Disulfide-linked reversible terminators
The present disclosure provides methods of sequencing polynucleotides and compounds, compositions useful for sequencing of polynucleotides. The chemical compounds include nucleotides and their analogs which possess a sugar moiety comprising a cleavable chemical group capping the 3′-OH group and a base that is attached to a label through a cleavable linker comprising a disulfide bond. In addition, both the disulfide bond and the cleavable chemical group are cleavable by a chemical reagent. Furthermore, after the disulfide bond is cleaved by the chemical reagent, there is no free thiol group linked to the base of the nucleotides according to the fragmentation reaction shown below as an example. Example compounds according to the present disclosure are shown as Formula (I): wherein w is 1-5; X is O, S, or BH 3 ; B is a nucleotide base or an analog thereof, L 1-3 are linkers; and D 1 is a label.
1. A nucleoside 5′-triphosphate analog according to formula (I):
or a salt or protonated form thereof, wherein:
X is O, S, or BH 3 ;
w is 1, 2, 3, 4, or 5;
base B is selected from the group consisting of
and Y is CH or N;
L 1 is a first linker group and L 1 is
n is 0 or 1;
R 1 is
R 2 is
wherein p is 0-3, q is 0-12, r is 1-3; and
Z is O or NH;
L 2 is a second linker group and L 2 is
and m is 2 or 3;
L 3 is a third linker group and L 3 is
Q 1 and Q 2 are independently selected from the group consisting of none,
and
R 3 and R 4 are independently
wherein s is 0-3, t is 0-12, and u is 1-3; and
D 1 is a label, wherein the label is a fluorophore and wherein the label is different for each different base B in a composition.
2. The nucleoside 5′-triphosphate analog of claim 1 , wherein:
w is 1;
X is O;
L 1 is
L 2 is
3. The nucleoside 5′-triphosphate analog of claim 1 , wherein D 1 is a fluorophore.
4. A method of sequencing a polynucleotide, comprising:
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 one, two, three, or four nucleoside 5′-triphosphate analogs of claim 1 , wherein each of the one, two, three, of four nucleoside 5′-triphosphate analogs has a different Base selected from the group consisting of
and Y is CH or N, thereby generating one or more sequencing products complementary to the target polynucleotide.
5. The method of claim 4 , wherein the one, two, three, or four nucleoside 5′-triphosphate analogs of claim 1 at a concentration of no more than 100 μM.
6. The method of claim 4 , wherein the one, two, three, or four nucleoside 5′-triphosphate analogs of claim 1 at a concentration of no more than 50 μM.
7. The method of claim 4 , wherein the one, two, three, or four nucleoside 5′-triphosphate analogs of claim 1 at a concentration of no more than 10 μM.
8. The method of claim 4 , further comprising: treating the one or more sequencing products with trialkylphosphine.
9. The method of claim 8 , wherein the trialkylphosphine is tris(2-carboxyethyl)phosphine.