enzyme is endonuclease IV.

15. The nucleotide according to claim 9, wherein the label is present and the label is a fluorescent dye.

16. The nucleotide according to claim 9, where the base is selected from the group consisting of naturally occurring:

C, A, T, and G.

17. A composition, comprising two or more nucleotides of claim 16, wherein each different nucleotide of claim 16 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.

18. The nucleotide according to claim 9, wherein a 2′

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

19. A reversible terminator compound selected from the group consisting of Formula:

20. The compound according to claim 19, wherein:

the compound is Formula (II), and

R has the structure

21. The compound according to claim 20, wherein:

X is O, and

the variable number of phosphates is 1.

31. The compound according to claim 19, wherein B is selected from the group consisting of naturally occurring guanine, cytosine, adenine, uracil and thymine.

32. The compound according to claim 19, wherein at least one linker group L has the structure depicted in Scheme I:

33. The compound according to claim 32, wherein the at least one linker group 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)_m—

NHC(O)—

(CH₂)_n—

NHC(O)—

,

34. The compound according to claim 32, wherein Q1-Q3 are independently one or more of the following:

35. The compound according to claim 33, wherein K is a halogen.

36. The compound according to claim 19, wherein D1 and/or D2 are fluorescent detectable labels.

37. The compound according to claim 19, wherein D1 and D2 are acceptor-donor FRET pair labels.

38. A composition, comprising two or more nucleotides of claim 31, wherein each reversible terminator of claim 31 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.

45. The compound according to claim 19, wherein R is chlorophenyl.

46. The compound according to claim 45, wherein at least one linker group L has the structure depicted in Scheme I:

47. The compound according to claim 46, wherein the at least one linker group 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)_m—

NHC(O)—

(CH₂)_n—

NHC(O)—

,

48. The compound according to claim 46, wherein Q1-Q3 are independently one or more of the following:

49. The compound according to claim 47, wherein K is a halogen.

50. The compound according to claim 45, wherein D1 and/or D2 are fluorescent detectable labels.

51. The compound according to claim 45, wherein D1 and D2 are acceptor-donor FRET pair labels.

22. A reversible terminator compound selected from the group consisting of Formula:

23. The compound according to claim 22, wherein Q1-Q3 are independently one or more of the following:

24. The compound according to claim 23, wherein:

the compound is Formula (III).

25. The compound according to claim 24, wherein:

X is O, and

the variable number of phosphates is 1.

39. The compound according to claim 22, wherein D1 and/or D2 are fluorescent detectable labels.

40. The compound according to claim 22, wherein D1 and D2 are acceptor-donor FRET pair labels.

41. The compound according to claim 22, wherein B is selected from the group consisting of naturally occurring guanine, cytosine, adenine, uracil and thymine.

42. A composition, comprising two or more nucleotides of claim 41, wherein each reversible terminator of claim 41 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.

43. The compound according to claim 22, wherein R is chlorophenyl that is optionally substituted.

44. The compound according to claim 22, wherein R is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl and hexyl.

26. A reversible terminator compound of Formula (II′

);

27. A reversible terminator compound of Formula (I-A):

28. The compound according to claim 27, wherein B is selected from the group consisting of naturally occurring guanine, cytosine, adenine, uracil and thymine.