NUCLEOSIDES AND NUCLEOTIDES WITH 3' BLOCKING GROUPS AND CLEAVABLE LINKERS
Embodiments of the present disclosure relate to nucleotide and nucleoside molecules with 3′ vinyl or isonitrile containing blocking groups and/or tetrazine or strained unsaturated ring containing cleavable linkers. Additionally, the present disclosure provides methods of using the nucleoside/nucleotide in oligonucleotide synthesis, and methods of sequencing using the nucleotide described herein.
1 . A nucleoside or nucleotide having the structure of Formula (I):
or a salt thereof, wherein:
B comprises a nucleobase;
R 1 is H, hydroxy, halo, unsubstituted or substituted C 1 -C 6 alkoxy, or a hydroxy protecting group;
R 2 is H, a hydroxy protecting group, or —OR 2 is a monophosphate, diphosphate, triphosphate or phosphorothioate;
X is an unsubstituted or substituted C 6 -C 10 arylene, or an unsubstituted or substituted 5 to 10 membered heteroarylene;
each of R 3a and R 3b is independently H, azido, halo, nitro, cyano, unsubstituted or substituted C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, or C 1 -C 6 haloalkoxy;
each of R 4 , R 4b and R 4c is independently H, halo, C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, C 1 -C 3 hydroxyalkyl, azido, cyano, unsubstituted or substituted phenyl, unsubstituted or substituted 5 to 6 membered heteroaryl, unsubstituted or substituted C 3 -C 7 cycloalkyl, or unsubstituted or substituted 3 to 7 membered heterocyclyl.
2 . The nucleoside or nucleotide of claim 1 , wherein the nucleoside or nucleotide has the structure of Formula (Ia):
or a salt thereof.
3 . The nucleoside or nucleotide of claim 1 , wherein at least one of R 4a , R 4b and R 4c is methyl, ethyl, n-propyl, isopropyl, fluoro, chloro, —CHF 2 , —CH 2 F, —CH 2 Cl, —CHCl 2 , or —CF 3 .
4 . The nucleoside or nucleotide of claim 1 , wherein each of R 4a , R 4b and R 4c is H.
5 . The nucleoside or nucleotide of claim 1 , wherein R 1 is H, hydroxy, or a hydroxy protecting group selected from the group consisting of
that covalently attached to the 2′ oxygen atom.
6 . The nucleoside or nucleotide of claim 1 , wherein B comprises a nucleobase covalently bounded to a detectable label, optionally through a linker.
7 . The nucleoside or nucleotide of claim 6 , B is
8 . The nucleoside or nucleotide of claim 1 , wherein the nucleoside or nucleotide is nucleotide having 2′ deoxyribose, and —OR 2 is triphosphate.
9 . An oligonucleotide or polynucleotide comprising a nucleotide of claim 8 incorporated thereof.
10 . A method of determining the sequence of a target single-stranded polynucleotide, comprising:
(a) incorporating a nucleotide claim 8 into a copy polynucleotide strand complementary to at least a portion of the target polynucleotide strand;
(b) performing one or more fluorescent measurements for determining the identity of the nucleotide incorporated into the copy polynucleotide strand; and
(c) removing the 3′ blocking group from the nucleotide incorporated into the copy polynucleotide strand.
11 . The method of claim 10 , wherein the removing of the 3′ blocking group of the incorporated nucleotide is achieved by a tetrazine reagent.
12 . A method of controlled synthesis of an oligonucleotide or polynucleotide, comprising:
contacting a nucleotide of claim 8 with the oligonucleotide or polynucleotide in the presence of a polymerase; and
incorporating the nucleotide to the 3′ end of the oligonucleotide or polynucleotide.
13 . The method of claim 12 , further comprising:
removing the 3′ blocking group of the incorporated nucleotide with a tetrazine reagent to generate a 3′ hydroxy group on the incorporated nucleotide, and
incorporating a second nucleotide.
14 . The method of claim 11 , wherein the tetrazine reagent is selected from the group consisting of
and unsubstituted or substituted variants thereof.
15 . A nucleoside or nucleotide comprising a nucleobase attached to a detectable label via a cleavable linker, wherein the nucleoside or nucleotide comprises a ribose or 2′ deoxyribose moiety and a 3′ blocking group, and wherein the cleavable linker comprises an unsubstituted or substituted C 7 -C 14 unsaturated carbocyclic ring or ring system or unsubstituted or substituted 7 to 14 membered unsaturated heterocyclic ring or ring system, each comprising at least one double bond or one triple bond in the ring or ring system.
16 .- 24 . (canceled)
25 . A method of determining the sequence of a target single-stranded polynucleotide, comprising:
(a) incorporating a nucleotide of claim 15 into a copy polynucleotide strand complementary to at least a portion of the target polynucleotide strand;
(b) performing one or more fluorescent measurements for determining the identity of the nucleotide incorporated into the copy polynucleotide strand; and
(c) removing the detectable label from the nucleotide incorporated into the copy polynucleotide strand.
26 . (canceled)
27 . (canceled)
28 . A method of controlled synthesis of an oligonucleotide or polynucleotide, comprising:
contacting a nucleotide of claim 15 with the oligonucleotide or polynucleotide in the presence of a polymerase; and
incorporating the nucleotide to the 3′ end of the oligonucleotide or polynucleotide.
29 . (canceled)
30 . A nucleoside or nucleotide comprising a nucleobase attached to a detectable label via a cleavable linker, wherein the nucleoside or nucleotide comprises a ribose or 2′ deoxyribose moiety and a 3′ blocking group, and wherein the cleavable linker comprises an unsubstituted or substituted tetrazine moiety.
31 .- 37 . (canceled)
38 . A method of determining the sequence of a target single-stranded polynucleotide, comprising:
(a) incorporating a nucleotide of claim 30 into a copy polynucleotide strand complementary to at least a portion of the target polynucleotide strand;
(b) performing one or more fluorescent measurements for determining the identity of the nucleotide incorporated into the copy polynucleotide strand; and
(c) removing the detectable label of the nucleotide incorporated into the copy polynucleotide strand.
39 . (canceled)
40 . (canceled)
41 . (canceled)
42 . A method of controlled synthesis of an oligonucleotide or polynucleotide, comprising:
contacting a nucleotide of claim 30 with the oligonucleotide or polynucleotide in the presence of a polymerase; and
incorporating the nucleotide to the 3′ end of the oligonucleotide or polynucleotide.
43 . (canceled)
44 . A nucleoside or nucleotide comprising a ribose or 2′ deoxyribose moiety and a 3′ blocking group, and a detectable label attached to the 3′ blocking group optionally via a linker, wherein the 3′ blocking group comprises an isonitrile moiety.
45 .- 50 . (canceled)
51 . A method of determining the sequence of a target single-stranded polynucleotide, comprising:
(a) incorporating a nucleotide of claim 44 into a copy polynucleotide strand complementary to at least a portion of the target polynucleotide strand;
(b) performing one or more fluorescent measurements for determining the identity of the nucleotide incorporated into the copy polynucleotide strand; and
(c) removing the 3′ blocking group from the nucleotide incorporated into the copy polynucleotide strand.
52 . (canceled)
53 . A method of controlled synthesis of an oligonucleotide or polynucleotide, comprising:
contacting a nucleotide of claim 44 with the oligonucleotide or polynucleotide in the presence of a polymerase; and
incorporating the nucleotide to the 3′ end of the oligonucleotide or polynucleotide.
54 . (canceled)
55 . (canceled)