Nucleotide analogs
View Patent ↗The invention generally relates to nucleotide analogs and methods of their use in sequencing-by-synthesis reactions. In certain embodiments, the invention provides a nucleotide analog including a detectable label attached to a nitrogenous base portion of a nucleotide analog by a cleavable linker, in which contact of the analog with at least one activating agent results in cleavage of the label and elimination of the linker, thereby producing a natural nucleotide, a 9-deaza-G, 9-deaza-A, or ψ-uridine.
1. A nucleotide analog comprising a detectable label attached to the nitrogenous base portion of the nucleotide by a cleavable linker,
wherein contact of the analog with a reducing agent results in release of the label and elimination of the linker, thereby producing a natural nucleotide, 9-deaza-G, 9-deaza-A, or ψ-uridine, characterized in that no portion of the label or the linker remains attached to the nitrogenous base after cleavage.
2. The analog according to claim 1 , wherein the
reducing agent is selected from dithiothreitol (DTT), β-mercaptoethanol, dithioerythretol (DTE), GSH, cysteine, cysteamine, tricarboxyethyl phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP), glutathione, and salts of sulfurous acid.
3. The analog according to
claim 1 , wherein the reducing agent is dithiothreitol (DTT).
4. The analog according to
claim 1 , wherein the reducing agent is tricarboxyethyl phosphine (TCEP).
5. The analog according to claim 1 , wherein the linker is a Staudinger linker.
6. The analog according to claim 1 , wherein the linker comprises a disulfide bond.
7. The analog according to claim 1 , wherein after cleavage of the linker by contact with the reducing agent, a portion of the linker temporarily remains attached to the analog and is thereafter spontaneously eliminated by a cyclization reaction, thereby producing the natural nucleotide, the 9-deaza-G, the 9-deaza-A, or the ψ-uridine.
8. The analog according to claim 1 , wherein the linker is attached to the nitrogenous base portion of the analog as follows:
to N3 of the base when the base is thymine or uracil;
to N4 of the base when the base is cytosine or adenine;
to N2 of the base when the base is guanine;
to N7 of the base when the base is 9-deaza-G or 9-deaza-A; or
to the N5 when the base is ψ-uridine.
9. The analog according to claim 1 , wherein the linker is attached to the nitrogenous base portion of the analog as follows:
to C5 of the base when the base is thymine, cytosine, or uracil;
to C7 when the base is adenine or guanine;
to N7 when the base is 9-deaza-guanine or 9-deaza-adenine; or
to N5 when the base is ψ-uridine.
10. The analog according to claim 1 , wherein the detectable label is a fluorescent label.
11. The analog according to claim 10 , wherein the fluorescent label is selected from cyanine, rhodamine, fluorescein, coumarin, BODIPY, alexa, Atto and conjugated multi-dyes.
12. The analog according to claim 1 , wherein the detectable label is a non-optically detectable label.
13. A nucleotide analog comprising a structure selected from:
wherein D is a group that comprises a detectable label.
14. A method of sequencing a nucleic acid, the method comprising: directly or indirectly anchoring a nucleic acid duplex to a surface, the duplex comprising a template portion and a primer portion hybridized thereto; exposing the duplex to a nucleotide analog according to claim 1 in the presence of a polymerase capable of catalyzing the addition of the nucleotide analog to the primer portion in a template-dependent manner; detecting incorporation of the nucleotide analog into the primer portion; contacting the nucleic acid duplex with at least one activating agent, thereby cleaving the detectable label and eliminating the linker to produce a natural nucleotide or a 9-deaza-G; and repeating the exposing, detecting, and contacting steps at least once.
15. The method according to claim 14 , further comprising: removing unincorporated nucleotide analog and polymerase in all or some repetitions of the exposing, detecting, and contacting steps.
16. The analog according to claim 1 , wherein the nitrogenous base is a purine, the linker comprises a disulfide bond (—S—S—), and the linker is attached to the purine through a nitrogen atom in a ring of the purine.
17. The analog according to claim 1 , comprising a structure selected from:
wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, and D is a group that comprises a detectable label.
18. The analog according to claim 1 , wherein the nitrogenous base is a purine, the linker comprises a disulfide bond, and the linker is attached to the purine through a carbon atom in a ring of the purine.
19. The analog according to claim 1 , comprising a structure selected from:
wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, and D is a group that comprises a detectable label.
20. The analog according to claim 1 , wherein the nitrogenous base is a pyrimidine, the linker comprises a disulfide bond, and the linker is attached to the pyrimidine through a nitrogen atom in the ring of the pyrimidine.
21. The analog according to claim 1 , comprising a structure selected from:
wherein X═O, NH, S, or CH 2 , and
wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, and D is a group that comprises a detectable label.
22. The analog according to claim 1 , wherein the nitrogenous base is a pyrimidine, the linker comprises a disulfide bond, and the linker is attached to the pyrimidine through a carbon atom in the ring of the pyrimidine.
23. The analog according to claim 1 , comprising a structure selected from:
wherein X═O, NH, S, or CH 2 ; Y═H or CH 3 ; and
wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, and D is a group that comprises a detectable label.
24. The analog according to claim 1 , wherein the linker comprises an azide group (—N 3 ).
25. The analog according to claim 1 , comprising a structure selected from:
wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, and D is a group that comprises a detectable label.
26. The analog according to claim 1 , wherein the linker comprises a sulfenamide bond (—S—N—).
27. The analog according to claim 1 , comprising a structure selected from:
wherein Y═H or CH 3 ; and
wherein R is a ribose or deoxyribose, P is a mono-, di- or triphosphate, L is a linking component, and D is a group that comprises a detectable label.
28. The analog according to claim 1 , comprising an inhibitor that is configured so that the analog may be added to a nucleic acid primer by a polymerase, whereupon it inhibits the polymerase from adding a further nucleotide to the primer.
29. The analog according to claim 28 , configured so that the inhibitor is released along with the detectable label upon cleavage of the cleavable linker.
30. The analog according to claim 1 , wherein the natural nucleotide is adenine, guanine, thymine, cytosine, or uracil.
31. The analog according to claim 1 , incorporated into an oligonucleotide.
32. A method of releasing a label from an analog according to claim 1 , comprising contacting the analog with a reducing agent, thereby producing a natural nucleotide, 9-deaza-G, 9-deaza-A, or ψ-uridine with no portion of the label or the linker remaining after cleavage.
33. A method of releasing a label from an analog according to claim 31 , comprising contacting the oligonucleotide with a reducing agent, thereby producing a natural nucleotide, 9-deaza-G, 9-deaza-A, or ψ-uridine from the analog with no portion of the label or the linker remaining after cleavage.
34. A method of sequencing a nucleic acid, comprising iteratively carrying out the steps of adding a nucleotide analog according to claim 1 in a template-dependent manner to a primer that is duplexed with the nucleic acid, detecting incorporation of the nucleotide analog into the duplex, and then contacting the duplex with a reducing agent, thereby producing a natural nucleotide, 9-deaza-G, 9-deaza-A, ψ-uridine from the added nucleotide analog with no portion of the label or the linker remaining after cleavage.
35. The method of claim 34 , wherein the nucleic acid primer duplex is attached to a surface.
36. The method of claim 34 , wherein no more than one nucleotide analog is added to the duplex in any single iteration.
37. The analog according to any one of claims 17 , 19 , 21 , 23 , 25 , and 27 , comprising an inhibitor that is configured so that the analog may be added to a nucleic acid primer by a polymerase, whereupon it inhibits the polymerase from adding a further nucleotide to the primer.
38. The analog according to claim 37 , configured so that the inhibitor is released along with the detectable label upon cleavage of the cleavable linker.