MCCAULEY PATRICK (GB)
DHARMARWARDANA MADUSHANI (GB)
NIRANTAR SAURABH (SG)
GOLYNSKIY MISHA (US)
YANG XIANGYUAN (SG)
NEELAKANDAN RAMESH (SG)
MACKWORTH BENEDICT (GB)
ANASTASI CAROLE (GB)
KARUNAKARAN AATHAVAN (US)
VESSERE GERY M (US)
BRACHER DAVID (US)
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WHAT IS CLAIMED IS: 1. A kit for sequencing application, comprising: a first type of unlabeled nucleotide comprising a first hapten; a second type of unlabeled nucleotide comprising a second hapten; a third type of unlabeled nucleotide; and a set of affinity reagents comprising: a first affinity reagent comprising a first hapten-binding partner that is capable of specific binding to the first type of unlabeled nucleotide; and a second affinity reagent comprising a second hapten-binding partner that is capable of specific binding to the second type of unlabeled nucleotide; wherein the first affinity reagent comprises one or more first detectable labels that are excitable by a first excitation light source, the second affinity reagent comprises one or more second detectable labels that are excitable by a second excitation light source, and wherein the first detectable label is spectrally distinguishable from the second detectable label; and wherein one of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm, and the other one of the first excitation light source and the second excitation light source has a wavelength of about 520 nm to about 540 nm. 2. The kit of claim 1, wherein the first hapten is covalently attached to the nucleobase of the first type of unlabeled nucleotide via a cleavable linker. 3. The kit of claim 1 or 2, wherein the second hapten is covalently attached to the nucleobase of the second type of unlabeled nucleotide via a cleavable linker. 4. The kit of any one of claims 1 to 3, wherein the third type of unlabeled nucleotide comprises a mixture of the third type of unlabeled nucleotide comprising the first hapten and the third type of unlabeled nucleotide comprising the second hapten, and wherein both the first affinity reagent and the second affinity reagent are capable of specific binding to the third type of unlabeled nucleotide. 5. The kit of claim 4, wherein each of the first hapten and the second hapten is covalently attached to the nucleobase of the third type of nucleotide via a cleavable linker. 6. The kit of claim 4 or 5, wherein the first hapten comprises a biotin moiety and the first hapten-binding partner comprises an avidin. 7. The kit of claim 6, wherein the avidin is streptavidin or neutravidin. 8. The kit of claim 6 or 7, wherein one or more biotin binding sites of the avidin are blocked by a biotin moiety-containing molecule or an analog thereof. 9. The kit of any one of claims 6 to 8, wherein the second hapten comprises a dinitrophenyl (DNP) moiety and the second hapten-binding partner comprises an anti-DNP antibody. 10. The kit of claim 9, wherein the third type of unlabeled nucleotide comprises a mixture of a third type of unlabeled nucleotide comprising a biotin moiety, and a third type of unlabeled nucleotide comprising a DNP moiety. 11. The kit of any one of claims 6 to 8, wherein the second hapten comprises a digoxigenin (DIG) moiety and the second hapten-binding partner comprises an anti-DIG antibody. 12. The kit of claim 11, wherein the third type of unlabeled nucleotide comprises a mixture of a third type of unlabeled nucleotide comprising a biotin moiety, and a third type of unlabeled nucleotide comprising a DIG moiety. 13. The kit of any one of claims 1 to 3, wherein the third type of unlabeled nucleotide comprises a third hapten, and the set of affinity reagents further comprises a third affinity reagent comprising a third hapten-binding partner that is capable of specific binding to the third hapten. 14. The kit of claim 13, wherein the third hapten is covalently attached to the nucleobase of the third type of unlabeled nucleotide via a cleavable linker. 15. The kit of claim 13 or 14, wherein the first hapten comprises a biotin moiety and the first hapten-binding partner comprises an avidin. 16. The kit of claim 15, wherein the avidin is streptavidin or neutravidin. 17. The kit of claim 15 or 16, wherein one or more biotin binding sites of the avidin are blocked by a biotin moiety-containing molecule or an analog thereof. 18. The kit of any one of claims 15 to 17, wherein one of the second hapten and the third hapten comprises a DNP moiety, and the other one of the second hapten and the third hapten comprises a DIG moiety. 19. The kit of claim 13 or 14, wherein one of the first hapten and the second hapten comprises a DNP moiety, and the other one of the first hapten and the second hapten comprises a DIG moiety. 20. The kit of claim 19, wherein the third hapten comprises a biotin moiety and the third hapten-binding partner comprises an avidin. 21. The kit of claim 20, wherein the avidin is streptavidin or neutravidin. 22. The kit of claim 20 or 21, wherein one or more biotin binding sites of the avidin are blocked by a biotin moiety-containing molecule or an analog thereof. 23. The kit of any one of claims 13 to 22, wherein the third affinity reagent comprises one or more first detectable labels and one or more second detectable labels. 24. The kit of claim 23, wherein the third affinity reagent comprises a multi-dye labeled protein assembly system according to any one of claims 29 to 42. 25. The kit of any one of claims 1 to 24, further comprises a fourth type of unlabeled nucleotide, wherein the fourth type of unlabeled nucleotide is not capable of specific binding to any of the affinity reagents. 26. The kit of claim 25, wherein each of the first type, second type, third type and fourth type of unlabeled nucleotide comprises a 3´ blocking group. 27. The kit of any one of claims 1 to 26, wherein the first detectable label is a dye having the structure of Formula (I): , wherein R1 is -C(O)OC1-C6 alkyl, -SO3H, -OH, optionally substituted 5 to 10 membered heteroaryl, optionally substituted 3 to 10 membered heterocyclyl, optionally substituted phenyl, or -ORx; Rx is optionally substituted 5 to 10 membered heteroaryl, optionally substituted 3 to 10 membered heterocyclyl, or optionally substituted phenyl; m is an integer of 1, 2 or 3; and n is an integer of 1, 2, 3, 4 or 5. 28. The kit of any one of claims 1 to 27, further comprising a DNA polymerase and one or more buffer compositions. 29. A protein assembly system, comprising: a first protein labeled with one or more first detectable labels; one or more hapten-containing linkers covalently attached to the first protein; and one or more hapten-binding second proteins bound to one or more hapten- containing linkers; wherein the one or more hapten-binding second proteins are labeled with one or more second detectable labels, and wherein the one or more first detectable labels are spectrally distinguishable from the one or more second detectable labels. 30. The protein assembly system of claim 29, wherein the one or more hapten-containing linkers are biotin moiety-containing linkers, and the one or more hapten-binding second proteins are biotin-binding proteins. 31. The protein assembly system of claim 29 or 30, wherein the one or more hapten- binding second proteins comprise an avidin. 32. The protein assembly system of claim 31, wherein the avidin is streptavidin or neutravidin. 33. The protein assembly system of claim 31 or 32, wherein one or more biotin binding sites of the avidin are blocked by a biotin moiety-containing molecule or an analog thereof. 34. The protein assembly system of any one of claims 29 to 33, wherein the first protein comprises an antibody. 35. The protein assembly system of claim 34, wherein the antibody is an anti-DNP antibody or an anti-DIG antibody. 36. The protein assembly system of any one of claims 29 to 35, wherein the one or more first detectable labels are excitable by a blue light, and the one or more second detectable labels are excitable by a green light. 37. The protein assembly system of any one of claims 29 to 35, wherein the one or more first detectable labels are excitable by a green light, and the one or more second detectable labels are excitable by a blue light. 38. The protein assembly system of claim 36 or 37, wherein the blue light has a wavelength from about 450 nm to about 460 nm. 39. The protein assembly system of claim 36 or 37, wherein the green light has a wavelength from about 520 nm to about 540 nm. 40. The protein assembly system of any one of claims 29 to 39, wherein the first protein is labeled with a plurality of first detectable labels. 41. The protein assembly system of any one of claims 30 to 40, wherein the protein assembly system comprises at least two biotin-binding proteins, each biotin-binding protein is labeled with a plurality of second detectable labels. 42. The protein assembly system of any one of claims 30 to 41, wherein the biotin moiety- containing linker comprises PEG repeating units (—OCH2CH2—)n, and wherein n is an integer from 2 to 30. 43. The protein assembly system of any one of claims 29 to 42, wherein the one or more first detectable labels comprise a coumarin dye having the structure of Formula (I): , wherein R1 is -C(O)OC1-C6 alkyl, -SO3H, -OH, optionally substituted 5 to 10 membered heteroaryl, optionally substituted 3 to 10 membered heterocyclyl, optionally substituted phenyl, or -ORx; Rx is optionally substituted 5 to 10 membered heteroaryl, optionally substituted 3 to 10 membered heterocyclyl, or optionally substituted phenyl; m is an integer of 1, 2 or 3; and n is an integer of 1, 2, 3, 4 or 5. 44. A nucleotide conjugate comprising the protein assembly system of any one of claims 29 to 43. 45. The nucleotide conjugate of claim 44, wherein the protein assembly system is bound to a hapten moiety of the nucleotide, and wherein the hapten moiety of the nucleotide is capable of specific binding to at least one of the proteins of the protein assembly system. 46. The nucleotide conjugate of claim 45, wherein the hapten moiety of the nucleotide comprises a DIG or a DNP moiety. 47. The nucleotide conjugate of any one of claims 44 to 46, wherein the hapten moiety of the nucleotide is covalently attached to the nucleobase via a cleavable linker. 48. An oligonucleotide or polynucleotide comprising the nucleotide conjugate of any one of claims 44 to 47 incorporated therein. 49. The oligonucleotide or polynucleotide of claim 48, wherein the oligonucleotide or polynucleotide is at least partially complementary and hybridized to a target polynucleotide immobilized on a surface of a solid support. 50. The oligonucleotide or polynucleotide of claim 48 or 49, wherein the solid support comprising a plurality of immobilized target polynucleotides, wherein the density of the immobilized target polynucleotides on the solid support is at least about 50,000/mm2. 51. A method of determining the sequences of a plurality of different target polynucleotides, comprising: (a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b) contacting the solid support with an aqueous solution comprising DNA polymerase and one or more of four different types of unlabeled nucleotides under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides; wherein each of the four types of nucleotides comprises a 3′ blocking group; the first type of unlabeled nucleotide comprises a first hapten; the second type of unlabeled nucleotide comprises a second hapten; (c) contacting the extended copy polynucleotides with a set of affinity reagents under conditions wherein an affinity reagent binds specifically to the incorporated unlabeled nucleotides to provide labeled extended copy polynucleotides; (d) imaging the solid support and performing one or more fluorescent measurements of the extended copy polynucleotides; and (e) removing the 3′ blocking group of the incorporated nucleotides; wherein the set of affinity reagents comprises: a first affinity reagent comprising a first hapten-binding partner that is capable of specific binding to the first type of unlabeled nucleotides; and a second affinity reagent comprising a second hapten-binding partner that is capable of specific binding to the second type of unlabeled nucleotides; wherein the first affinity reagent comprises one or more first detectable labels that are excitable by a first excitation light source, the second affinity reagent comprises one or more second detectable labels that are excitable by a second excitation light source, and wherein the one or more first detectable labels are spectrally distinguishable from the one or more second detectable labels; and wherein one of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm, and the other one of the first excitation light source and the second excitation light source has a wavelength of about 520 nm to about 540 nm. 52. The method of claim 51, wherein the first hapten is covalently attached to the nucleobase of the first type of unlabeled nucleotide via a cleavable linker. 53. The method of claim 51 or 52, wherein the second hapten is covalently attached to the nucleobase of the second type of unlabeled nucleotide via a cleavable linker. 54. The method of any one of claims 51 to 53, wherein the third type of unlabeled nucleotide comprises a mixture of the third type of unlabeled nucleotide comprising the first hapten and the third type of unlabeled nucleotide comprising the second hapten, and wherein both the first affinity reagent and the second affinity reagent are capable of specific binding to the third type of unlabeled nucleotide. 55. The method of claim 54, wherein each of the first hapten and the second hapten is covalently attached to the nucleobase of the third type of nucleotide via a cleavable linker. 56. The method of claim 54 or 55, wherein the first hapten comprises a biotin moiety and the first hapten-binding partner comprises avidin. 57. The method of claim 56, wherein the avidin is streptavidin or neutravidin. 58. The method of claim 56 or 57, wherein one or more biotin binding sites of the avidin are blocked by a biotin moiety-containing molecule or an analog thereof. 59. The method of any one of claims 56 to 58, wherein the second hapten comprises a DNP moiety and the second hapten-binding partner comprises an anti-DNP antibody. 60. The method of claim 59, wherein the third type of unlabeled nucleotide comprises a mixture of a third type of unlabeled nucleotide comprising a biotin moiety, and a third type of unlabeled nucleotide comprising a DNP moiety. 61. The method of any one of claims 56 to 58, wherein the second hapten comprises a DIG moiety and the second hapten-binding partner comprises an anti-DIG antibody. 62. The method of claim 61, wherein the third type of unlabeled nucleotide comprises a mixture of a third type of unlabeled nucleotide comprising a biotin moiety, and a third type of unlabeled nucleotide comprising a DIG moiety. 63. The method of any one of claims 51 to 53, wherein the third type of unlabeled nucleotide comprises a third hapten, and the set of affinity reagents further comprises a third affinity reagent comprising a third hapten-binding partner that is capable of specific binding to the third hapten. 64. The method of claim 63, wherein the third hapten is covalently attached to the nucleobase of the third type of unlabeled nucleotide via a cleavable linker. 65. The method of claim 63 or 64, wherein the first hapten comprises a biotin moiety and the first hapten-binding partner comprises an avidin. 66. The method of claim 65, wherein one of the second hapten and the third hapten comprises a DNP moiety, and the other one of the second hapten and the third hapten comprises a DIG moiety. 67. The method of claim 63 or 64, wherein one of the first hapten and the second hapten comprises a DNP moiety, and the other one of the first hapten and the second hapten comprises a DIG moiety. 68. The method of claim 67, wherein the third hapten comprises a biotin moiety and the third hapten-binding partner comprises an avidin. 69. The method of claim 65 or 68, wherein the avidin is streptavidin or neutravidin. 70. The method of claim 68 or 69, wherein one or more biotin binding sites of the avidin are blocked by a biotin moiety-containing molecule or an analog thereof. 71. The method of any one of claims 63 to 70, wherein the third affinity reagent comprises one or more first detectable labels and one or more second detectable labels. 72. The method of claim 71, wherein the third affinity reagent comprises a multi-dye labeled protein assembly system according to any one of claims 29 to 42. 73. The method of any one of claims 51 to 72, further comprises a fourth type of unlabeled nucleotide, wherein the fourth type of unlabeled nucleotide is not capable of specific binding to any of the affinity reagents. 74. The method of any one of claims 51 to 73, wherein step (e) also removes the detectable labels of the incorporated nucleotides. 75. The method of claim 74, wherein the detectable labels and the 3ʹ blocking groups of the incorporated nucleotides are removed in a single chemical reaction. 76. The method of any one of claims 51 to 75, further comprising (f) washing the solid support with an aqueous wash solution. 77. The method of claim 76, wherein steps (b) to (f) are repeated at least 50, 100, 150, 200, 250, 300, 400 or 500 cycles to determine the target polynucleotide sequences. 78. The method of any one of claims 51 to 77, wherein the four types of nucleotides comprise dATP, dCTP, dGTP and dTTP or dUTP, or non-natural nucleotide analogs thereof. 79. The method of any one of claims 51 to 78, wherein the density of the immobilized target polynucleotides on the solid support is at least about 50,000/mm2. 80. The method of any one of claims 51 to 79, wherein the method reduces or eliminates sequence context effect. 81. A labeled nanoparticle, comprising: a polymer matrix comprising a plurality of detectable labels, wherein the backbone of the polymer comprises one or more cleavable moieties, and wherein the labeled nanoparticle is degradable into smaller polymeric chains upon cleavage of the cleavable moieties. 82. The labeled nanoparticle of claim 81, wherein the plurality of detectable labels comprise fluorophores excitable by a light source having a wavelength between a wavelength of about 450 nm to about 460 nm. 83. The labeled nanoparticle of claim 81 or 82, wherein the smaller polymeric chains of the degraded nanoparticle are water soluble. 84. The labeled nanoparticle of any one of claims 81 to 83, wherein the cleavable moieties are chemically degradable, enzymatically degradable, thermally degradable, or photo degradable. 85. The labeled nanoparticle of any one of claims 81 to 84, wherein the cleavable moieties comprise a disulfide, an azido, or an allyl moiety, or combinations thereof. 86. The labeled nanoparticle of any one of claims 81 to 85, wherein the polymer comprises a cross-linked or a dendritic polymer, or a liposome. 87. The labeled nanoparticle of any one of claims 81 to 86, wherein the polymer comprises polyacrylamide, polyglycolic acid (PGA), polyvinyl alcohol (PVA). 88. The labeled nanoparticle of any one of claims 81 to 88, wherein the plurality of detectable labels are covalently attached to the polymer matrix. 89. The labeled nanoparticle of any one of claims 81 to 88, wherein the plurality of detectable labels are encapsulated or physically confined within the polymer matrix. 90. The labeled nanoparticle of any one of claims 81 to 89, further comprising one or more proteins attached thereto. |
, ed analogs thereof. [0139] In some embodiments, the fluorescent dyes described herein may be further modified to introduce one or more substituents (such as -SO 3 H, -OH, -C(O)OH, -C(O)OR, where R is unsubstituted or substituted C1-C6 alkyl) to improve the hydrophilicity of the dyes when the dyes are conjugated with the antibody/protein (i.e., hapten-binding partner) described herein, while maintaining the signal intensity of the dye. In some such embodiments, the first detectable label described in the kits and protein assembly system of the present disclosure may be a coumarin dye having the structure of Formula (I): wherein m is an integer of 1, 2 or 3; n is an integer of 1, 2, 3, 4 or 5; R1 is -C(O)OC 1 -C 6 alkyl, -SO 3 H, -OH, optionally substituted 5 to 10 membered heteroaryl, optionally substituted 3 to 10 membered heterocyclyl, optionally substituted phenyl, or -ORx, wherein Rx is optionally substituted 5 to 10 membered heteroaryl, optionally substituted 3 to 10 membered heterocyclyl, or optionally substituted phenyl. In some embodiments, m is 1. In some embodiments, n is 1. In some other embodiments, n is 3. In further embodiments, m is 1 and n is 3. In one embodiment, R1 is -C(O)OtBu. In another embodiment, R1 is -SO3H. In another embodiment, R1 is furyl (e.g., 2-furyl). In another embodiment, R1 . Further non-limiting embodiments of the coumarin dyes include: O H . [0140] When the dye forms a covalent bond with the affinity reagent (e.g., avidin, protein or antibody), the carboxy group of the dye may react with an amino functional group of the affinity reagent to form an amide bond. For example, the dye of Formula (I) comprises the moiety after being covalently bound to the affinity reagent. 3ʹ Blocking Groups [0141] The nucleotide described herein may also have a 3´ blocking group covalently attached to the deoxyribose sugar of the nucleotide. Various 3´ blocking group are disclosed in WO2002/029003, WO2004/018497 and WO2014/139596. For example, the blocking group may be azidomethyl (-CH 2 N 3 ) or substituted azidomethyl (e.g., -CH(CHF 2 )N 3 or CH(CH 2 F)N 3 ), or allyl, each connecting to the 3´-oxygen atom of the deoxyribose moiety. In some embodiments, the 3´ blocking group is azidomethyl, forming 3ʹ-OCH 2 N 3 with the 3ʹ carbon of the ribose or deoxyribose. [0142] Additional 3ʹ blocking groups are disclosed in U.S. Publication No. 2020/0216891 A1, which is incorporated by reference in its entirety. Non-limiting examples of the acetal blocking group , , each covalently attached to the 3ʹ carbon of the deoxyribose. Deprotection of the 3ʹ Blocking Groups [0143] In some embodiments, the 3´ hydroxy protecting group such as azidomethyl may be removed or deprotected by using a water-soluble phosphine reagent to generate a free 3´- OH. Non-limiting examples include tris(hydroxymethyl)phosphine (THMP), tris(hydroxyethyl)phosphine (THEP) or tris(hydroxypropyl)phosphine (THP or THPP).3ʹ-acetal blocking groups described herein may be removed or cleaved under various chemical conditions. For 3´ blocking groups that contain an allyl moiety, non-limiting cleaving condition includes a Pd(II) complex, such as Pd(OAc)2 or allylPd(II) chloride dimer, in the presence of a phosphine ligand, for example tris(hydroxymethyl)phosphine (THMP), or tris(hydroxypropyl)phosphine (THP or THPP). For those blocking groups containing an alkynyl group (e.g., an ethynyl), they may also be removed by a Pd(II) complex (e.g., Pd(OAc)2 or allyl Pd(II) chloride dimer) in the presence of a phosphine ligand (e.g., THP or THMP). Palladium Cleavage Reagents [0144] In some other embodiments, the 3´ blocking group such as allyl or AOM as described herein may be cleaved by a palladium catalyst. In some such embodiments, the Pd catalyst is water soluble. In some such embodiments, is a Pd(0) complex (e.g., Tris(3,3′,3″- phosphinidynetris(benzenesulfonato)palladium(0) nonasodium salt nonahydrate). In some instances, the Pd(0) complex may be generated in situ from reduction of a Pd(II) complex by reagents such as alkenes, alcohols, amines, phosphines, or metal hydrides. Suitable palladium sources include Na 2 PdCl 4 , Li 2 PdCl 4 , Pd(CH 3 CN) 2 Cl 2, (PdCl(C 3 H 5 )) 2 , [Pd(C 3 H 5 )(THP)]Cl, [Pd(C3H5)(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd(Acac)2, PdCl2(COD), Pd(TFA)2, Na 2 PdBr 4 , K 2 PdBr 4 , PdCl 2 , PdBr 2 , and Pd(NO 3 ) 2 . In one such embodiment, the Pd(0) complex is generated in situ from Na 2 PdCl 4 or K 2 PdCl 4 . In another embodiment, the palladium source is allyl palladium(II) chloride dimer [(PdCl(C3H5))2]. In some embodiments, the Pd(0) complex is generated in an aqueous solution by mixing a Pd(II) complex with a phosphine. Suitable phosphines include water soluble phosphines, such as tris(hydroxypropyl)phosphine (THP), tris(hydroxymethyl)phosphine (THMP), 1,3,5-triaza-7-phosphaadamantane (PTA), bis(p- sulfonatophenyl)phenylphosphine dihydrate potassium salt, tris(carboxyethyl)phosphine (TCEP), and triphenylphosphine-3,3´,3´´-trisulfonic acid trisodium salt. [0145] In some embodiments, the palladium catalyst is prepared by mixing [(Allyl)PdCl]2 with THP in situ. The molar ratio of [(Allyl)PdCl]2 and the THP may be about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10. In one embodiment, the molar ratio of [(Allyl)PdCl] 2 to THP is 1:10. In some other embodiment, the palladium catalyst is prepared by mixing a water soluble Pd reagent such as Na2PdCl4 or K2PdCl4 with THP in situ. The molar ratio of Na2PdCl4 or K2PdCl4 and THP may be about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10. In one embodiment, the molar ratio of Na2PdCl4 or K2PdCl4 to THP is about 1:3. In another embodiment, the molar ratio of Na2PdCl4 or K2PdCl4 to THP is about 1:3.5. In yet another embodiment, the molar ratio of Na2PdCl4 or K2PdCl4 to THP is about 1:2.5. In some further embodiments, one or more reducing agents may be added, such as ascorbic acid or a salt thereof (e.g., sodium ascorbate). In some embodiments, the cleavage mixture may contain additional buffer reagents, such as a primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In some further embodiments, the buffer reagent comprises ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylethanolamine (DMEA), 2- diethylethanolamine (DEEA), N,N,N′,N′-tetramethylethylenediamine(TEMED), or N,N,N′,N′- tetraethylethylenediamine (TEEDA), or 2-piperidine ethanol (also known as (2- hydroxyethyl)piperidine, having the structure , or combinations thereof. In one embodiment, the buffer reagent comprises or embodiment, the buffer reagent comprises or is (2-hydroxyethyl)piperidine. In another embodiment, the buffer reagent contains one or more inorganic salts such as a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In one embodiment, the inorganic salt is a sodium salt. Cleavable Linkers [0146] In some embodiments, the hapten moiety of nucleotide described herein is covalently attached to the nucleobase of the nucleotide via a cleavable linker. Use of the term “cleavable linker” is not meant to imply that the whole linker is required to be removed. The cleavage site can be located at a position on the linker that ensures that part of the linker remains attached to the dye and/or substrate moiety after cleavage. Cleavable linkers may be, by way of non-limiting example, electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavable under reductive conditions (for example disulfide or azide containing linkers), oxidative conditions, cleavable via use of safety-catch linkers and cleavable by elimination mechanisms. The use of a cleavable linker to attach the dye compound to a substrate moiety ensures that the label can, if required, be removed after detection, avoiding any interfering signal in downstream steps. [0147] Useful linker groups may be found in PCT Publication No. WO2004/018493 (herein incorporated by reference), examples of which include linkers that may be cleaved using water-soluble phosphines or water-soluble transition metal catalysts formed from a transition metal and at least partially water-soluble ligands. In aqueous solution the latter form at least partially water-soluble transition metal complexes. Such cleavable linkers can be used to connect bases of nucleotides to labels such as the dyes set forth herein. [0148] Particular linkers include those disclosed in PCT Publication No. WO2004/018493 (herein incorporated by reference) such as those that include moieties of the formulae:
(wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C1-C10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). In some aspect, the linkers connect the bases of nucleotides to labels such as, for example, the dye compounds described herein. [0149] Additional examples of linkers include those disclosed in U.S. Publication No. 2016/0040225 (herein incorporated by reference), such as those include moieties of the formulae: * * (wherein * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). The linker moieties illustrated herein may comprise the whole or partial linker structure between the nucleotides/nucleosides and the labels. [0150] Additional examples of linkers are disclosed in U.S. Publication No. , B Z O K , is 1, 2, 3, 4, 5; k is 1; Z is –N3 (azido), –O-C1-C6 alkyl, –O-C2-C6 alkenyl, or –O-C2-C6 alkynyl; and Hap comprises a hapten moiety described herein, which may contain additional linker structure. One of ordinary skill in the art understands that the hapten moiety described herein is covalently bound to the linker by reacting a functional group of the hapten compound (e.g., carboxyl) with a functional group of the linker (e.g., amino). In one embodiment, the cleavable linker comprises linker moiety) where Z is –O-allyl. For the purpose of the present disclosure, the nucleotide may contain multiple cleavable linkers repeating units (e.g., k is 1, 2, 3, 45, 6, 7, 8, 9 or 10). [0151] An hapten moiety may be attached to any position on the nucleotide base, for example, through a linker. In particular embodiments, Watson-Crick base pairing can still be carried out for the resulting analog. Particular nucleobase labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base. As described above a linker group may be used to covalently attach a dye to the nucleotide. [0152] In particular embodiments, the unlabeled nucleotide may be enzymatically incorporable and enzymatically extendable. Accordingly, a linker moiety may be of sufficient length to connect the nucleotide to the compound such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by a nucleic acid replication enzyme. Thus, the linker can also comprise a spacer unit, such as one or more PEG unit(s) (-OCH 2 CH 2 -) n , where n is an integer of 1-20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The spacer distances, for example, the nucleotide base from a cleavage site or label. [0153] A hapten containing unlabeled nucleosides or nucleotides described herein may have the formula: [0154] where Hap is a hapten mo ety descrbed herein; B is a nucleobase, such as, for example uracil, thymine, cytosine, adenine, 7-deaza adenine, guanine, 7-deaza guanine, and the like; L is an optional linker which may or may not be present; R' can be H, or -OR' is monophosphate, diphosphate, triphosphate, thiophosphate, a phosphate ester analog, –O– attached to a reactive phosphorous containing group, or –O– protected by a blocking group; R'' is H or OH; and R''' is H, a 3' hydroxy blocking group described herein, or -OR''' forms a phosphoramidite. Where -OR''' is phosphoramidite, R' is an acid-cleavable hydroxy protecting group which allows subsequent monomer coupling under automated synthesis conditions. In some further , In some further embodiments, the nucleobase comprises the , , , or . [0155] In yet another alternative embodiment, there is no blocking group on the 3´ carbon of the pentose sugar and the labeled avidin attached to the base via a linker, for example, can be of a size or structure sufficient to act as a block to the incorporation of a further nucleotide. Thus, the block can be due to steric hindrance or can be due to a combination of size, charge and structure, whether or not the dye is attached to the 3´ position of the sugar. [0156] The use of a blocking group allows polymerization to be controlled, such as by stopping extension when an unlabeled nucleotide is incorporated. If the blocking effect is reversible, for example, by way of non-limiting example by changing chemical conditions or by removal of a chemical block, extension can be stopped at certain points and then allowed to continue. [0157] In a particular embodiment, the linker (between the hapten moiety and nucleotide) and blocking group are both present and are separate moieties. In particular embodiments, the linker and blocking group are both cleavable under the same or substantially similar conditions. Thus, deprotection and deblocking processes may be more efficient because only a single treatment will be required to remove both the dye compound and the blocking group. However, in some embodiments a linker and blocking group need not be cleavable under similar conditions, instead being individually cleavable under distinct conditions. [0158] Non-limiting exemplary unlabeled nucleotides as described herein include: wherein L represents a linker, including a cleavable linker described herein; R represents a ribose or deoxyribose moiety as described above, or a ribose or deoxyribose moiety with the 5´ position substituted with mono-, di- or tri- phosphates; Hap represents a hapten moiety described herein. [0159] In some embodiments, non-limiting exemplary unlabeled nucleotide containing a hapten moiety covalently attached via a cleavable linker are shown below: , , , wherein PG stands for the 3ʹ blocking groups described herein; p is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and m is 0, 1, 2, 3, 4, or 5. In one embodiment, –O–PG is AOM. In another embodiment, –O–PG is –O–azidomethyl. In one embodiment, k is 5. In some further embodiments, p is 1, 2 or 3; and m is 5. refers to the connection point of the hapten moiety with the cleavable linker as a result of a reaction between an amino group of the linker moiety and the carboxyl group of the hapten. In further embodiments, the nucleotide may be attached to the hapten moiety via more than one of the same cleavable linkers (such as LN3- LN3, sPA-sPA, AOL-AOL). In other embodiments, the nucleotide may be attached to the hapten moiety via two or more different cleavable linkers (such sPA-LN3, sPA-sPA-LN3, sPA-LN3- LN3, etc.). In any embodiments of the labeled nucleotide described herein, the nucleotide is a nucleotide triphosphate. In further embodiments, the nucleotide has a 2´ deoxyribose. Methods of Sequencing [0160] Additional aspect of the present disclosure relates to a method for determining the sequences of a plurality of different target polynucleotides in parallel, comprising: (a) contacting a solid support with a solution comprising sequencing primers under hybridization conditions, wherein the solid support comprises a plurality of different target polynucleotides immobilized thereon; and the sequencing primers are complementary to at least a portion of the target polynucleotides; (b) contacting the solid support with an aqueous solution comprising DNA polymerase and one or more of four different types of unlabeled nucleotides (e.g., an incorporation mixture containing A, C, G, T or U) under conditions suitable for DNA polymerase-mediated primer extension, and incorporating one type of nucleotides into the sequencing primers to produce extended copy polynucleotides; wherein each of the four types of nucleotides comprises a 3′ blocking group; the first type of unlabeled nucleotide comprises a first hapten; the second type of unlabeled nucleotide comprises a second hapten; (c) contacting the extended copy polynucleotides with a set of affinity reagents under conditions wherein one affinity reagent binds specifically to the incorporated unlabeled nucleotides to provide labeled extended copy polynucleotides; (d) imaging the solid support and performing one or more fluorescent measurements of the extended copy polynucleotides; and (e) removing the 3′ blocking group of the incorporated nucleotides; wherein the set of affinity reagents comprises: a first affinity reagent comprising a first hapten-binding partner that is capable of specific binding to the first type of unlabeled nucleotides; and a second affinity reagent comprising a second hapten-binding partner that is capable of specific binding to the second type of unlabeled nucleotides; wherein the first affinity reagent comprises one or more first detectable labels that are excitable by a first excitation light source, the second affinity reagent comprises one or more second detectable labels that are excitable by a second excitation light source, and wherein the one or more first detectable labels are spectrally distinguishable from the one or more second detectable labels; and wherein one of the first excitation light source and the second excitation light source has a wavelength of about 450 nm to about 460 nm, and the other one of the first excitation light source and the second excitation light source has a wavelength of about 520 nm to about 540 nm. [0161] In some embodiments of the sequencing method described herein, the first hapten is covalently attached to the nucleobase of the first type of unlabeled nucleotide via a cleavable linker. In some further embodiments, the second hapten is covalently attached to the nucleobase of the second type of unlabeled nucleotide via a cleavable linker. Two-Tag Sequencing [0162] In some embodiments of the sequencing method described herein, the third type of unlabeled nucleotide comprises a mixture of the third type of unlabeled nucleotide comprising the first hapten and the third type of unlabeled nucleotide comprising the second hapten, and wherein both the first affinity reagent and the second affinity reagent are capable of specific binding to the third type of unlabeled nucleotide. In some further embodiments, each of the first hapten and the second hapten is covalently attached to the nucleobase of the third type of nucleotide via a cleavable linker. In this instance, the incorporation of the first type of nucleotide is determined by a signal state in the first fluorescent measurement and a dark state in the second fluorescent measurement. The incorporation of the second type of nucleotide is determined by a signal state in the first fluorescent measurement and a dark state in the second fluorescent measurement. The incorporation of the third type of nucleotide is determined by a signal state in the first fluorescent measurement and a signal state in the second fluorescent measurement. The incorporation of the fourth type of nucleotide is determined by a dark state in the first fluorescent measurement and a dark state in the second fluorescent measurement. The sequencing system described herein may also be referred to as two-tag system as there are two affinity reagents labeled with spectrally distinguishable detectable labels respectively. [0163] In some embodiments of the two-tag sequencing method described herein, the first hapten comprises a biotin moiety and the first hapten-binding partner comprises an avidin. In a further embodiment, the avidin is streptavidin. In a further embodiment, the avidin is neutravidin. In further embodiments, one or more biotin binding sites of the avidin are blocked by a biotin moiety-containing molecule or an analog thereof. In some embodiments, the second hapten comprises a DNP moiety and the second hapten-binding partner comprises an anti-DNP antibody. In some embodiments, the third type of unlabeled nucleotide comprises a mixture of a third type of unlabeled nucleotide comprising a biotin moiety, and a third type of unlabeled nucleotide comprising a DNP moiety. In other embodiments, the second hapten comprises a DIG moiety and the second hapten-binding partner comprises an anti-DIG antibody. In other embodiments, the third type of unlabeled nucleotide comprises a mixture of a third type of unlabeled nucleotide comprising a biotin moiety, and a third type of unlabeled nucleotide comprising a DIG moiety. Three-Tag Sequencing [0164] In some other embodiments of the method described herein, the third type of unlabeled nucleotide comprises a third hapten, and the set of affinity reagents further comprises a third affinity reagent comprising a third hapten-binding partner that is capable of specific binding to the third hapten. In some such embodiments, the third hapten is covalently attached to the nucleobase of the third type of unlabeled nucleotide via a cleavable linker. In this instance, the incorporation of the first type of nucleotide is determined by a signal state in the first fluorescent measurement and a dark state in the second fluorescent measurement. The incorporation of the second type of nucleotide is determined by a signal state in the first fluorescent measurement and a dark state in the second fluorescent measurement. The incorporation of the third type of nucleotide is determined by a signal state in the first fluorescent measurement and a signal state in the second fluorescent measurement. The incorporation of the fourth type of nucleotide is determined by a dark state in the first fluorescent measurement and a dark state in the second fluorescent measurement. The sequencing system described herein may also be referred to as three-tag system as there are three affinity reagents labeled with spectrally distinguishable detectable labels respectively. [0165] In some embodiments of the three-tag sequencing method described herein, the first hapten comprises a biotin moiety and the first hapten-binding partner comprises avidin. In a further embodiment, the avidin is streptavidin. In a further embodiment, the avidin is neutravidin. In further embodiments, one or more biotin binding sites of the avidin are blocked by a biotin moiety-containing molecule or an analog thereof. In some embodiments, one of the second hapten and the third hapten comprises a DNP moiety, and the other one of the second hapten and the third hapten comprises a DIG moiety. For example, the second hapten comprises a DNP moiety and the second hapten-binding partner comprises an anti-DNP antibody. The third hapten comprises a DIG moiety and the third hapten-binding partner comprises an anti-DIG antibody. [0166] In some other embodiments of the three-tag sequencing method described herein, one of the first hapten and the second hapten comprises a DNP moiety, and the other one of the first hapten and the second hapten comprises a DIG moiety. For example, the first hapten comprises a DNP moiety and the first hapten-binding partner comprises an anti-DNP antibody. The second hapten comprises a DIG moiety and the second hapten-binding partner comprises an anti-DIG antibody. In some further embodiments, the third hapten comprises a biotin moiety and the third hapten-binding partner comprises avidin. In a further embodiment, the avidin is streptavidin. In a further embodiment, the avidin is neutravidin. In some further embodiments, one or more biotin binding sites of the avidin are blocked by a biotin moiety-containing molecule or an analog thereof. [0167] In some embodiments of the three-tag sequencing method described herein, the third affinity reagent comprises one or more first detectable labels and one or more second detectable labels. For example, the third affinity reagent may comprise or is a protein assembly system described herein. In some embodiments, the protein assembly system used in the three-tag method comprises: a protein (e.g., an antibody) labeled with one or more first detectable labels; one or more biotin moieties containing linkers covalently attached to the protein; and one or more biotin-binding proteins (e.g., avidins such as streptavidin or neutravidin) bound to the one or more biotin moieties via noncovalent interactions; wherein the biotin-binding proteins are labeled with one or more second detectable labels, and wherein the first detectable labels are spectrally distinguishable from the second detectable labels. In further embodiments, the remaining biotin binding sites of the biotin-binding proteins (e.g., avidins such as streptavidin or neutravidin) in the protein assembly system are further blocked with a biotin moiety-containing molecule or an analog thereof such that the protein assembly system may only bind to the hapten moiety specific to the protein (e.g., the antibody). [0168] Though certain haptens and hapten-binding partners have been discussed in relation to two-tag or three-tag sequencing schemes, other hapten and hapten-binding partners may be used, such as those exemplified in Table 1. [0169] In any embodiments of the sequencing method described herein, the incorporation mixture further comprises a fourth type of unlabeled nucleotide, wherein the fourth type of unlabeled nucleotide is not capable of specific binding to any of the affinity reagents. Furthermore, the fourth type of unlabeled nucleotide cannot be excited by either the first or the second light source. [0170] The term “signal state” when used in reference to an imaging event, refers to the state of a polynucleotide incorporating an unlabeled nucleotide and subsequently conjugate with a labeled affinity reagent described herein, in which a specific emission signal is produced by such imaging event, and the emission signal is measured, detected or collected. For example, one or more detectable labels of the affinity reagent specifically bound to the incorporated unlabeled nucleotide may be excited by a light source (e.g., a laser) at a specific wavelength and emits a fluorescent signal that is collected or detected in the single emission detection channel/filter, indicating a “signal state” in such imaging event and fluorescent measurement. [0171] The term “dark state,” when used in reference to an imaging event, refers to the state of a polynucleotide incorporating an unlabeled nucleotide, and subsequently conjugate with a labeled affinity reagent described herein, in which no specific emission signal is produced by such imaging event, or no emission signal is measured, collected, and/or detected. [0172] In one example of the method described herein, “C” nucleotide conjugate is determined by a signal state in the first fluorescent measurement and a dark state in the second fluorescent measurement; “T” nucleotide conjugate is determined by a dark state in the first fluorescent measurement and a signal state in the second fluorescent measurement; “A” nucleotide conjugate is determined by a signal state in the first fluorescent measurement and a signal state in the second fluorescent measurement; and “G” nucleotide conjugate is determined by a dark state in the first fluorescent measurement and a dark state in the second fluorescent measurement. In another example, “T” nucleotide conjugate is determined by a signal state in the first fluorescent measurement and a dark state in the second fluorescent measurement; “C” nucleotide conjugate is determined by a dark state in the first fluorescent measurement and a signal state in the second fluorescent measurement; “A” nucleotide conjugate is determined by a signal state in the first fluorescent measurement and a signal state in the second fluorescent measurement; and “G” nucleotide conjugate is determined by a dark state in the first fluorescent measurement and a dark state in the second fluorescent measurement. [0173] In some embodiments of the sequencing method described herein, step (e) also removes the detectable labels of the incorporated nucleotides. In some such embodiments, the detectable labels and the 3ʹ blocking group are removed in a single step (e.g., under the same chemical reaction condition). In other embodiments, the detectable labels and the 3ʹ blocking group are removed in two separate steps (e.g., the detectable labels and the 3ʹ blocking group are removed under two separate chemical reaction conditions). In some embodiments, the detectable labels are removed by cleaving the cleavable linker in which the hapten moiety is covalent attached to the nucleotide. In some further embodiments, the method further comprises (f) washing the solid support with an aqueous wash solution. [0174] In some embodiments, the imaging step (d) is performed by two light sources (e.g., a laser) operating at different wavelengths. In particular, one light source may have a wavelength between about 450 nm to about 460 nm, and the other light source may have a wavelength between about 510 nm to about 540 nm, or between about 520 nm to about 535 nm. As such, step (d) comprises two separate imaging events and two fluorescent measurements. [0175] In further embodiments of the sequencing method described herein, steps (b) through (f) are performed in repeated cycles (e.g., at least 30, 50, 100, 150, 200, 250, 300, 400, or 500 times) and the method further comprises sequentially determining the sequence of at least a portion of the target polynucleotide based on the identity of each sequentially incorporated nucleotide conjugates. In some such embodiments, steps (b) through (f) are repeated at least 50 cycles. [0176] In some embodiments of the method described herein, the incorporation of the nucleotide conjugates is performed by a mutant of 9°N polymerase, such as those disclosed in WO 2005/024010, U.S. Publication Nos. 2020/0131484 A1, 2020/0181587 A1, and U.S. Ser. Nos. 63/412,241 and 63/433,971, each of which is incorporated by reference in its entirety. Exemplary polymerases include but not limited to Pol 812, Pol 1901, Pol 1558 or Pol 963. The amino acid sequences of Pol 812, Pol 1901, Pol 1558 or Pol 963 DNA polymerases are described, for example, in U.S. Patent Publication Nos.2020/0131484 A1 and 2020/0181587 A1. [0177] In some embodiments of the method described herein, the one or more four different types of nucleotides in the incorporation mixture described in step (b) include nucleotide types selected from the group consisting of A, C, G, T and U, and non-natural nucleotide analogs thereof. In further embodiments, the incorporation mixture comprises four different types of nucleotide conjugates (A, C, G, and T or U), or non-natural nucleotide analogs thereof. In further embodiments, the four different types of nucleotide conjugates are dATP, dCTP, dGTP and dTTP or dUTP, or non-natural nucleotide analogs thereof. In further embodiments, each of the four types of nucleotide conjugates in the incorporation mixture contains a 3ʹ blocking group. [0178] In any embodiments of the method described herein, the density of the immobilized different target polynucleotides (i.e., clusters) on the solid support is about or at least about 50,000/mm2, 100,000/mm2, 150,000/mm2, 200,000/mm2, 250,000/mm2, 300,000/mm2, 350,000/mm2, or 400,000/mm2. [0179] Though certain haptens and hapten-binding partners have been discussed in relation to two-tag sequencing schemes, other hapten and hapten-binding partners may be used. Table 1 lists non-limiting examples of haptens and hapten-binding partners. In further embodiments, the incorporation mixture used in the sequencing method may also include one or more labeled nucleotide(s). For example, the labeled nucleotide(s) may contain a fluorescent label that cannot be excitable by either the first or the second light source. [0180] In any embodiments of the method described herein, the first detectable label may be a blue dye described herein (e.g., the coumarin dye of Formula (I), (I-1), (I-2), (I-3) or (I- 4). In some further embodiments, the second detectable label may be a green dye (e.g., a cyanine dye) described herein. [0181] In any embodiments of methods of sequencing described herein, the method can reduce or eliminate sequence context or sequence specific effect. Two-channel base- calling relies upon the ability to discriminate bases by their intensity in two emission color channels. This intensity modulation adds noise to the system and can cause miscalls when the sequence-specific intensity modulation shift a given cluster’s intensity towards a decision boundary. Using a single ffN (such as ffA) which is subsequently conjugated with a protein labeled with two colored dyes that can produce color in two channels reduce or eliminate sequence-specific intensity shifts and thereby improve base calling accuracy. General description on sequencing by synthesis [0182] In a specific embodiment, a synthetic step is carried out and may optionally comprise incubating a template or target polynucleotide strand with a reaction mixture comprising fluorescently labeled nucleotides of the disclosure. A polymerase can also be provided under conditions which permit formation of a phosphodiester linkage between a free 3' hydroxy group on a polynucleotide strand annealed to the template or target polynucleotide strand and a 5' phosphate group on the labeled nucleotide. Thus, a synthetic step can include formation of a polynucleotide strand as directed by complementary base pairing of nucleotides to a template/target strand. [0183] In all embodiments of the methods, the detection step may be carried out while the polynucleotide strand into which the labeled nucleotides are incorporated is annealed to a template/target strand, or after a denaturation step in which the two strands are separated. Further steps, for example chemical or enzymatic reaction steps or purification steps, may be included between the synthetic step and the detection step. In particular, the polynucleotide strand incorporating the labeled nucleotide(s) may be isolated or purified and then processed further or used in a subsequent analysis. By way of example, polynucleotide strand incorporating the labeled nucleotide(s) as described herein in a synthetic step may be subsequently used as labeled probes or primers. In other embodiments, the product of the synthetic step set forth herein may be subject to further reaction steps and, if desired, the product of these subsequent steps purified or isolated. [0184] Suitable conditions for the synthetic step will be well known to those familiar with standard molecular biology techniques. In one embodiment, a synthetic step may be analogous to a standard primer extension reaction using nucleotide precursors, including the labeled nucleotides as described herein, to form an extended polynucleotide strand (primer polynucleotide strand) complementary to the template/target strand in the presence of a suitable polymerase enzyme. In other embodiments, the synthetic step may itself form part of an amplification reaction producing a labeled double stranded amplification product comprised of annealed complementary strands derived from copying of the primer and template polynucleotide strands. Other exemplary synthetic steps include nick translation, strand displacement polymerization, random primed DNA labeling, etc. A particularly useful polymerase enzyme for a synthetic step is one that is capable of catalyzing the incorporation of the labeled nucleotides as set forth herein. A variety of naturally occurring or mutant/modified polymerases can be used. By way of example, a thermostable polymerase can be used for a synthetic reaction that is carried out using thermocycling conditions, whereas a thermostable polymerase may not be desired for isothermal primer extension reactions. Suitable thermostable polymerases which are capable of incorporating the labeled nucleotides according to the disclosure include those described in WO 2005/024010 or WO06120433, each of which is incorporated herein by reference. In synthetic reactions which are carried out at lower temperatures such as 37 °C, polymerase enzymes need not necessarily be thermostable polymerases, therefore the choice of polymerase will depend on a number of factors such as reaction temperature, pH, strand-displacing activity and the like. [0185] In specific non-limiting embodiments, the disclosure encompasses methods of nucleic acid sequencing, re-sequencing, whole genome sequencing, single nucleotide polymorphism scoring, any other application involving the detection of the modified nucleotide or nucleoside labeled with dyes set forth herein when incorporated into a polynucleotide. [0186] SBS generally involves sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction using a polymerase or ligase in order to form an extended polynucleotide chain complementary to the template/target nucleic acid to be sequenced. The identity of the base present in one or more of the added nucleotide(s) can be determined in a detection or “imaging” step. The identity of the added base may be determined after each nucleotide incorporation step. The sequence of the template may then be inferred using conventional Watson-Crick base-pairing rules. The use of the nucleotides labeled with dyes set forth herein for determination of the identity of a single base may be useful, for example, in the scoring of single nucleotide polymorphisms, and such single base extension reactions are within the scope of this disclosure. [0187] In an embodiment of the present disclosure, the sequence of a template/target polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent strand complementary to the template polynucleotide to be sequenced through the detection of fluorescent label(s) attached to the incorporated nucleotide(s). Sequencing of the template polynucleotide can be primed with a suitable primer (or prepared as a hairpin construct which will contain the primer as part of the hairpin), and the nascent chain is extended in a stepwise manner by addition of nucleotides to the 3' end of the primer in a polymerase-catalyzed reaction. [0188] In particular embodiments, each of the different nucleotide triphosphates (A, T, G and C) may be labeled with a unique fluorophore and also comprises a blocking group at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides may be unlabeled (dark). The polymerase enzyme incorporates a nucleotide into the nascent chain complementary to the template/target polynucleotide, and the blocking group prevents further incorporation of nucleotides. Any unincorporated nucleotides can be washed away and the fluorescent signal from each incorporated nucleotide can be “read” optically by suitable means, such as a charge-coupled device using light source excitation and suitable emission filters. The 3'- blocking group and fluorescent dye compounds can then be removed (deprotected) (simultaneously or sequentially) to expose the nascent chain for further nucleotide incorporation. [0189] The method, as exemplified above, utilizes the incorporation of fluorescently labeled, 3' blocked nucleotides A, G, C, and T into a growing strand complementary to the immobilized polynucleotide, in the presence of DNA polymerase. The polymerase incorporates a base complementary to the target polynucleotide but is prevented from further addition by the 3' blocking group. The label of the incorporated nucleotide can then be determined, and the blocking group removed by chemical cleavage to allow further polymerization to occur. The nucleic acid template to be sequenced in a SBS reaction may be any polynucleotide that it is desired to sequence. The nucleic acid template for a sequencing reaction will typically comprise a double stranded region having a free 3' hydroxy group that serves as a primer or initiation point for the addition of further nucleotides in the sequencing reaction. The region of the template to be sequenced will overhang this free 3' hydroxy group on the complementary strand. The overhanging region of the template to be sequenced may be single stranded but can be double- stranded, provided that a “nick” is present on the strand complementary to the template strand to be sequenced to provide a free 3' OH group for initiation of the sequencing reaction. In such embodiments, sequencing may proceed by strand displacement. In certain embodiments, a primer bearing the free 3' hydroxy group may be added as a separate component (e.g., a short oligonucleotide) that hybridizes to a single-stranded region of the template to be sequenced. Alternatively, the primer and the template strand to be sequenced may each form part of a partially self-complementary nucleic acid strand capable of forming an intra-molecular duplex, such as for example a hairpin loop structure. Hairpin polynucleotides and methods by which they may be attached to solid supports are disclosed in PCT Publication Nos. WO0157248 and WO2005/047301, each of which is incorporated herein by reference. Nucleotides can be added successively to a growing primer, resulting in synthesis of a polynucleotide chain in the 5' to 3' direction. The nature of the base which has been added may be determined, particularly but not necessarily after each nucleotide addition, thus providing sequence information for the nucleic acid template. Thus, a nucleotide is incorporated into a nucleic acid strand (or polynucleotide) by joining of the nucleotide to the free 3' hydroxy group of the nucleic acid strand via formation of a phosphodiester linkage with the 5' phosphate group of the nucleotide. [0190] The nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid molecule comprised of deoxynucleotides and ribonucleotides. The nucleic acid template may comprise naturally occurring and/or non-naturally occurring nucleotides and natural or non- natural backbone linkages, provided that these do not prevent copying of the template in the sequencing reaction. [0191] In certain embodiments, the nucleic acid template to be sequenced may be attached to a solid support via any suitable linkage method known in the art, for example via covalent attachment. In certain embodiments template polynucleotides may be attached directly to a solid support (e.g., a silica-based support). However, in other embodiments of the disclosure the surface of the solid support may be modified in some way so as to allow either direct covalent attachment of template polynucleotides, or to immobilize the template polynucleotides through a hydrogel or polyelectrolyte multilayer, which may itself be non-covalently attached to the solid support. [0192] Arrays in which polynucleotides have been directly attached to a support (for example, silica-based supports such as those disclosed in WO00/06770 (incorporated herein by reference), wherein polynucleotides are immobilized on a glass support by reaction between a pendant epoxide group on the glass with an internal amino group on the polynucleotide. In addition, polynucleotides can be attached to a solid support by reaction of a sulfur-based nucleophile with the solid support, for example, as described in W02005/047301 (incorporated herein by reference). A still further example of solid-supported template polynucleotides is where the template polynucleotides are attached to hydrogel supported upon silica-based or other solid supports, for example, as described in WO00/31148, WO01/01143, WO02/12566, WO03/014392, U.S. Pat. No.6,465,178 and WO00/53812, each of which is incorporated herein by reference. [0193] A particular surface to which template polynucleotides may be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and in WO2005/065814, which is incorporated herein by reference. Specific hydrogels that may be used include those described in WO2005/065814 and U.S. Pub. No. 2014/0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl) acrylamide-co- acrylamide)). [0194] DNA template molecules can be attached to beads or microparticles, for example, as described in U.S. Pat. No. 6,172,218 (which is incorporated herein by reference). Attachment to beads or microparticles can be useful for sequencing applications. Bead libraries can be prepared where each bead contains different DNA sequences. Exemplary libraries and methods for their creation are described in Nature, 437, 376-380 (2005); Science, 309, 5741, 1728- 1732 (2005), each of which is incorporated herein by reference. Sequencing of arrays of such beads using nucleotides set forth herein is within the scope of the disclosure. [0195] Template(s) that are to be sequenced may form part of an “array” on a solid support, in which case the array may take any convenient form. Thus, the method of the disclosure is applicable to all types of high-density arrays, including single-molecule arrays, clustered arrays, and bead arrays. Nucleotides labeled with dye compounds of the present disclosure may be used for sequencing templates on essentially any type of array, including but not limited to those formed by immobilization of nucleic acid molecules on a solid support. [0196] However, nucleotides labeled with dye compounds of the disclosure are particularly advantageous in the context of sequencing of clustered arrays. In clustered arrays, distinct regions on the array (often referred to as sites, or features) comprise multiple polynucleotide template molecules. Generally, the multiple polynucleotide molecules are not individually resolvable by optical means and are instead detected as an ensemble. Depending on how the array is formed, each site on the array may comprise multiple copies of one individual polynucleotide molecule (e.g., the site is homogenous for a particular single- or double-stranded nucleic acid species) or even multiple copies of a small number of different polynucleotide molecules (e.g., multiple copies of two different nucleic acid species). Clustered arrays of nucleic acid molecules may be produced using techniques generally known in the art. By way of example, WO 98/44151 and WO00/18957, each of which is incorporated herein, describe methods of amplification of nucleic acids wherein both the template and amplification products remain immobilized on a solid support in order to form arrays comprised of clusters or “colonies” of immobilized nucleic acid molecules. The nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for sequencing using nucleotides labeled with dye compounds of the disclosure. [0197] Nucleotides labeled with dye compounds of the present disclosure are also useful in sequencing of templates on single molecule arrays. The term “single molecule array” or “SMA” as used herein refers to a population of polynucleotide molecules, distributed (or arrayed) over a solid support, wherein the spacing of any individual polynucleotide from all others of the population is such that it is possible to individually resolve the individual polynucleotide molecules. The target nucleic acid molecules immobilized onto the surface of the solid support can thus be capable of being resolved by optical means in some embodiments. This means that one or more distinct signals, each representing one polynucleotide, will occur within the resolvable area of the particular imaging device used. [0198] Single molecule detection may be achieved wherein the spacing between adjacent polynucleotide molecules on an array is at least 100 nm, more particularly at least 250 nm, still more particularly at least 300 nm, even more particularly at least 350 nm. Thus, each molecule is individually resolvable and detectable as a single molecule fluorescent point, and fluorescence from said single molecule fluorescent point also exhibits single step photobleaching. [0199] The terms “individually resolved” and “individual resolution” are used herein to specify that, when visualized, it is possible to distinguish one molecule on the array from its neighboring molecules. Separation between individual molecules on the array will be determined, in part, by the particular technique used to resolve the individual molecules. The general features of single molecule arrays will be understood by reference to published applications WO00/06770 and WO 01/57248, each of which is incorporated herein by reference. Although one use of the labeled nucleotides of the disclosure is in SBS reactions, the utility of such nucleotides is not limited to such methods. In fact, the labeled nucleotides described herein may be used advantageously in any sequencing methodology which requires detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide. [0200] In particular, nucleotides labeled with dye compounds of the disclosure may be used in automated fluorescent sequencing protocols, particularly fluorescent dye-terminator cycle sequencing based on the chain termination sequencing method of Sanger and co-workers. Such methods generally use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides in a primer extension sequencing reaction. So-called Sanger sequencing methods, and related protocols (Sanger-type), utilize randomized chain termination with labeled dideoxynucleotides. EXAMPLES [0201] Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims. General procedure for determining degree of protein labeling [0202] The degree of protein labeling by a dye may be estimated via absorbance measurements. First, if present, nonconjugated dye must be removed from a sample including dye-labeled protein. The nonconjugated dye should be completely, or substantially removed from the sample for accurate determination of the protein:dye ratio. The dye may be removed by dialysis or gel filtration, for example. [0203] After removing nonconjugated dye, the absorbance of the protein:dye conjugate may be measured at a wavelength of 280 nm, A 280 . The 280 nm absorbance A 280 may be measured using a spectrophotometer. The cuvette of the spectrophotometer may have a 1 cm path length. If an initial absorbance measurement exceeds 2.0, the sample may be diluted by a dilution factor DF necessary to lower the absorbance below 2.0 before remeasuring. Additionally, an absorbance Amax of the protein:dye conjugate may be measured at the wavelength λmax at which the dye is maximally absorbent. [0204] Molarity of the protein in the sample may be calculated according to the following: ^ − (^ × CF n concentration M ^^^ ^^^ ) protei ( ) = × ^^ ^ wherein ε is the protein molar extinction coefficient and CF is the correction factor that adjusts for the amount of absorbance at 280 nm caused by the dye. The molar protein concentration can be used to calculate the protein:dye molar ratio: ^ of the labeled protein dye per mole protein = ^^^ ^ × ^^ ^ × [protein concentration ( M ) ] wherein ε’ is the molar extinction coefficient of the fluorescent dye. Example 1. Preparation of blue and green dyes labeled streptavidin [0205] To prepare blue dye-labeled streptavidin, 1 equivalent of coumarin dye A- PEG12-COOH was co-evaporated with 1 mL of DMF and dried under vacuum. Then, 10 equivalents of N, N-diisopropylethylamine and 1.1 equivalents of TSTU were added. The reaction was stirred at room temperature for 30 min to form the activated ester of the coumarin dye A- PEG12-COOH. Then 10 µL of the activated ester was added to 1.0 mL of 2.0 mg/mL streptavidin (SA) in pH 6.5 potassium phosphate buffer. The resulting solution was stirred at room temperature for 1 hour and the reaction mixture was purified by a PD minitrap G-25 size exclusion column using pH 6.5, 10 mM potassium phosphate buffer to afford a resulting streptavidin (coumarin A- PEG12) conjugate. Coumarin dye A was covalently attached to streptavidin through the reaction of the activated ester of the carboxyl group and the lysine residue of the streptavidin to form amide bonding. The number of coumarin dye A covalently attached to streptavidin was calculated based on the general procedure for determining degree of protein labeling by compare the ratio of absorption wavelength of protein and dye. The resulting protein dye conjugate was determined as SA(coumarin dye A-PEG 12 ) 4.5 . [0206] To prepare green dye labeled streptavidin, 1 equivalent of cyanine dye B was co-evaporated with 1 mL of DMF and dried under vacuum. Then 10 equivalent of N, N- Diisopropylethylamine and 1.1 equivalent of TSTU were added. The reaction was stirred at room temperature for 30 min to form the activated ester of the cyanine dye B. The resulting conjugate 5.0 µM SA(coumarin dye A-PEG12)4.5 in pH 6.5 potassium phosphate was then mixed with 10 µL of activated ester of cyanine dye B and stirred at room temperature for 1 hour and the reaction mixture was purified by a PD minitrap G-25 size exclusion columns using pH 8.0, 10 mM tris buffer to obtain dual labeled streptavidin (labeled with both blue coumarin dye A and green cyanine dye B). The degree of cyanine dye B labeling was calculated based on the same general procedure as described above as SA(coumarin dye A-PEG 12 ) 4.5 (cyanine dye B) 1.4 . The complex was characterized by nanodrop photo spectrometer. [0207] Coumarin A is disclosed in U.S. Publication No.2022/0033900 A1, having the structure moiety when conjugated with a protein (e.g., streptavidin) or antibody (e.g., anti-DNP) through the PEG linker. This coumarin dye is excitable by a blue light at about 450 nm to about 460 nm (also called a “blue dye”). Cyanine dye B is disclosed in International Publication No. WO2014/135221 A1, having the structure moiety when conjugated with streptavidin. This cyanine dye is excitable by a green light at about 520 nm to about 540 nm (also called a “green dye”). Example 2. Preparation of a dual-protein assembly system [0208] First, 1 equivalent of coumarin dye A-PEG 12 -COOH was co-evaporated with 1 mL of DMF and dried under vacuum. Then 10 equivalent of N, N-diisopropylethylamine and 1.1 equivalent of TSTU were added. The reaction was stirred at room temperature for 30 min to form the activated ester of the coumarin dye A-PEG 12 -COOH. Then 20 µL of the activated ester of the coumarin dye A-PEG12-COOH was added to 0.5 mL of 2.0 mg/mL (12.5 µM) anti-DNP antibody in pH 6.5 potassium phosphate buffer. The resulting solution was stirred at room temperature for 1 hour and the reaction mixture was purified by a PD minitrap G-25 size exclusion column using pH 6.5, 10 mM potassium phosphate buffer to obtain coumarin dye A labeled anti-DNP. The degree of dye labeling was calculated based on the general procedure described above as anti- DNP(coumarin dye A-PEG 12 ) 7 . Then, 1 mL of 6.2 µM anti-DNP(coumarin dye A-PEG 12 ) 7 was mixed with 10 µL of 2.35 mg/mL of NHS-PEG 12 -Biotin in DMA solution and stirred at room temperature for 1 hour and the reaction mixture was purified by a PD minitrap G-25 size exclusion column using pH 8.0, 10 mM tris buffer to obtain anti-DNP(coumarin dye A-PEG12)7-Biotin with 3.0 µM solution concentration. [0209] The preparation of cyanine dye B labeled streptavidin SA(cyanine dye B)7 was similar to the procedure described in U.S. Publication No. 2013/0079232 A1, which is incorporated by reference in its entirety. The degree of dye labeling was calculated based on the general procedure described above as SA(cyanine dye B)7. The dual protein system was then assembled by adding 48 µL of 3.0 µM of anti-DNP(coumarin dye A-PEG12)7-Biotin and 31.5 µL of 13.7 µM SA(cyanine dye B) 7 (3 molar equivalent) in 20.5 µL of pH 8.010 mM tris buffer to form anti-DNP(coumarin dye A-PEG 12 ) 7 -Biotin-[SA(cyanine dye B) 7 ] 3. The dual protein complex was characterized by nanodrop photo spectrometer. Example 3. Two-tag SBS with unlabeled nucleotides [0210] In this example, SBS was conducted using a two-dye scheme with universal ffNs having two different hapten moieties. The unlabeled ffNs used in the incorporation mix included dark ffG, biotin-linked ffA (ffA-sPA-LN3-biotin), DNP-linked ffA (ffA-sPA-LN3- DNP), DNP-linked ffT (ffT-LN3-DNP), biotin-linked ffC (ffC-LN3-LN3-biotin), and SO7181- linked ffC (ffC-LN3-SO7181). SO7181 is a commercially available red dye. An affinity reagent mixture used in the secondary labeling after the incorporation of the unlabeled nucleotides included 0.1 µM anti-DNP antibody labeled with coumarin dye A (anti-DNP(coumarin dye A)4), and 0.1 µM of streptavidin labeled with cyanine dye B. The affinity reagent mixture was introduced to incorporated ffNs and allowed to bind at 60 °C for 60 seconds. The SBS run was conducted on a MiSeq™ using blue/green light excitations (first blue light at about 460 nm, then green light at about 532 nm). [0211] FIG.3A is a two-dye SBS signal intensity scatterplot generated from a reading at incorporation cycle 26 of that SBS run. Channel 1 (x-axis) plots intensity in the green channel while Channel 2 (y-axis) plots intensity in the blue channel. For that run, the average error rate over 150 cycles was 1.1%, the average phasing was 0.07%, and the average signal remaining was 61%. [0212] A second SBS run was conducted under similar conditions with reversed labeled protein tags. It included 0.1 µM anti-DNP antibody labeled with cyanine dye B, and 0.1 µM of streptavidin labeled with coumarin dye A. FIG. 3B is a two-dye SBS intensity plot generated from a reading at incorporation cycle 26 of that run. Channel 1 (x-axis) plots intensity in the blue channel while Channel 2 (y-axis) plots intensity in the green channel. For this run, the error rate was 0.54%, the phasing was 0.13%, and the signal remaining was 63%. Example 4. Three-tag SBS with unlabeled nucleotides [0213] In this example, SBS was conducted using a three-tag system with a set of unlabeled nucleotides having three different hapten moieties. The ffNs used included dark ffG, DNP-linked ffA (ffA-sPA-LN3-DNP), biotin-linked ffC (ffC-LN3-LN3-biotin), and DIG-linked ffT (ffT-sPA-LN3-DIG). The affinity reagent mixture (i.e., labeled protein mixture) included a dual-labeled streptavidin complex SA(coumarin dye A-PEG 12 ) 4.5 (cyanine dye B) 1.4 as described in Example 1, including, on average, 4.5 molar equivalent coumarin dye A moieties (blue dye) and 1.4 molar equivalent cyanine dye B (green dye); an anti-DIG antibody labeled with cyanine dye B (anti-DIG(cyanine dye B) 8 ) and an anti-DNP antibody labeled with coumarin dye A (anti- DNP(coumarin dye A)4). The labeling protein mix was introduced to incorporated ffNs and allowed to bind at 60 °C for 60 seconds. The SBS run was conducted on a MiSeq™ using blue/green light excitations. [0214] FIG. 4A is a two-dye SBS signal intensity scatterplot generated from incorporation cycle 1 of that run. Channel 1 (x-axis) plots intensity in the green channel while Channel 2 (y-axis) plots intensity in the blue channel. For that run, error rate at cycle 150 was 0.79%, phasing was 0.059%, and signal remaining was 86%. [0215] A second SBS run with a different affinity reagent mixture was conducted under similar conditions. The ffNs in the incorporation mixture included: dark ffG, DNP-linked ffA (ffA-sPA-LN3-DNP), biotin-linked ffC (ffC-LN3-LN3-biotin), and DIG-linked ffT (ffT-sPA- LN3-DIG). The affinity reagent mixture (i.e., labeled protein mixture) included a dual protein assembly system described in Example 2 – anti-DNP(coumarin dye A-PEG 12 ) 7 -Biotin- [SA(cyanine dye B)7]3. The anti-DNP antibody was covalently labeled with coumarin dye A moieties (blue dye). In addition, the anti-DNP antibody was linked via biotin linker to three molar equivalent of cyanine dye B (green dye) labeled streptavidin. These streptavidin molecules were on average labeled with seven cyanine dye B moieties and any remaining biotin-binding sites were blocked such that this protein assembly cannot bind to biotin-linked ffC. The labeling protein mix also included an anti-DIG antibody labeled with cyanine dye B (anti-DIG(cyanine dye B) 8 ) and streptavidin labeled with coumarin dye A (SA(coumarin dye A) 4 ). [0216] FIG. 4B is a two-dye SBS signal intensity scatterplot generated from incorporation cycle 1 of that second run. Channel 1 (x-axis) plots intensity in the blue channel while Channel 2 (y-axis) plots intensity in the green channel. Furthermore, it was observed that there was no Channel 1-Channel 2 anticorrelated sequence context behavior of the A cloud (typically observed in two-channel SBS when ffA is labeled with either a blue or a green dye) in the scatterplot because of the single entity labels for each of the ffA, ffC and ffT. Example 5. Synthesis of Hydrophilic Coumarin Dyes of Formula (I) and Fluorescent Intensity Solution Data [0217] 3-Amino-4-hydroxybenzoic acid (4.05 g, 26.5 mmol, 1.0 eq) was suspended in EtOH (50 ml) and cooled to 0°C. Ethyl 3-ethoxy-3-iminopropionate hydrochloride (5.69 g, 29.1 mmol, 1.1 eq) was added and the flask allowed to warm to RT, followed by heating under reflux for 16 hrs. The reaction mixture was cooled to RT and the precipitate formed collected by filtration, washing with EtOH. The target Compound 1 was collected as an off-white powder (5.99 g, 24.1 mmol, 91 %). 1H NMR (400 MHz, DMSO) δ 13.12 (s, <1H), 8.26 (d, J = 1.6 Hz, 1H), 8.03 (dd, J = 8.5, 1.7 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 4.27 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H). [0218] 4-Fluoro-2-hydroxybenzaldehyde (3.35 g, 23.9 mmol, 1.0 eq) and Compound 1 (5.97 g, 23.9 mmol, 1.0 eq) were suspended in EtOH, followed by addition of AcOH (836 µl, 12.0 mmol, 0.5 eq) and piperidine (974 µl, 12.0 mmol, 0.5 eq). The reaction was heated under reflux for 16hrs (orange suspension formed which dissolved on heating). The reaction mixture was cooled to RT and the precipitate collected by filtration, washed with EtOH and dried under high vacuum to afford the Compound 2 as a pale-yellow solid (5.05 g, 15.5 mmol, 65 %). 1H NMR (400 MHz, DMSO) δ 9.13 (s, 1H), 8.29 (d, J = 1.5 Hz, 1H), 8.15 – 8.03 (m, 2H), 7.80 (d, J = 8.5 Hz, 1H), 7.52 (dd, J = 9.6, 2.5 Hz, 1H), 7.38 (td, J = 8.8, 2.5 Hz, 1H). Synthesis of Dye I-1 [0219] Compound 3 (53.5 mg, 190 µmol, 2.0 eq) and Compound 2 (30.9 mg, 95 µmol, 1.0 eq) were suspended in DMSO (2 ml). DIPEA (165 µl, 950 µmol, 10.0 eq) was added and the reaction heated at 110 °C for 16 hrs. (Orange suspension to dark orange solution). Half the volume of solvent was distilled off under high vacuum at 60°C and the residue diluted with a 1:1 mixture of water and acetonitrile (10 ml). The product was purified by Prep HPLC (eluting at 35% acetonitrile/water) to afford Dye I-1 (31.3 µmol, 33 %). λ abs = 450nm, λ fl = 495 nm. I-2 [0220] A solution of 3-amino-1-propane sulfonic acid (0.75 g, 5.39 mmol, 1.0 eq) in water (5 ml) was heated to 60°C. Aqueous NaOH (2 M, 3 ml) was added, followed by a solution of 3-bromopropane sulfonic acid sodium salt (1.40 g, 6.22 mmol, 1.15 eq) in water (5 ml) dropwise and stirred at 60 °C for 16 hrs. The reaction mixture was evaporated and redissolved in a minimum volume of water and purified by C18 column chromatography (0-10% Acetonitrile/Water) to give Compound 4 as a crude mixture with undesired primary amine and tertiary amine (unreactive in next step). g mg, eq) were suspended in DMSO (40 ml). DIPEA (4.0 ml, 23.1 mmol, 10.0 eq) was added and the reaction heated at 110°C for 16 hrs. (Orange suspension to dark orange solution). Half the volume of solvent was distilled off under high vac at 60°C and the residue diluted with water (40 ml) and filtered. Dye I-2 was purified by Prep HPLC (eluting at 20% acetonitrile/water) to afford dye I-2 (187 µmol, 8%). λabs = 449nm, λfl = 494 nm. General Synthesis of Amine-modified Coumarin Dyes [0222] Amine R1-NH-R2 (2.0 eq) and Compound 2 (1.0 eq) were suspended in DMSO. DIPEA (10.0 eq) was added, and the reaction heated at 110 °C for 6-16 hrs. (Orange suspension to dark orange solution). Half the volume of solvent was distilled off under high vac at 60 °C and the residue diluted with water and filtered. The product was purified by Prep HPLC (eluting at 20 - 40% acetonitrile/water) to afford amine-modified coumarin dyes (8-40 % yield). Using the general synthetic method described herein, dye I-4 was prepared with 40% yield. λabs = 451nm, λfl = 500 nm. [0223] The fluorescent intensity of dye I-1 and dye I-2 were tested in solution against the reference coumarin dye C, which is a standard blue dye used in Illumina’s two-channel SBS with good stability and fluorescent intensity. In addition, protein/antibody (such as streptavidin, anti-DIG, anti-DNP) labeled with multiple dye I-1 and dye I-2 were also prepared following similar procedure described in Example 1, and their fluorescent intensities were also tested in solution. As shown in FIGs.5A and 5B, labeled streptavidin, labeled anti-DIG, and labeled anti- DNP demonstrated greater fluorescent intensities in solution compared with ffA labeled with coumarin dye C at the same concentration. Example 6. MiSeq® SBS Using Hydrophilic Coumarin Dyes of Formula (I) [0224] ffC-sPA-dye I-1 and ffC-sPA-dye I-2 were tested on Illumina’s MiSeq™ platform with blue and green LED operating at 460 nm and 532 nm, against the standard ffC-sPA- coumarin dye C. Sequencing recipe was using 22 °C scanning, no variable dosage, no re-use, 25s incorporation time and 10s cleavage time. In addition to the labeled ffC, additional components of the incorporation mix include: (1) a set of nucleotides comprising dark G, ffC-SO7181, ffA- LN3-BL-coumarin dye C, ffA-BL-NR550S0 (a known green dye), ffT-LN3-cyanine dye B; (2) DNA polymerase Pol 1901; and (3) a glycine buffer. [0225] FIGs. 6A-6C are scatterplot of blue/green two-channel MiSeq® SBS runs at cycle 5, comparing the effect of changing ffC-sPA-coumarin dye C with ffC-sPA-dye I-1 or ffC- sPA-dye I-2. Both ffC-sPA-dye I-1 or ffC-sPA-dye I-2 showed an increase in brightness in sequencing compared to the standard ffC labeled with coumarin dye C. [0226] When exposed to increased dosage during sequencing, ffN mixes incorporating ffC-sPA-dye I-2 and ffC-spA-dye I-4, both retained more signal than corresponding ffC-spA- coumarin dye C (FIGs.7A and 7B). This may indicate that the growing DNA strand was damaged less by these ffNs during each cycle of the sequencing process.
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