5-methylcytosine detection, compositions and methods therefor
View Patent ↗Compositions and methods for detecting 5-methylcytosine in a nucleic acid are disclosed. A 5-methylcytosine discriminator, which is a deoxyribonucleosidetriphosphate comprising a cytosine-pairing moiety such as a guanosine and a moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine is described. The discriminator is able to base pair with a cytosine but not a 5-methylcytosine. A 5-methylcytosine comprised by a target nucleotide can be detected in a reaction using a DNA polymerase and a primer hybridized immediately adjacent to the target nucleotide. In the reaction, pyrophosphate released upon incorporation of a dNTP complementary to a target nucleotide is detected. Lack of incorporation of the discriminator, but incorporation of a dGTP, can indicate that the target nucleotide is a 5-methylcytosine.
1. A 5-methylcytosine discriminator comprising:
a cytosine-pairing moiety; and
an alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine;
wherein the 5-methylcytosine discriminator further comprising a linker extending between the cytosine-pairing moiety and the alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.
2. A 5-methylcytosine discriminator according to claim 1 , wherein the linker is a planar linker.
3. A 5-methylcytosine discriminator according to claim 2 , wherein the planar linker comprises a plurality of fused rings.
4. A 5-methylcytosine discriminator according to claim 3 , wherein the plurality of fused rings comprises one or more aromatic rings.
5. A 5-methylcytosine discriminator according to claim 3 , wherein the plurality of fused rings comprises from three to about ten fused rings.
6. A 5-methylcytosine discriminator according to claim 5 , wherein the plurality of fused rings comprises from four to about eight fused rings.
7. A 5-methylcytosine discriminator according to claim 5 , wherein the plurality of fused rings comprises three fused rings.
8. A 5-methylcytosine discriminator according to claim 6 , wherein the plurality of fused rings comprises four fused rings.
9. A 5-methylcytosine discriminator according to claim 2 , wherein the cytosine-pairing moiety and the planar linker are rigidly attached.
10. A 5-methylcytosine discriminator according to claim 2 , wherein the cytosine-pairing moiety and the planar linker are co-planar.
11. A 5-methylcytosine discriminator according to claim 9 , further comprising an amide bond extending between the cytosine-pairing moiety and the linker.
12. A 5-methylcytosine discriminator according to claim 1 , comprising a structure:
wherein:
R 1 comprises the cytosine-pairing moiety; and R 2 comprises the alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.
13. A 5-methylcytosine discriminator according to claim 1 , comprising a structure:
wherein:
R 3 comprises the cytosine-pairing moiety; and R 4 comprises the alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.
14. A 5-methylcytosine discriminator according to claim 1 , wherein the alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.6 Angstroms.
15. A 5-methylcytosine discriminator according to claim 1 , wherein the alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.2 Angstroms.
16. A 5-methylcytosine discriminator according to claim 1 , wherein the alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 0.75 Angstroms.
17. A 5-methylcytosine discriminator according to claim 1 , wherein the cytosine-pairing moiety is a guanine moiety.
18. A 5-methylcytosine discriminator according to claim 1 , further comprising a pentose moiety.
19. A 5-methylcytosine discriminator according to claim 18 , wherein the pentose moiety is a 2′-deoxyribose moiety.
20. A 5-methylcytosine discriminator according to claim 18 , wherein the pentose moiety is a 2′,3′-dideoxyribose moiety.
21. A 5-methylcytosine discriminator according to claim 18 , further comprising three phosphate moieties.
22. A 5-methylcytosine discriminator according to claim 1 , comprising the structure:
23. A 5-methylcytosine discriminator according to claim 1 , comprising the structure:
24. A 5-methylcytosine discriminator according to claim 1 , further comprising a label.
25. A 5-methylcytosine discriminator according to claim 24 , wherein the label is a fluorophore selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.
26. A 5-methylcytosine discriminator according to claim 25 , wherein the fluorophore is VIC or FAM.
27. A 5-methylcytosine discriminator according to claim 24 , wherein the label is a hapten.
28. A 5-methylcytosine discriminator according to claim 27 , wherein the hapten is biotin or digoxygenin.
29. A 5-methylcytosine discriminator according to claim 24 , wherein the label is a radioisotope selected from 32 P, 33 P, 35 S, 3 H and 14 C.
30. A kit for detecting 5-methylcytosine, the kit comprising:
a) a 5-methylcytosine discriminator comprising: a cytosine-pairing moiety; and
an alkyl moiety which hinders hydrogen bonding between the cytosine pairing moiety and a 5-methylcytosine;
wherein the 5-methylcytosine discriminator further comprising a linker extending between the cytosine-pairing moiety and the alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine; and
b) packaging.
31. A kit for detecting 5-methylcytosine according to claim 30 , wherein the 5-methylcytosine discriminator further comprises a planar linker comprising a plurality of fused aromatic rings extending between the cytosine-pairing moiety and the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.
32. A kit for detecting 5-methylcytosine according to claim 31 , wherein the plurality of fused rings comprises three fused rings.
33. A kit for detecting 5-methylcytosine according to claim 31 , wherein the plurality of fused rings comprises four fused rings.
34. A kit for detecting 5-methylcytosine according to claim 31 , wherein the cytosine-pairing moiety and the planar linker are co-planar and wherein the 5- methylcytosine discriminator further comprises an amide bond extending between the cytosine- pairing moiety and the linker.
35. A kit for detecting 5-methylcytosine according to claim 30 , wherein the 5-methylcytosine discriminator further comprises a structure:
wherein:
R 1 comprises the cytosine-pairing moiety; and R 2 comprises the alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.
36. A kit for detecting 5-methylcytosine according to claim 30 , wherein the 5-methylcytosine discriminator further comprises a structure:
wherein:
R 3 comprises the cytosine-pairing moiety; and R 4 comprises the alkyl moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.
37. A kit for detecting 5-methylcytosine according to claim 30 , wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.6 Angstroms.
38. A kit for detecting 5-methylcytosine according to claim 30 , wherein the moiety which hinders hydrogen bonding between the cytosine pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.2 Angstroms.
39. A kit for detecting 5-methylcytosine according to claim 30 , wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 0.75 Angstroms.
40. A kit for detecting 5-methylcytosine according to claim 30 , wherein the cytosine-pairing moiety is a guanine moiety.
41. A kit for detecting 5-methylcytosine according to claim 30 , wherein the 5-methylcytosine discriminator comprises the structure:
42. A kit for detecting 5-methylcytosine according to claim 30 , wherein the 5-methylcytosine discriminator comprises the structure:
43. A kit for detecting 5-methylcytosine according to claim 30 , wherein the 5-methylcytosine discriminator further comprises a label.
44. A kit for detecting 5-methylcytosine according to claim 43 , wherein the label is a fluorophore selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9,and dR6G.
45. A kit for detecting 5-methylcytosine according to claim 44 , wherein the fluorophore is FAM or VIC.
46. A kit for detecting 5-methylcytosine according to claim 43 , wherein the label is a radioisotope selected from 32 P, 33 P, 35 S, 3 H and 14 C.
47. A kit for detecting 5-methylcytosine according to claim 43 , wherein the label is a hapten selected from biotin and digoxygenin.
48. A kit for detecting 5-methylcytosine according to claim 43 , further comprising a dGTP comprising a second label or a ddGTP comprising a second label.
49. A 5-methylcytosine discriminator according to claim 1 , wherein the alkyl moiety is a methyl moiety.