Cleavable disulfide linkers and uses thereof
Disclosed herein, inter alia, are methods and cleavable compounds that minimize byproduct formation following cleavage.
1. A method of incorporating a compound into a primer, the method comprising:
(i) contacting a primer with a compound, wherein said primer is hybridized to a nucleic acid molecule in a cell or tissue, wherein the compound comprises a detectable label attached thereto via a divalent linker, L 100 , wherein L 100 comprises the formula:
wherein R 5 and R 6 are independently unsubstituted alkyl, —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 , —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , substituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and W 1 and W 2 are —O—; and
(ii) incorporating with a polymerase the compound into the primer.
2. The method of claim 1 , wherein the cell or tissue is within a flow cell.
3. The method of claim 1 , wherein the cell or tissue is immobilized to a well of a microplate.
4. The method of claim 1 , prior to step (i), hybridizing a polynucleotide probe to an RNA molecule and amplifying the polynucleotide probe to form the nucleic acid molecule.
5. The method of claim 4 , further comprising hybridizing the primer to the nucleic acid molecule.
6. The method of claim 1 , further comprising cleaving the divalent linker with a reducing agent.
7. The method of claim 1 , further comprising detecting the detectable label of the incorporated compound following step (ii).
8. The method of claim 7 , further comprising repeating steps (i) and (ii) to sequence the nucleic acid molecule.
9. The method of claim 7 , wherein detecting comprises detecting an emission wavelength from the detectable label.
10. The method of claim 1 , wherein the compound has the formula:
wherein R 3 is a reversible terminator moiety, hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R 4 is a detectable moiety;
L 100 has the formula:
wherein
L 1 has the formula -L 101 -L 102 -L 103 -;
L 101 , L 102 , and L 103 are independently a bond, —NH—, —O—, —C(O)—, —C(O) NH—,
—NHC(O)—, —NHC(O) NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;
L 2 has the formula -L 201 -L 202 -L 203 -; and
L 201 , L 202 , and L 203 are independently a bond, —NH—, —O—, —C(O)—, —C(O) NH—,
—NHC(O)—, —NHC(O) NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.
11. The method of claim 1 , wherein R 5 and R 6 are independently substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
12. The method of claim 1 , wherein R 5 and R 6 are unsubstituted alkyl.
13. The method of claim 1 , wherein R 5 and R 6 are unsubstituted methyl, unsubstituted ethyl, unsubstituted isopropyl, or unsubstituted n-propyl.
14. The method of claim 1 , wherein L 100 has the formula:
15. The method of claim 1 , wherein L 100 has the formula:
16. The method of claim 1 , wherein L 100 has the formula:
17. The method of claim 1 , wherein the compound has the formula:
18. The method of claim 1 , wherein the compound has the formula:
19. The method of claim 10 , wherein R 3 is a reversible terminator moiety comprising:
20. The method of claim 10 , wherein R 3 is
wherein R 7 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; and
R 8 is substituted or unsubstituted alkyl.