Nucleotide cleavable linkers and uses thereof
Disclosed herein, inter alia, are compounds, compositions, and methods of use thereof for sequencing a nucleic acid.
1. A compound having the formula:
wherein
B is a divalent nucleobase;
R 1 is a polyphosphate moiety, a monophosphate moiety, or a nucleic acid moiety;
R 2 is a hydrogen or —OH;
R 3 is an —O-polymerase-compatible cleavable moiety, —OH, —ONH 2 , —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, or —OCH 2 F;
L 101 , L 103 , L 104 , and L 105 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;
R 100 is —SR 102 or —CN;
R 102 is substituted or unsubstituted alkyl, hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R 102a is hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , 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; and
R 4 is a detectable moiety.
2. The compound of claim 1 , wherein R 3 is an —O-polymerase-compatible cleavable moiety.
3. The compound of claim 1 , wherein the polymerase-compatible cleavable moiety is -(substituted or unsubstituted alkylene)-SS-(unsubstituted alkyl).
4. The compound of claim 1 , wherein the polymerase-compatible cleavable moiety is -(halo-substituted or unsubstituted C 1 -C 3 alkylene)-SS-(unsubstituted C 1 -C 4 alkyl).
5. The compound of claim 1 , wherein the polymerase-compatible cleavable moiety is:
6. The compound of claim 1 , wherein the polymerase-compatible cleavable moiety is:
7. The compound of claim 1 , wherein R 1 is a monophosphate moiety or a polyphosphate moiety.
8. The compound of claim 1 , wherein
B is a divalent cytosine or a derivative thereof, divalent guanine or a derivative thereof, divalent adenine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, divalent hypoxanthine or a derivative thereof, divalent xanthine or a derivative thereof, divalent 7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine or a derivative thereof, or divalent 5-hydroxymethylcytosine or a derivative thereof.
9. The compound of claim 1 , wherein;
L 101 , L 103 , L 104 , and L 105 are independently a bond, 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; and
R 102 and R 102a are independently hydrogen or unsubstituted alkyl.
10. The compound of claim 1 , wherein
L 101 is a substituted or unsubstituted C 1 -C 4 alkylene or substituted or unsubstituted 8 to 20 membered heteroalkylene;
L 103 is a bond or substituted or unsubstituted 2 to 10 membered heteroalkylene;
L 104 is a bond, substituted or unsubstituted 4 to 18 membered heteroalkylene, or substituted or unsubstituted phenylene;
L 105 is a bond or substituted or unsubstituted 4 to 18 membered heteroalkylene;
R 102 is unsubstituted C 1 -C 4 alkyl; and
R 102a is hydrogen or unsubstituted methyl.
11. The compound of claim 1 , wherein
L 101 , L 103 , and L 105 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 104 is unsubstituted phenylene; and
R 102 and R 102a are independently hydrogen or unsubstituted alkyl.
12. The compound of claim 1 , wherein
L 101 is a substituted or unsubstituted C 1 -C 4 alkylene or substituted or unsubstituted 8 to 20 membered heteroalkylene;
L 103 is a bond or substituted or unsubstituted 2 to 10 membered heteroalkylene;
L 104 is an unsubstituted phenylene;
L 105 is a bond or substituted or unsubstituted 4 to 18 membered heteroalkylene;
R 102 is unsubstituted C 1 -C 4 alkyl; and
R 102a is hydrogen or unsubstituted methyl.
13. The compound of claim 1 , wherein -(L 101 )-OC(SSR 102 )(R 102a )-(L 103 )-(L 104 )-(L 105 )- is
14. The compound of claim 1 , wherein -(L 101 )-OC(SSR 102 )(R 102a )-(L 103 )-(L 104 )-(L 105 )- is
15. The compound of claim 1 , wherein R 4 is a fluorescent dye moiety.
16. A method for sequencing a nucleic acid, comprising:
(i) incorporating in series with a nucleic acid polymerase, within a reaction vessel, one of four different compounds into a primer to create an extension strand, wherein said primer is hybridized to said nucleic acid and wherein each of the four different compounds comprises a unique detectable label;
(ii) detecting said unique detectable label of each incorporated compound, so as to thereby identify each incorporated compound in said extension strand, thereby sequencing the nucleic acid;
wherein each of said four different compounds is independently a compound of claim 1 .
17. A method of incorporating a compound into a primer, the method comprising combining a polymerase, a primer hybridized to nucleic acid template and the compound within a reaction vessel and allowing said polymerase to incorporate said compound into said primer thereby forming an extended primer, wherein said compound is a compound of claim 1 .
18. A nucleic acid polymerase complex comprising a nucleic acid polymerase, wherein said nucleic acid polymerase is bound to a compound of claim 1 .
19. The compound of claim 1 , having the formula
wherein R 3 is an —O-polymerase compatible cleavable moiety selected from the group consisting of
20. The compound of claim 19 , wherein
L 101 is —CCCH 2 —NHC(O)—(CH 2 ) c (OCH 2 CH 2 ) b — wherein c is 1 and b is 1;
L 103 is —(CH 2 CH 2 O) d — wherein d is 1, 2 or 3;
L 104 is an unsubstituted phenylene;
L 105 is C(O)NH—(CH 2 ) g —NH wherein g is 0, 1, 2 or 3; and
R 102 is hydrogen or unsubstituted C 1 -C 4 alkyl.