Multivalent binding compositions having reactive groups

Abstract

The present disclosure provides nucleotide conjugates that are configured to include a core that is attached to a plurality of nucleotide arms, each of which is modular and includes (i) a core attachment portion, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit. The nucleotide unit of each nucleotide arm can bind to a polymerase that is complexed with a nucleic acid template and a nucleic acid primer. The nucleotide unit can bind to the 3' end of the primer at a position opposite the complementary nucleotide in the template strand. Under suitable conditions, the nucleotide unit of the nucleotide conjugate binds to the primer strand but does not undergo polymerase-catalyzed incorporation. The binding event is detectable, and the specific base of the nucleotide unit can be identified. The nucleotide conjugates described herein are useful for nucleic acid sequencing methods, particularly massively parallel sequencing methods utilized in next-generation sequencing platforms.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 328,663, filed April 7, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, which is provided as a file titled 52933-750_601_SL.XML, created April 6, 2023, and 753,818 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Nucleic acid sequencing can be used to obtain information in a variety of biomedical contexts, including diagnostics, prognostics, biotechnology, and forensic biology. Various sequencing methods have been developed, including Maxam-Gilbert sequencing and chain termination reaction (CTR), de novo sequencing methods including shotgun sequencing and bridge PCR, and next-generation sequencing methods including polony sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, HeliScope single-molecule sequencing, and SMRTR sequencing. Despite advances in DNA sequencing, many challenges remain. Summary of the Invention

[0004] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence comprises a D141A mutation and a D143A mutation. In some embodiments, the amino acid sequence further comprises a Y410A mutation, a L409S mutation, a Y261A mutation, a P411G mutation, a F406I mutation, a P411A mutation, a Y7A mutation, a Y493I mutation, a Y493T mutation, a V513I mutation, a L409A mutation, a A485S mutation, a Y410G mutation, a I521H mutation, or a K507L mutation, or any combination thereof with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence is (a) a Y410A mutation with reference to SEQ ID NO: 391; (b) a L409S mutation, a Y410A mutation, and a Y261A mutation with reference to SEQ ID NO: 391; (c) a L409S mutation, a Y410A mutation, a P411G mutation, and a Y261A mutation with reference to SEQ ID NO: 391; (d) a Y261A mutation, a F406I mutation, a L409S mutation, a Y410A mutation, and a P411A mutation with reference to SEQ ID NO: 391; (e) a Y7A mutation, a Y261A mutation, and a Y410A mutation with reference to SEQ ID NO: 391; (f) a Y261A mutation, a Y410A mutation, and a Y493I mutation with reference to SEQ ID NO: 391; (g) a Y261A mutation, a Y410A mutation, and a Y493T mutation with reference to SEQ ID NO: 391; (h) a Y7A mutation, a Y261A mutation, a Y410A mutation, and a Y493T mutation with reference to SEQ ID NO: 391; (i) a Y7A mutation, a Y261A mutation, a L409S mutation, and a Y410A mutation with reference to SEQ ID NO: 391; (j) a Y261A mutation, a L409A mutation, a Y410A mutation, and a P411A mutation with reference to SEQ ID NO: 391; (k) a Y261A mutation, a L409S mutation, a Y410A mutation, and a P411G mutation with reference to SEQ ID NO: 391; (l) a Y261A mutation, a L409S mutation, and a Y410A mutation with reference to SEQ ID NO: 391; (m) a Y261A mutation, a L409S mutation, and a Y410G mutation with reference to SEQ ID NO: 391; (n) a Y261A mutation, a L409A mutation, a Y410A mutation, a P411A mutation, and an A485S mutation with reference to SEQ ID NO: 391;(o) Y261 mutation, L409S mutation, Y410A mutation, P411G mutation, and A485S mutation with reference to SEQ ID NO: 391; (p) Y261A mutation, L409S mutation, Y410A mutation, and A485S mutation with reference to SEQ ID NO: 391; (q) Y261A mutation, L409S mutation, Y410G mutation, and A485S mutation with reference to SEQ ID NO: 391; (r) Y261A mutation, L409A mutation, Y410A mutation, P411A mutation, and I521H mutation with reference to SEQ ID NO: 391; (s) SEQ ID NO: 39 In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, a Y410A mutation, a P411G mutation, and an I521H mutation with reference to SEQ ID NO: 391, (t) a Y261A mutation, a L409S mutation, a Y410A mutation, and an I521H mutation with reference to SEQ ID NO: 391, (u) a Y261A mutation, a L409S mutation, a Y410G mutation, and an I521H mutation with reference to SEQ ID NO: 391, or (v) a Y261A mutation, a L409S mutation, a Y410A mutation, an A485S mutation, a K507L mutation, and an I521H mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises any one of SEQ ID NOs: 392-413. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 392-413. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0005] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence comprises a D158A mutation and an E160A mutation. In some embodiments, the amino acid sequence further comprises a L431A mutation, a Y432A mutation, a P433I mutation, an A507S mutation, a K506Q mutation, a P433A mutation, a I543H mutation, a L431S mutation, a P433G mutation, a K529L mutation, or a Y432G mutation, or any combination thereof with reference to SEQ ID NO: 414.In some embodiments, the amino acid sequence comprises: (a) a L431A mutation, a Y432A mutation, a P433I mutation, and an A507S mutation with reference to SEQ ID NO: 414; (b) a L431A mutation, a Y432A mutation, a P433I mutation, and a K506Q mutation with reference to SEQ ID NO: 414; (c) a L431A mutation, a Y432A mutation, a P433I mutation, a K506Q mutation, and an A507S mutation with reference to SEQ ID NO: 414; (d) a L431A mutation, a Y432A mutation, a P433I mutation, a K506Q mutation, and an A507S mutation with reference to SEQ ID NO: 414; (e) an L431A mutation, a Y432A mutation, a P433A mutation, and an A507S mutation with reference to SEQ ID NO: 414; (f) an L431A mutation, a Y432A mutation, a P433A mutation, and an I543H mutation with reference to SEQ ID NO: 414; (g) an L431S mutation, a Y432A mutation, and a P433G mutation with reference to SEQ ID NO: 414; (h) an L431S mutation with reference to SEQ ID NO: 414; Y432A mutation, P433G mutation, and A507S mutation, (i) L431S mutation, Y432A mutation, P433G mutation, and I543H mutation with reference to SEQ ID NO: 414, (j) L431S mutation and Y432A mutation with reference to SEQ ID NO: 414, (l) L431S mutation, Y432A mutation, and A507S mutation with reference to SEQ ID NO: 414, (m) L431S mutation, Y432A mutation, A507S mutation with reference to SEQ ID NO: 414, K (n) an L431S mutation, a Y432A mutation, and an I543H mutation with reference to SEQ ID NO: 414; (o) an L431S mutation and a Y432G mutation with reference to SEQ ID NO: 414; (p) an L431S mutation, a Y432G mutation, and an A507S mutation with reference to SEQ ID NO: 414; or (q) the L431S mutation, the Y432G mutation, and the I543H mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises any one of SEQ ID NOs: 415-430. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 415-430. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase.In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0006] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence comprises a D141A mutation and an E143A mutation. In some embodiments, the amino acid sequence further comprises a Y412A mutation, an L411A mutation, a P413A mutation, an A488S mutation, an I524H mutation, an L411S mutation, a P413G mutation, a K510L mutation, or a Y412G mutation, or any combination thereof with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence comprises: (a) a Y412A mutation with reference to SEQ ID NO: 431; (b) a L411A mutation, a Y412A mutation, and a P413A mutation with reference to SEQ ID NO: 431; (c) a L411A mutation, a Y412A mutation, a P413A mutation, and an A488S mutation with reference to SEQ ID NO: 431; (d) a L411A mutation, a Y412A mutation, a P413A mutation, and an I524H mutation with reference to SEQ ID NO: 431; (e) a L411S mutation, a Y412A mutation, and a P413G mutation with reference to SEQ ID NO: 431; (f) a L411S mutation, a Y412A mutation, a P413G mutation, and an A488S mutation with reference to SEQ ID NO: 431; (g) a L411S mutation, a Y412A mutation, a P413G mutation, and an I524H mutation with reference to SEQ ID NO: 431; (i) the L411S mutation, the Y412A mutation, and the A488S mutation with reference to SEQ ID NO: 431; (j) the L411S mutation, the Y412A mutation, the A488S mutation, the K510L mutation, and the I524H mutation with reference to SEQ ID NO: 431; (k) the L411S mutation, the Y412A mutation, and the I524H mutation with reference to SEQ ID NO: 431; (l) the L411S mutation and the Y412G mutation with reference to SEQ ID NO: 431; (m) the L411S mutation, the Y412G mutation, and the A488S mutation with reference to SEQ ID NO: 431; or (n) the L411S mutation, the Y412G mutation, and the I524H mutation with reference to SEQ ID NO: 431.In some embodiments, the amino acid sequence further comprises any one of SEQ ID NOs: 432-445. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 432-445. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0007] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the amino acid sequence comprises two or more of a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 447. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 447. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0008] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the amino acid sequence comprises two or more of a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 449. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 449. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0009] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the amino acid sequence comprises two or more of a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 451. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 451. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0010] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the amino acid sequence comprises two or more of a D170A mutation, an E172A mutation, and a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 453. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 453. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0011] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the amino acid sequence comprises two or more of a D170A mutation, an E172A mutation, and a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 455. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 455. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0012] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the amino acid sequence comprises two or more of a D173A mutation, an E175A mutation, a Y452A mutation, and a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 457. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 457. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0013] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the amino acid sequence comprises two or more of a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 459. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 459. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0014] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the amino acid sequence comprises two or more of a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 461. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 461. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0015] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the amino acid sequence further comprises an amino acid deletion from amino acid position I496 to amino acid position S1033, or any portion of the amino acid sequence, with reference to SEQ ID NO: 462. In some embodiments, the amino acid sequence comprises two or more of a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 463. In some embodiments, the amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 463. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0016] In some embodiments, aspects disclosed herein are methods of nucleic acid analysis, comprising providing a formulation comprising (i) a synthetic polypeptide disclosed herein, (ii) a primed nucleic acid sequence, and (ii) a nucleotide unit complementary to a nucleotide of the primed nucleic acid, wherein the formulation is provided under conditions sufficient to form a binding complex comprising the synthetic polypeptide, the nucleotide unit, and the primed nucleic acid sequence. In some embodiments, the method comprises a nucleic acid sequencing method. In some embodiments, the method comprises sequencing by synthesis. In some embodiments, the formulation further comprises one or more compositions, wherein one composition of the one or more compositions comprises (a) a core and (b) at least two nucleotide arms, wherein one nucleotide arm of the at least two nucleotide arms comprises (i) a core attachment moiety attached to the core, (ii) a spacer attached to the core attachment moiety, (iii) a linker attached to the spacer, and (iv) a nucleotide unit attached to the linker. In some embodiments, the linker is

[0017] [ka]

[0018] [ka] wherein n is 1 to 6 and m is 0 to 10.

[0019] In some embodiments herein, a kit is disclosed, the kit comprising one or more containers containing a synthetic polypeptide and a detectable nucleotide unit, and instructions for introducing the synthetic polypeptide and the nucleotide unit into a primed nucleic acid sequence containing a nucleotide complementary to the nucleotide unit under conditions sufficient to form a binding complex containing the synthetic polypeptide, the nucleotide unit, and the primed nucleic acid sequence. In some embodiments, the kit further comprises one or more compositions, wherein one of the one or more compositions comprises a core and at least two nucleotide arms, wherein one of the at least two nucleotide arms comprises a core attachment moiety attached to the core, a spacer attached to the core attachment moiety, a linker attached to the spacer, and a nucleotide unit attached to the linker. In some embodiments, the linker is

[0020] [ka]

[0021] [ka] wherein n is 1 to 6 and m is 0 to 10.

[0022] In some embodiments, the aspects disclosed herein are systems comprising a synthetic polypeptide, a primed nucleic acid sequence, and a nucleotide unit that is detectable and complementary to a nucleotide in the primed nucleic acid sequence, the system being configured to form a binding complex comprising the primed nucleic acid sequence, the synthetic polypeptide, and the nucleotide unit. In some embodiments, the system further comprises one or more compositions, wherein one composition of the one or more compositions comprises a core and at least two nucleotide arms, wherein one nucleotide arm of the at least two nucleotide arms comprises a core attachment moiety attached to the core, a spacer attached to the core attachment moiety, a linker attached to the spacer, and a nucleotide unit attached to the linker, and the binding complex comprises the primed nucleic acid sequence, the synthetic polypeptide, and the one composition. In some embodiments, the system further comprises two or more copies of the primed nucleic acid sequence and two or more synthetic polypeptides, the two or more synthetic polypeptides being configured to form a multivalent binding complex comprising two or more nucleotide units of the one composition, two or more copies of the primed nucleic acid sequence, and two or more synthetic polypeptides. In some embodiments, the linker is

[0023] [ka]

[0024] [ka] wherein n is 1 to 6 and m is 0 to 10.

[0025] An aspect disclosed herein is, in some embodiments, a system comprising: (i) a synthetic polypeptide disclosed herein; (ii) a primed nucleic acid sequence; and (iii) a nucleotide unit complementary to a nucleotide of the primed nucleic acid, provided under conditions sufficient to form a binding complex comprising the synthetic polypeptide, the nucleotide unit, and the primed nucleic acid sequence.

[0026] Aspects disclosed herein, in some embodiments, are systems comprising (i) a synthetic polypeptide disclosed herein and (ii) a nucleotide. In some embodiments, the nucleotide comprises a blocking group. In some embodiments, the nucleotide does not comprise a blocking group. In some embodiments, the nucleotide comprises a label. In some embodiments, the nucleotide is unlabeled. In some embodiments, the blocking group is linked to the 3' carbon of the sugar moiety of the nucleotide, and the blocking group reacts with a chemical compound to remove the blocking group from the nucleotide and generate a nucleotide comprising an OH moiety at the 3' carbon of the sugar moiety of the nucleotide. In some embodiments, the blocking group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group. In some embodiments, (a) the alkyl, alkenyl, alkynyl, or aryl group of the blocking group reacts with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ); (b) the aryl or benzyl group of the blocking group reacts with H and palladium on carbon (Pd / C); (c) the amine, amide, keto, isocyanate, phosphate, thiol, or aryl group of the blocking group reacts with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH)))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ); (d) the disulfide group reacts with a phosphine or thiol group containing beta-mercaptoethanol or dithiothreitol (DTT); (d) the carbonate group of the blocking group reacts with potassium carbonate (K2CO3) in methanol (MeOH), triethylamine in pyridine, or Zn in acetic acid (AcOH); and (e) the urea or silyl group of the blocking group reacts with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the azide of the blocking group comprises an azide, azide, or azidomethyl group. In some embodiments, the azide, azide, or azidomethyl group reacts with a chemical containing a phosphine compound.In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety, hi some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

[0027] Aspects disclosed herein include, in some embodiments, a composition comprising: (a) a core; and (b) at least two nucleotide arms, wherein one nucleotide arm of the at least two nucleotide arms comprises: (i) a core attachment moiety attached to the core; (ii) a spacer attached to the core attachment moiety; (iii) a linker attached to the spacer; and (iv) a nucleotide unit attached to the linker; wherein the linker is

[0028] [ka]

[0029] [ka] wherein n is 1 to 6 and m is 0 to 10. In some embodiments, the linker is

[0030] [ka] In some embodiments, the linker comprises:

[0031] [ka] In some embodiments, the linker comprises:

[0032] [ka] In some embodiments, the linker comprises:

[0033] [ka] In some embodiments, the linker comprises:

[0034] [ka] wherein n is 1 to 6 and m is 0 to 10. In some embodiments, the linker comprises:

[0035] [ka] wherein n is 1 to 6 and m is 0 to 10. In some embodiments, the linker comprises:

[0036] [ka] wherein n is 1 to 6 and m is 0 to 10. In some embodiments, the linker comprises:

[0037] [ka] wherein n is 1 to 6 and m is 0 to 10. In some embodiments, the linker comprises:

[0038] [ka] In some embodiments, the linker comprises:

[0039] [ka] In some embodiments, the linker comprises:

[0040] [ka] In some embodiments, the linker comprises:

[0041] [ka] Includes.

[0042] In some embodiments, the linker is

[0043] [ka] In some embodiments, the at least two nucleotide arms comprise between 3 and 20 nucleotide arms. In some embodiments, the core comprises a polypeptide. In some embodiments, the polypeptide comprises streptavidin or avidin. In some embodiments, the core attachment moiety comprises biotin. In some embodiments, the composition further comprises a fluorescent label attached to the core or the nucleotide arm. In some embodiments, the spacer is

[0044] [ka] wherein m is 20 to 500 and o is 1 to 10. In some embodiments, the nucleotide arm further comprises a reactive group attached to the nucleotide unit, the reactive group being configured to react with a drug. In some embodiments, the reactive group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group. In some embodiments, (a) an alkyl, alkenyl, alkynyl, or aryl group of the reactive group reacts with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ); (b) an aryl or benzyl group of the reactive group reacts with H and palladium on carbon (Pd / C); and (c) an amine, amide, keto, isocyanate, phosphate, thiol, or disulfide group of the reactive group. (d) the carbonate group of the reactive group reacts with potassium carbonate (K2CO3) in methanol (MeOH), triethylamine in pyridine, or Zn in acetic acid (AcOH); and (e) the urea or silyl group of the reactive group reacts with tetrabutylammonium fluoride, hydrogen fluoride pyridine (HF-pyridine), ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the azido group of the reactive group comprises an azide, azide, or azidomethyl group. In some embodiments, the agent comprises a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP). In some embodiments, the nucleotide units of at least two nucleotide arms comprise the same nucleobase type.In some embodiments, the nucleotide unit comprises a blocking group linked to the 3' carbon of the sugar moiety of the nucleotide unit, and the blocking group reacts with a chemical compound to remove the blocking group from the nucleotide unit and generate a nucleotide unit comprising an OH moiety at the 3' carbon of the sugar moiety of the nucleotide unit. In some embodiments, the blocking group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group. In some embodiments, (a) the alkyl, alkenyl, alkynyl, or aryl group of the blocking group reacts with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ); (b) the aryl or benzyl group of the blocking group reacts with H and palladium on carbon (Pd / C); and (c) the amine, amide, keto, isocyanate, phosphate, or thiol group of the blocking group reacts with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). (d) the carbonate group of the blocking group reacts with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH); and (e) the urea or silyl group of the blocking group reacts with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the azide of the blocking group comprises an azide, azide, or azidomethyl group. In some embodiments, the azide, azide, or azidomethyl group reacts with a chemical compound comprising a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

[0045] An aspect disclosed herein is, in some embodiments, a formulation comprising at least two compositions disclosed herein, wherein the at least two compositions comprise a first composition and a second composition, and wherein the nucleotide units of the first composition comprise a different nucleobase than the nucleotide units of the second composition.

[0046] Aspects disclosed herein include, in some embodiments, a formulation comprising at least two compositions, the at least two compositions comprising a first composition and a second composition, wherein (a) the nucleotide units of the first composition comprise a first blocking group linked to the 3' carbon of the sugar moiety, the first blocking group being reacted with a chemical compound to remove the first blocking group and generate an OH moiety on the 3' carbon of the sugar moiety of the nucleotide units of the first composition, and (b) the nucleotide units of the second composition comprise a second blocking group linked to the 3' carbon of the sugar moiety, the second blocking group being reacted with a chemical compound to remove the second blocking group and generate an OH moiety on the 3' carbon of the sugar moiety of the nucleotide units of the second composition, wherein the nucleotide units of the first composition are different from the nucleotide units of the second composition.

[0047] An aspect disclosed herein is, in some embodiments, a formulation comprising at least two compositions disclosed herein, wherein the at least two compositions comprise a first composition and a second composition, and wherein the linker of the first composition is different from the linker of the second composition.

[0048] An aspect disclosed herein is, in some embodiments, a formulation comprising at least two compositions disclosed herein, wherein the at least two compositions comprise a first composition and a second composition, wherein the linker of the first composition comprises a first reactive group and the linker of the second composition comprises a second reactive group, and the second reactive group is different from the first reactive group.

[0049] An aspect disclosed herein is, in some embodiments, a formulation comprising at least two compositions disclosed herein, the at least two compositions comprising a first composition and a second composition, the first composition comprising a first fluorophore and the second composition comprising a second fluorophore, wherein the first fluorophore emits light at a wavelength different from the wavelength of light emitted from the second fluorophore.

[0050] An aspect disclosed herein is, in some embodiments, a formulation comprising at least two compositions disclosed herein, wherein the at least two compositions comprise a first composition and a second composition, wherein the first composition comprises a fluorescent label and the second composition is unlabeled.

[0051] In some embodiments, aspects disclosed herein are kits for processing nucleic acid molecules, the kit comprising: (a) a composition disclosed herein or a formulation disclosed herein; and (b) instructions for using the composition in a nucleotide identification reaction. In some embodiments, the kit further comprises an agent that reacts with a reactive group in a linker of the composition. In some embodiments, the kit further comprises an agent that reacts with a reactive group at the 3' carbon of the sugar moiety in a nucleotide unit of the composition. In some embodiments, the kit further comprises a reagent for use in a nucleotide coupling reaction. In some embodiments, the reagent comprises a cation. In some embodiments, the kit further comprises a solution containing a cation, one or more polymerizing enzymes, one or more primer sequences, one or more unlabeled nucleotides, or any combination of (i) to (iv).

[0052] Aspects disclosed herein, in some embodiments, are systems comprising: (a) a composition disclosed herein; (b) two or more copies of a primed nucleic acid sequence, the two or more copies of the primed nucleic acid sequence comprising nucleotides complementary to nucleotide units of the composition; and (c) two or more polymerases, the system being configured to form a multivalent binding complex comprising the two or more primed nucleic acid sequences, the two or more polymerases, and the composition. In some embodiments, the multivalent binding complexes are formed under conditions such that nucleotide units of the composition are not incorporated into the two or more copies of the primed nucleic acid sequence. In some embodiments, the two or more copies of the nucleic acid sequence and the two or more copies of the nucleic acid sequence primer molecule are immobilized to a support under conditions sufficient to immobilize the multivalent binding complexes to the support. In some embodiments, a plurality of multivalent binding complexes are immobilized on the support, and the density of the plurality of multivalent binding complexes immobilized on the support is greater than or equal to 1000 bp per square millimeter (mm 2 ) per 10 2 ~10 9 In some embodiments, the plurality of multivalent binding complexes on the support are in fluid communication with each other and with a solution of reagents under conditions sufficient for the plurality of multivalent binding complexes to react with the solution of reagents in a massively parallel manner.

[0053] An aspect disclosed herein is, in some embodiments, a method of nucleic acid analysis comprising introducing a composition disclosed herein to a primed nucleic acid sequence under conditions sufficient to form a binding complex comprising (i) a nucleotide unit of the composition and (ii) a nucleotide in the primed nucleic acid sequence, wherein the nucleotide is complementary to the nucleotide unit of the composition.

[0054] An aspect disclosed herein is, in some embodiments, a method of nucleic acid analysis comprising introducing a composition disclosed herein to two or more copies of a primed nucleic acid sequence under conditions sufficient to form a multivalent binding complex comprising (i) two or more nucleotide units of the composition and (ii) two or more nucleotides in the two or more copies of the primed nucleic acid sequence, wherein the two or more nucleotides are complementary to the two or more nucleotide units of the composition.

[0055] Aspects disclosed herein are, in some embodiments, synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0056] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0057] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0058] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0059] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0060] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0061] Aspects disclosed herein are, in some embodiments, synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0062] Aspects disclosed herein are, in some embodiments, synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0063] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0064] Aspects disclosed herein, in some embodiments, are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0065] Aspects disclosed herein are, in some embodiments, a synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0066] Aspects disclosed herein are, in some embodiments, synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0067] Aspects disclosed herein are, in some embodiments, synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

[0068] Incorporation by Reference All publications, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, this specification is intended to supersede and / or replace any such conflicting material. [Brief explanation of the drawings]

[0069] The novel features of the present method and system are set forth with particularity in the appended claims. The features and advantages of the present method and system will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the method and system are utilized, and the accompanying drawings.

[0070] [Figure 1]1 is a schematic diagram of a non-limiting example of a nucleotide conjugate comprising a generic core conjugated to multiple nucleotide arms, according to some embodiments herein. In some embodiments, each nucleotide arm comprises a spacer, a linker, and a nucleotide unit. [Figure 2] 1 is a schematic diagram of a non-limiting example of a nucleotide conjugate comprising a dendrimer core conjugated to multiple nucleotide arms, according to some embodiments herein, in which each nucleotide arm comprises a spacer, a linker, and a nucleotide unit. [Figure 3] 1 shows a schematic diagram of a non-limiting example of a nucleotide conjugate according to some embodiments herein, comprising a core attached / linked to multiple nucleotide arms, each nucleotide arm comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit. [Figure 4] 1 is a schematic diagram of a non-limiting example of a nucleotide arm comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit, according to some embodiments herein. [Figure 5A] FIG. 1 shows chemical structures of non-limiting examples of spacers at the top and linkers at the bottom, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker, according to some embodiments herein. [Figure 5B] FIG. 1 shows chemical structures of non-limiting examples of linkers, including linkers 1-6, according to some embodiments herein. [Figure 5C] FIG. 1 shows chemical structures of non-limiting examples of linkers, including linkers 7-9, according to some embodiments herein. [Figure 5D] FIG. 1 shows chemical structures of non-limiting examples of linkers including linkers 10 and 11 connected to nucleotide units, according to some embodiments herein. [Figure 5E]FIG. 1 shows chemical structures of non-limiting examples of linkers including linkers 12 and 13 connected to nucleotide units, according to some embodiments herein. [Figure 5F] FIG. 1 shows chemical structures of non-limiting examples of linkers including linkers 14-16 connected to nucleotide units, according to some embodiments herein. [Figure 6A] FIG. 1 shows the chemical structures of non-limiting examples of nucleotide arms, including a spacer connected to a linker and a linker connected to a nucleotide unit, according to some embodiments herein. [Figure 6B] FIG. 1 shows the chemical structures of non-limiting examples of nucleotide arms, including a spacer connected to a linker and a linker connected to a nucleotide unit, according to some embodiments herein. [Figure 7A] FIG. 1 shows chemical structures of non-limiting examples of linkers tethered to nucleotides by propargylamine attachment, according to some embodiments herein. [Figure 7B] 10A-10C depict additional chemical structures of non-limiting examples of linkers tethered to nucleotides by propargylamine attachment, according to some embodiments herein. [Figure 7C] 10A-10C depict additional chemical structures of non-limiting examples of linkers tethered to nucleotides by propargylamine attachment, according to some embodiments herein. [Figure 7D] 10A-10C depict additional chemical structures of non-limiting examples of linkers tethered to nucleotides by propargylamine attachment, according to some embodiments herein. [Figure 7E] 10A-10C depict additional chemical structures of non-limiting examples of linkers tethered to nucleotides by propargylamine attachment, according to some embodiments herein. [Figure 7F] 10A-10C depict additional chemical structures of non-limiting examples of linkers tethered to nucleotides by propargylamine attachment, according to some embodiments herein. [Figure 7G]10A-10C depict additional chemical structures of non-limiting examples of linkers tethered to nucleotides by propargylamine attachment, according to some embodiments herein. [Figure 8A] 1 shows the chemical structure of a non-limiting example of a biotinylated nucleotide arm comprising a biotin moiety, a spacer, a linker (e.g., an 11-atom linker), and a nucleotide unit, according to some embodiments herein. In this non-limiting example, the nucleotide unit is connected to the linker by a propargylamine attachment at the 5-position of the pyrimidine base, or the 7-position of the purine base, or the 1-position of the pyrimidine base, or the 9-position of the purine base. [Figure 8B] 1 shows the chemical structure of a non-limiting example of a biotinylated nucleotide arm comprising a biotin moiety, a spacer, a linker (e.g., Linker 6), and a nucleotide unit, according to some embodiments herein. In this non-limiting example, the nucleotide unit is connected to the linker by a propargylamine attachment at the 5-position of the pyrimidine base, or the 7-position of the purine base, or the 1-position of the pyrimidine base, or the 9-position of the purine base. [Figure 9] 1 is a bar graph showing the results of capture assays performed by reacting various fluorescently labeled nucleotide conjugates with the corresponding correct DNA templates. [Figure 10] 1 is a bar graph showing the results of a capture assay in which increasing concentrations of various fluorescently labeled nucleotide conjugates were reacted with the corresponding correct DNA template. [Figure 11]

[0023] Figure 1 provides four graphs showing the results of a capture assay comparing the signal intensity of fluorescently labeled nucleotide conjugates having nucleotide arms containing either the N3 linker, Linker 6, Linker 8, or propargyl linker. The nucleotide conjugates were labeled with CF680 or CF532 fluorophores. Two different concentrations of nucleotide conjugate were tested (20 nM and 80 nM). The graphs show the capture time in seconds (x-axis) and P90 signal intensity (y-axis). [Figure 12]

[0023] Figure 1 provides four graphs showing the results of a capture assay comparing the signal intensity of fluorescently labeled nucleotide conjugates having nucleotide arms containing either the N3 linker, Linker 6, Linker 8, or propargyl linker. The nucleotide conjugates were labeled with AF647 or CF570 fluorophores. Two different concentrations of nucleotide conjugate were tested (20 nM and 80 nM). The graphs show the capture time in seconds (x-axis) and P90 signal intensity (y-axis). [Figure 13] Figure 1 provides three graphs showing the results of a real-time imaging capture kinetics assay comparing the signal intensity of fluorescently labeled nucleotide conjugates with nucleotide arms containing linkers 6 or one of 10-16. Three different concentrations of nucleotide conjugates were tested (15 nM, 7.5 nM, and 2.5 nM). The graphs show capture time in seconds (x-axis) and signal intensity (y-axis). [Figure 14] 14 is a graph showing the results of binding kinetics studies for fluorescently labeled nucleotide conjugates having nucleotide arms containing linkers 6 or one of 10-16. The graph shows nucleotide conjugate concentration (x-axis, nM) and rate (y-axis). The legend shown in Figure 14 is also applicable to Figure 13. [Figure 15] FIG. 1 is a bar graph showing the binding constants (K) determined for fluorescently labeled nucleotide conjugates having nucleotide arms containing linkers 6 or one of 10-16. [Figure 16] 2 shows a 2.8 Å model determined from X-ray crystallography of a sequencing polymerase co-crystallized with a template molecule hybridized to a primer having a 3′-terminal di-deoxyribonucleotide and a nucleotide conjugate comprising a nucleotide arm having a linker 6 and a dCTP nucleotide unit. The model shows two magnesium ions (spheres) in the active site of the polymerase. [Figure 17]FIG. 1 is a diagram of the amino acid sequence of a non-limiting example of a streptavidin subunit (SEQ ID NO: 466). [Figure 18] FIG. 1 is a diagram of the amino acid sequence of a non-limiting example of a streptavidin subunit (SEQ ID NO: 467). [Figure 19] FIG. 1 is a diagram of the amino acid sequence of a non-limiting example of a streptavidin subunit (SEQ ID NO: 468). [Figure 20] FIG. 1 is a diagram of the amino acid sequence of a non-limiting example of a streptavidin subunit (SEQ ID NO: 469). [Figure 21] FIG. 1 is a diagram of the amino acid sequence of a non-limiting example of a streptavidin subunit (SEQ ID NO: 470). [Figure 22] FIG. 1 is a diagram of the amino acid sequence of a non-limiting example of an avidin subunit (SEQ ID NO: 471). [Figure 23] FIG. 1 is a diagram of the amino acid sequence of a non-limiting example of an avidin subunit (SEQ ID NO: 472). [Figure 24] FIG. 1 is a diagram of the amino acid sequence (SEQ ID NO: 1) of the wild-type DNA polymerase of Candidatus altiarchaeales archaeon (GenBank accession number RLI89578.1). [Figure 25] FIG. 1 is a diagram of the amino acid sequence (SEQ ID NO: 464) of the wild-type DNA polymerase of Candidatus altiarchaeales archaeon (GenBank Accession No. OYT41123.1). [Figure 26] FIG. 1 is a diagram of the amino acid sequence of the N-terminal domain of the DNA polymerase of Candidatus altiarchaeales archaeon RLI 89578.1 (SEQ ID NO: 269). [Figure 27] FIG. 1 is a diagram of the amino acid sequence of the exonuclease domain of the DNA polymerase of Candidatus altiarchaeales archaeon RLI 89578.1 (SEQ ID NO: 270). [Figure 28]FIG. 1 is a diagram of the amino acid sequence of the palm(1) domain of the DNA polymerase of Candidatus altiarchaeales archaeon RLI 89578.1 (SEQ ID NO: 271). [Figure 29] FIG. 1 is a diagram of the amino acid sequence of the fingers domain of the DNA polymerase of Candidatus altiarchaeales archaeon RLI 89578.1 (SEQ ID NO: 272). [Figure 30] FIG. 1 is a diagram of the amino acid sequence of the palm(2) domain of the DNA polymerase of Candidatus altiarchaeales archaeon RLI 89578.1 (SEQ ID NO: 273). [Figure 31] FIG. 1 is a diagram of the amino acid sequence of the thumb domain of the DNA polymerase of Candidatus altiarchaeales archaeon RLI 89578.1 (SEQ ID NO: 274). [Figure 32] FIG. 1 is a diagram of the amino acid sequence of 9°N polymerase (SEQ ID NO: 280). [Figure 33] FIG. 2 is a diagram of the amino acid sequence of 9°N polymerase UniProtKB-Q56366 (DPOL_THES9) (SEQ ID NO: 281). [Figure 34] FIG. 1 is a diagram of the amino acid sequence of THERMINATOR polymerase (SEQ ID NO: 282). [Figure 35] FIG. 1 is a diagram of the amino acid sequence of Pyrococcus abyssi DNA polymerase (SEQ ID NO: 286). [Figure 36] FIG. 1 is a diagram of the amino acid sequence of VENT polymerase (SEQ ID NO: 283). [Figure 37] FIG. 1 is a diagram of the amino acid sequence of DEEP VENT polymerase (SEQ ID NO: 284). [Figure 38] FIG. 1 is a diagram of the amino acid sequence of Pfu polymerase (SEQ ID NO: 285). [Figure 39] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase of Geobacillus stearothermophilus (SEQ ID NO: 275). [Figure 40] FIG. 1 is a diagram of the amino acid sequence of RB69 polymerase (SEQ ID NO: 287). [Figure 41A] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase of Thermococci archaeon (SEQ ID NO: 391) with the framework sequence of RLF 89458.1. [Figure 41B] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase of Thermococci archaeon (SEQ ID NO: 465) with the backbone sequence of RLF 78286.1. [Figure 42] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase of Thermoplasmata archaeon (SEQ ID NO: 414) with the backbone sequence of RLF 60390.1. [Figure 43] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase of Thermococcus sp. 2319x1 (SEQ ID NO: 431) with the backbone sequence of WP 175059460.1. [Figure 44] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase of Thermococcus litoralis (SEQ ID NO: 446) with the backbone sequence of ADK 47977.1. [Figure 45] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase of the archaeal BMS3Abin16 (SEQ ID NO: 448) with the backbone sequence of GBE 17769.1. [Figure 46] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase of the archaeal BMS3Bbin16 (SEQ ID NO: 450) with the backbone sequence of GBE 55812.1. [Figure 47] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase of Candidatus Hadarchaeum yellowstonense (SEQ ID NO: 452) with the framework sequence of KUO 42443.1. [Figure 48] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase having the backbone sequence of KXB 02540.1 (SEQ ID NO: 454). [Figure 49]FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase (SEQ ID NO: 456) with the backbone sequence of MBC 7218772.1. [Figure 50] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase (SEQ ID NO: 458) with the backbone sequence of RMF 90817.1. [Figure 51] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase (SEQ ID NO: 460) with the framework sequence of WP 058946753.1. [Figure 52] FIG. 1 is a diagram of the amino acid sequence of the wild-type DNA polymerase (SEQ ID NO: 462) with the framework sequence of WP 167886206.1. [Figure 53] FIG. 1 is a block diagram depicting an exemplary machine comprising a computer system (100) for nucleic acid processing. [Figure 54] FIG. 1 is a diagram of an embodiment of an application provision system. [Figure 55] FIG. 2 is a diagram of another embodiment of an application provision system. DETAILED DESCRIPTION OF THE INVENTION

[0071] Some embodiments disclosed herein include compositions, systems, and kits for use in processing and / or analyzing nucleic acid sequences. Methods for processing and / or analyzing nucleic acid sequences using the compositions, systems, and kits disclosed herein are also provided. In some embodiments, the inventive concepts disclosed herein utilize nucleotide conjugates comprising a core to which is attached one or more nucleotide units that are complementary to nucleotides in the nucleic acid sequence to be processed or analyzed. Such nucleotide conjugates are capable of forming multivalent binding complexes comprising the nucleotide conjugate attached to the nucleotide of a nucleic acid sequence and, optionally, a polymerizing enzyme. In some embodiments, the inventive concepts disclosed herein utilize synthetic polymerizing enzymes with one or more mutations in their amino acid sequences that enhance binding to the disclosed nucleotides or nucleotide units compared to an otherwise identical polymerizing enzyme lacking the one or more mutations.

[0072] composition In some embodiments, a composition is disclosed herein. In embodiments, the composition can be used in any of the methods, formulations, or systems disclosed herein. In some embodiments, the composition can be used in the nucleic acid processing, analysis, identification, or detection methods disclosed herein. In some embodiments, the composition can be used with a polypeptide disclosed herein. In some embodiments, the composition can be or comprise a nucleotide conjugate disclosed herein. In some embodiments, the composition can be capable of forming a binding complex with a primed nucleic acid sequence and a polypeptide disclosed herein. In some embodiments, the composition can be capable of forming a multivalent binding complex with two or more primed nucleic acid sequences and two or more polypeptides disclosed herein.

[0073] The present disclosure provides compositions, systems, methods, and kits that include nucleotide conjugates. In some embodiments, the nucleotide conjugates can include a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can include the same type of nucleotide unit. For example, the nucleotide conjugates can include a core (e.g., a streptavidin core or an avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the attached arms have a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0074] The present disclosure provides compositions, systems, methods, and kits that include nucleotide conjugates. In some embodiments, the nucleotide conjugates can include a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can include different types of nucleotide units. For example, the nucleotide conjugates can include a core (e.g., a streptavidin core or an avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where at least a first attached arm can have a first nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, and a second attached arm can have a second nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, where the first nucleotide unit and the second nucleotide unit are different.

[0075] The present disclosure provides compositions, systems, methods, and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise the same type of spacer. For example, the nucleotide conjugates can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the attached arms have the same spacer. In some embodiments, the spacer can be selected from any of the spacers described herein (e.g., FIG. 5A (top)).

[0076] The present disclosure provides compositions, systems, methods, and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise different types of spacers. For example, the nucleotide conjugates can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where at least a first attached arm can have a first type of spacer and a second attached arm can have a second type of spacer, and the first spacer unit and the second spacer unit are different. In some embodiments, the first and second types of linkers can be selected from any of the spacers described herein (e.g., FIG. 5A (top)).

[0077] The present disclosure provides compositions, systems, methods, and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise the same type of linker. For example, the nucleotide conjugates can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the attached arms have the same linker. In some embodiments, the linker can be selected from any of the linkers described herein (e.g., Figure 5A (bottom) and Figures 5B-5F).

[0078] The present disclosure provides compositions, systems, methods, and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise different types of linkers. For example, the nucleotide conjugates can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where at least a first attached arm can have a first type of linker and a second attached arm can have a second type of linker, and the first and second linker units are different. In some embodiments, the first and second types of linkers can be selected from any of the linkers described herein (e.g., Figure 5A (bottom) and Figures 5B-5F).

[0079] The present disclosure provides compositions, systems, methods, and kits that include nucleotide conjugates. In some embodiments, the nucleotide conjugates can include a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can include the same type of spacer and linker. For example, the nucleotide conjugates can include a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the attached arms have the same spacer and linker. In some embodiments, the spacer and linker can be selected from any of the spacers and linkers described herein.

[0080] The present disclosure provides compositions, systems, methods, and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise the same type of reactive group. For example, the nucleotide conjugates can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the attached arms have the same reactive group. In some embodiments, the reactive group can comprise an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0081] In some embodiments, the reactive groups in the linker can react with chemical reagents. For example, alkyl, alkenyl, alkynyl, and allyl reactive groups can react with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl reactive groups can react with H and palladium on carbon (Pd / C). Amine, amide, keto, isocyanate, phosphate, thiol, and disulfide reactive groups can react with phosphines or thiol groups containing β-mercaptoethanol or dithiothritol (DTT). Carbonate reactive groups can react with potassium carbonate (KCO) in methanol (MeOH), triethylamine in pyridine, or Zn in acetic acid (AcOH). The reactive groups urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0082] In some embodiments, the nucleotide arms can have the same type of reactive group in the linker, in which case the reactive group can comprise an azide, azide, or azidomethyl group. In some embodiments, the azide, azide, or azidomethyl group in the linker can react with a chemical. In some embodiments, the chemical can comprise a phosphine compound. In some embodiments, the phosphine compound can comprise a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound can comprise tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0083] The present disclosure provides compositions, systems, methods, and kits that include nucleotide conjugates. In some embodiments, the nucleotide conjugates can include a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can include different types of reactive groups in the linker. For example, the nucleotide conjugates can include a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where at least a first attached arm can have a first type of reactive group in the first linker unit, and a second attached arm can have a second type of reactive group in the second linker unit, and the first reactive group and the second reactive group are different.

[0084] In some embodiments, the first reactive group in the first linker unit and the second reactive group in the second linker unit can be selected from the group consisting of alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azide groups, amine groups, amide groups, keto groups, isocyanate groups, phosphate groups, thio groups, disulfide groups, carbonate groups, urea groups, and silyl groups, in any combination.

[0085] In some embodiments, the first and second reactive groups can react with chemicals. For example, alkyl, alkenyl, alkynyl, and allyl reactive groups can react with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl reactive groups can react with H and palladium on carbon (Pd / C). Amine, amide, keto, isocyanate, phosphate, thiol, and disulfide reactive groups can react with phosphines or thiol groups containing β-mercaptoethanol or dithiothritol (DTT). Carbonate reactive groups can react with potassium carbonate (KCO) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The reactive groups urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0086] In some embodiments, the nucleotide arms can have different types of reactive groups in the linker, where the reactive group can include an azide, azide, or azidomethyl group. In some embodiments, the azide, azide, or azidomethyl group in the linker can react with a chemical. In some embodiments, the chemical can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0087] The present disclosure provides compositions, systems, methods, and kits that include nucleotide conjugates. In some embodiments, the nucleotide conjugates can include a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can include nucleotide units with the same type of sugar 3'OH group. For example, the nucleotide conjugates can include a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, in which case all the attached arms have nucleotide units with the same type of sugar 3'OH group.

[0088] The present disclosure provides compositions, systems, methods, and kits comprising nucleotide conjugates. In some embodiments, a nucleotide conjugate can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise nucleotide units having the same type of sugar 3' blocking group (e.g., chain terminating moiety). For example, a nucleotide conjugate can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, in which case all of the attached arms can have nucleotide units with the same type of sugar 3' blocking group. In some embodiments, the sugar 3' blocking group can comprise an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the sugar 3' blocking group can comprise a 3'-O-alkylhydroxylamino group, a 3'-phosphorothioate group, a 3'-O-malonyl group, or a 3'-O-benzyl group. In some embodiments, the sugar 3' blocking group can comprise an azide, azido, or azidomethyl group.

[0089] In some embodiments, sugar 3' blocking groups can be reacted with chemical reagents. For example, alkyl, alkenyl, alkynyl, and allyl sugar 3' blocking groups can be reacted with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl sugar 3' blocking groups can be reacted with H and palladium on carbon (Pd / C). Amine, amide, keto, isocyanate, phosphate, thiol, and disulfide sugar 3' blocking groups can be reacted with phosphines or thiol groups containing β-mercaptoethanol or dithiothritol (DTT). Carbonate sugar 3' blocking groups can be reacted with potassium carbonate (KCO) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The sugar 3' blocking groups, urea and silyl, can be reacted with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0090] In some embodiments, sugar 3' blocking groups (e.g., azide, azide, and azidomethyl) can be reacted with a chemical entity. In some embodiments, the chemical entity can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0091] The present disclosure provides compositions, systems, methods, and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates can comprise a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can comprise nucleotide units with different sugar 3' blocking groups. For example, the nucleotide conjugates can comprise a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where at least a first attached arm can have a first nucleotide unit with a first 3' blocking group, and a second attached arm can have a second nucleotide unit with a second 3' blocking group, where the first 3' blocking group and the second 3' blocking group are different.

[0092] In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl groups, in any combination. In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of 3'-O-alkylhydroxylamino, 3'-phosphorothioate, 3'-O-malonyl, or 3'-O-benzyl groups, in any combination. In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of azide, azido, or azidomethyl groups, in any combination.

[0093] In some embodiments, the first and second 3'-blocking groups can react with chemical reagents. For example, alkyl, alkenyl, alkynyl, and allyl 3'-blocking groups can react with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl 3'-blocking groups can react with H and palladium on carbon (Pd / C). Amine, amide, keto, isocyanate, phosphate, thiol, and disulfide 3'-blocking groups can react with phosphines or thiol groups containing β-mercaptoethanol or dithiothritol (DTT). Carbonate 3'-blocking groups can react with potassium carbonate (KCO) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The 3' blocking groups urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0094] In some embodiments, the first and second 3' blocking groups (e.g., azide, azide, and azidomethyl) can be reacted with a chemical entity. In some embodiments, the chemical entity can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0095] The present disclosure provides compositions, systems, methods, and kits that include nucleotide conjugates. In some embodiments, the nucleotide conjugates can include a core attached to multiple nucleotide arms. In some embodiments, the multiple nucleotide arms can include nucleotide units with a first sugar 3'OH blocking group. In some embodiments, the multiple nucleotide arms can include nucleotide units with a second sugar 3'OH blocking group. In some cases, the first and second 3'OH blocking groups can be different. For example, a nucleotide conjugate can include a core (e.g., a streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, where (a) at least a first arm can include a first nucleotide unit having a sugar moiety that includes a 3' OH group, (b) at least a second arm can include a second nucleotide unit having a first 3' blocking group, and (c) at least a third arm can include a third nucleotide unit having a second blocking group, wherein the first 3' blocking group and the second 3' blocking group are different from each other.

[0096] In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl groups, in any combination. In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of 3'-O-alkylhydroxylamino, 3'-phosphorothioate, 3'-O-malonyl, or 3'-O-benzyl groups, in any combination. In some embodiments, the first 3' blocking group in the first nucleotide unit and the second 3' blocking group in the second nucleotide unit can be selected from the group consisting of azide, azido, or azidomethyl groups, in any combination.

[0097] In some embodiments, the first and second 3'-blocking groups can react with chemical reagents. For example, alkyl, alkenyl, alkynyl, and allyl 3'-blocking groups can react with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl 3'-blocking groups can react with H and palladium on carbon (Pd / C). Amine, amide, keto, isocyanate, phosphate, thiol, and disulfide 3'-blocking groups can react with phosphines or thiol groups containing β-mercaptoethanol or dithiothritol (DTT). Carbonate 3'-blocking groups can react with potassium carbonate (KCO) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The 3' blocking groups urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0098] In some embodiments, the first and second 3' blocking groups (e.g., azide, azide, and azidomethyl) can be reacted with a chemical entity. In some embodiments, the chemical entity can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0099] The present disclosure provides compositions, systems, methods, and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates can have a core. In some embodiments, the core can be labeled with at least one detectable reporter moiety to form a labeled core. In some embodiments, the labeled core attached to two or more nucleotide arms can comprise a labeled nucleotide conjugate. In some embodiments, a streptavidin or avidin core can be labeled with one to six or more reporter moieties. In some embodiments, the reporter moiety can comprise a fluorophore.

[0100] A mixture of nucleotide conjugates having different units in the nucleotide arms, which can be distinguished from the different nucleotide conjugates. In some embodiments, the core of a first nucleotide conjugate is labeled with a reporter moiety, thereby distinguishing it from a second labeled (or unlabeled) nucleotide conjugate. For example, the units in the nucleotide arms of a labeled first nucleotide conjugate can be different from the units in the nucleotide arms of a labeled second nucleotide conjugate. Any unit in the first nucleotide conjugate (e.g., a spacer, a linker, a reactive group, a nucleotide base, a sugar 3'OH, a 3' blocking group, or a combination thereof) can be different from the corresponding unit in the second nucleotide conjugate, and the first and second reporter moieties correspond to distinct units. In some embodiments, the first and second reporter moieties can be spectrally distinguishable from each other. In some embodiments, the core of the first nucleotide conjugate can be labeled with a first reporter moiety corresponding to the base in the attached nucleotide arm (e.g., dATP, dGTP, dCTP, dTTP, or dUTP), and the core of the second nucleotide conjugate can be labeled with a second reporter moiety corresponding to the base in the attached nucleotide arm (e.g., dATP, dGTP, dCTP, dTTP, or dUTP), where the base in the first nucleotide conjugate and the base in the second nucleotide conjugate are different. In some embodiments, the first and second reporter moieties are spectrally distinguishable from each other. In some embodiments, detection of the first reporter moiety indicates a binding event, incorporation event, or a combination of a binding event and an incorporation event of the first nucleotide conjugate having the first base, and detection of the second reporter moiety indicates a binding event, incorporation event, or a combination of a binding event and an incorporation event of the second nucleotide conjugate having the second base. The binding event can be the binding of a nucleotide conjugate to a multiplex polymerase.The incorporation event can be the incorporation of a nucleotide unit onto the 3' end of an extendible primer in a multiplex polymerase, where the nucleotide unit is part of a nucleotide conjugate.

[0101] Synthetic Polypeptides In some embodiments, synthetic polypeptides are disclosed herein. In some embodiments, the synthetic polypeptides are purified or isolated. In some embodiments, the synthetic polypeptides are polymerizing enzymes. In some embodiments, the polymerizing enzymes include nucleic acid polymerases or polymerizing portions thereof. In some embodiments, the polymerizing enzymes include DNA polymerases or polymerizing portions thereof. In some embodiments, the polymerizing enzymes include RNA polymerases or polymerizing portions thereof. In some embodiments, the synthetic polypeptides may be used in formulations, systems, or methods disclosed herein. For example, the synthetic polypeptides may be used in methods of nucleic acid sequence analysis, sequencing, identification, or processing, such as those disclosed herein.

[0102] The synthetic polypeptides disclosed herein comprise an amino acid sequence. In some embodiments, the amino acid sequence is about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% identical to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. In some embodiments, the amino acid sequence comprises one or more mutations. In some embodiments, the one or more mutations improve binding to a nucleotide or nucleotide unit disclosed herein compared to an otherwise identical synthetic polypeptide lacking the one or more mutations. In some embodiments, binding is improved by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an otherwise identical synthetic polypeptide lacking the one or more mutations. In some embodiments, binding is improved by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to an otherwise identical synthetic polypeptide lacking the one or more mutations. Affinity (e.g., strength of nucleotide binding by a synthetic polypeptide) can be determined by measuring the dissociation constant (K) of the binding complex formed between the nucleotide and the synthetic polypeptide using a suitable technique, such as that provided in Wu, J., de Paz, A., Zamft, BM et al., "DNA binding strength increases the processivity and activity of a Y-Family DNA polymerase." Sci Rep 7, 4756 (2017), which is incorporated herein by reference in its entirety. d ) can be measured by calculating

[0103] The one or more mutations may be a substitution, insertion, deletion, or chemical modification of one or more amino acids in the amino acid sequence. The amino acids disclosed herein may also be referred to by the one-letter or three-letter codes set forth in Table 6 below.

[0104] [Table 1]

[0105] Missense amino acid substitutions are sometimes designated A1#A2, where A1 is the amino acid at amino acid position # that is substituted with amino acid A2. For example, W26C indicates that amino acid 26 (tryptophan, W) is changed to cysteine ​​(C) in the base sequence. In contrast, nonsense substitutions are designated A1#X, where A1 is the amino acid at amino acid position # that is substituted with the stop codon X. Deletions are designated by "del" after the amino acids adjacent to the deletion site. For example, K29del indicates the deletion of a lysine (K) at amino acid position 29 in the base sequence. Insertions are designated by "ins" between the amino acids adjacent to the insertion site, followed by the inserted amino acid. For example, K29_M30insQSK indicates the insertion of the amino acid sequence QSK between the lysine (K) at amino acid position 29 and the methionine (M) at amino acid position 30 in the base sequence. In some embodiments, the base sequence is any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 452, 454, 456, 458, 460, and 462.

[0106] In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 71% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 72% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 73% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 74% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 76% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 77% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 78% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 79% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:1, wherein the amino acid sequence comprises one or more mutations listed in Table 1A for SEQ ID NO:1.

[0107] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391.Disclosed herein in some embodiments are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:391, wherein the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO:391.

[0108] In some embodiments, the amino acid sequence comprises a D141A mutation and a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y410A mutation, a L409S mutation, a Y261A mutation, a P411G mutation, a F406I mutation, a P411A mutation, a Y7A mutation, a Y493I mutation, a Y493T mutation, a V513I mutation, a L409A mutation, an A485S mutation, a Y410G mutation, a I521H mutation, or a K507L mutation, or any combination thereof with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y410A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a L409S mutation, a Y410A mutation, and a Y261A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises an L409S mutation, a Y410A mutation, a P411G mutation, and a Y261A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, an F406I mutation, an L409S mutation, a Y410A mutation, and a P411A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y7A mutation, a Y261A mutation, and a Y410A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a Y410A mutation, and a Y493I mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a Y410A mutation, and a Y493T mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a Y410A mutation, and a V513I mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y7A mutation, a Y261A mutation, a L409S mutation, and a Y410A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409A mutation, a Y410A mutation, and a P411A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, a Y410A mutation, and a P411G mutation with reference to SEQ ID NO: 391.In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, and a Y410A mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, and a Y410G mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409A mutation, a Y410A mutation, a P411A mutation, and an A485S mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, a Y410A mutation, a P411G mutation, and an A485S mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, a Y410A mutation, a P411G mutation, and an A485S mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, a Y410G mutation, and an A485S mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409A mutation, a Y410A mutation, a P411A mutation, and an I521H mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, a Y410A mutation, a P411G mutation, and an I521H mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, a Y410A mutation, and an I521H mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, a Y410A mutation, and an I521H mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, a L409S mutation, a Y410A mutation, a A485S mutation, a K507L mutation, and a I521H mutation with reference to SEQ ID NO: 391. In some embodiments, the amino acid sequence further comprises any one of SEQ ID NOs: 392-413.

[0109] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414.Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO:414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO:414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO:414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO:414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO:414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO:414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:414, wherein the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO:414.

[0110] In some embodiments, the amino acid sequence comprises a D158A mutation and an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431A mutation, a Y432A mutation, a P433I mutation, an A507S mutation, a K506Q mutation, a P433A mutation, a I543H mutation, a L431S mutation, a P433G mutation, a K529L mutation, or a Y432G mutation, or any combination thereof with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431A mutation, a Y432A mutation, a P433I mutation, and a A507S mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431A mutation, a Y432A mutation, a P433I mutation, and a K506Q mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431A mutation, a Y432A mutation, a P433I mutation, a K506Q mutation, and an A507S mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431A mutation, a Y432A mutation, and a P433A mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431A mutation, a Y432A mutation, a P433A mutation, and an A507S mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431A mutation, a Y432A mutation, a P433A mutation, and an I543H mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431S mutation, a Y432A mutation, and a P433G mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431S mutation, a Y432A mutation, a P433G mutation, and an A507S mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431S mutation, a Y432A mutation, a P433G mutation, and an I543H mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431S mutation and a Y432A mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431S mutation, a Y432A mutation, and an A507S mutation with reference to SEQ ID NO: 414.In some embodiments, the amino acid sequence further comprises a L431S mutation, a Y432A mutation, an A507S mutation, a K529L mutation, and an I543H mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431S mutation, a Y432A mutation, and an I543H mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431S mutation and a Y432G mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431S mutation, a Y432G mutation, and an A507S mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises a L431S mutation, a Y432G mutation, and an I543H mutation with reference to SEQ ID NO: 414. In some embodiments, the amino acid sequence further comprises any one of SEQ ID NOs: 415-430.

[0111] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431.Disclosed herein in some embodiments are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO:431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO:431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO:431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO:431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO:431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO:431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:431, wherein the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO:431.

[0112] In some embodiments, the amino acid sequence comprises a D141A mutation and an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a Y412A mutation, a L411A mutation, a P413A mutation, an A488S mutation, a I524H mutation, a L411S mutation, a P413G mutation, a K510L mutation, or a Y412G mutation, or any combination thereof with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a Y412A mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411A mutation, a Y412A mutation, and a P413A mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411A mutation, a Y412A mutation, a P413A mutation, and a A488S mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411A mutation, a Y412A mutation, a P413A mutation, and an I524H mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411S mutation, a Y412A mutation, and a P413G mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411S mutation, a Y412A mutation, a P413G mutation, and an A488S mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411S mutation, a Y412A mutation, a P413G mutation, and an I524H mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411S mutation and a Y412A mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411S mutation, a Y412A mutation, and an A488S mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411S mutation, a Y412A mutation, an A488S mutation, a K510L mutation, and an I524H mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411S mutation, a Y412A mutation, and an I524H mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence comprises a L411S mutation and a Y412G mutation with reference to SEQ ID NO: 431.In some embodiments, the amino acid sequence further comprises a L411S mutation, a Y412G mutation, and an A488S mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises a L411S mutation, a Y412G mutation, and an I524H mutation with reference to SEQ ID NO: 431. In some embodiments, the amino acid sequence further comprises any one of SEQ ID NOs: 432-445.

[0113] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO:446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO:446.Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the amino acid sequence comprises two or more of a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the amino acid sequence comprises a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the amino acid sequence further comprises SEQ ID NO:447.

[0114] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448.Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the amino acid sequence comprises two or more of a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the amino acid sequence comprises three or more of a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 449.

[0115] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450.Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the amino acid sequence comprises two or more of a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the amino acid sequence comprises three or more of a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 451.

[0116] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452.Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the amino acid sequence comprises two or more of a D170A mutation, an E172A mutation, and a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the amino acid sequence comprises a D170A mutation, an E172A mutation, and a Y449A mutation with reference to SEQ ID NO: 452. In some embodiments, the amino acid sequence further comprises SEQ ID NO:453.

[0117] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454.Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the amino acid sequence comprises two or more of a D170A mutation, an E172A mutation, and a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the amino acid sequence comprises a D170A mutation, an E172A mutation, and a Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the amino acid sequence further comprises SEQ ID NO:455.

[0118] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456.Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the amino acid sequence comprises two or more of a D173A mutation, an E175A mutation, a Y452A mutation, and a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the amino acid sequence comprises three or more of a D173A mutation, an E175A mutation, a Y452A mutation, and a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, and a C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 457.

[0119] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458.Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the amino acid sequence comprises two or more of a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the amino acid sequence comprises three or more of a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, and a Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 459.

[0120] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460.Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the amino acid sequence comprises two or more of a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the amino acid sequence comprises a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the amino acid sequence further comprises SEQ ID NO:461.

[0121] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462.Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein in some embodiments is a synthetic polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the amino acid sequence further comprises an amino acid deletion from amino acid position 1496 to amino acid position S1033, or any portion of the amino acid sequence, with reference to SEQ ID NO: 462. In some embodiments, the amino acid sequence comprises two or more of a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the amino acid sequence comprises a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the amino acid sequence comprises (i) an amino acid deletion from amino acid position I496 to amino acid position S1033, or any portion of that amino acid sequence, with reference to SEQ ID NO: 462, and (ii) two or more of a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the amino acid sequence comprises (i) an amino acid deletion from amino acid position I496 to amino acid position S1033, or any portion of that amino acid sequence, with reference to SEQ ID NO: 462, and (ii) a D141A mutation, an E143A mutation, and a Y412A mutation with reference to SEQ ID NO: 462. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 463.

[0122] Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 71% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 72% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 73% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 74% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 76% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 77% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 78% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein is a synthetic polypeptide comprising an amino acid sequence having at least 79% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 81% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 82% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, a synthetic polypeptide comprises an amino acid sequence having at least 83% sequence identity to any one of SEQ ID NOs: 1-472.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 84% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 86% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 87% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 88% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 89% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 91% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 93% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 94% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 96% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NOs: 1-472.In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-472.

[0123] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462.Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. In some embodiments, the amino acid sequence provides a binding constant of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any value therebetween, (i) between the amino acid sequence and the primed nucleic acid sequence, (ii) between the amino acid sequence and the nucleotide, or (iii) both (i) and (ii).

[0124] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 81% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 82% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 83% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 84% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 86% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 87% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462.Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 88% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 89% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. In some embodiments, the amino acid sequence provides a binding constant of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any value therebetween, (i) between the amino acid sequence and the primed nucleic acid sequence, (ii) between the amino acid sequence and the nucleotide, or (iii) both (i) and (ii).

[0125] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 91% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 92% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 93% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 94% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 96% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462.Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460, and 462. In some embodiments, the amino acid sequence provides a binding constant of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any value therebetween, (i) between the amino acid sequence and the primed nucleic acid sequence, (ii) between the amino acid sequence and the nucleotide, or (iii) both (i) and (ii).

[0126] In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerizing enzyme. In some embodiments, the polymerizing enzyme comprises a nucleic acid polymerase or a polymerizing portion thereof. In some embodiments, the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

[0127] Disclosed herein are compositions comprising a nucleic acid sequence encoding any of the synthetic polypeptides disclosed herein. Disclosed herein are compositions comprising a DNA sequence encoding any of the synthetic polypeptides disclosed herein. Disclosed herein are compositions comprising a messenger RNA sequence encoding any of the synthetic polypeptides disclosed herein. Disclosed herein are vectors comprising a nucleic acid sequence encoding any of the synthetic polypeptides disclosed herein. In some embodiments, the vector may comprise a plasmid, a viral vector, a non-viral vector, a bacterial vector, a yeast vector, a baculovirus vector, a plant vector, or a mammalian vector. In some embodiments, the vector may comprise a DNA vector. Disclosed herein are cells comprising a nucleic acid encoding any of the synthetic polypeptides disclosed herein. Disclosed herein are cells comprising a vector comprising a nucleic acid sequence encoding any of the synthetic polypeptides disclosed herein. In some embodiments, the cells are transduced with a nucleic acid or vector disclosed herein. In some embodiments, the cells express any of the synthetic polypeptides disclosed herein. In some embodiments, the synthetic polypeptide is isolated or extracted from the cell.

[0128] In some embodiments, the synthetic polypeptide is a polymerizing enzyme such as a polymerase. Non-limiting examples of polymerases of the present disclosure include Klenow DNA polymerase, Thermus aquaticus DNA polymerase I (Taq polymerase), KlenTaq polymerase, and bacteriophage T7 DNA polymerase; human α, δ, and ε DNA polymerases; bacteriophage polymerases such as T4 and phi29 bacteriophage DNA polymerase; Bacillus subtilis DNA polymerase III and Escherichia coli (E. coli) DNA polymerase IIIα and ε; reverse transcriptases such as M-type or O-type HIV reverse transcriptase, avian myeloblastosis virus reverse transcriptase, or Moloney murine leukemia virus (MMLV) reverse transcriptase, or telomerase. In some embodiments, the polymerase can include Klenow polymerase. In some embodiments, the polymerizing enzyme is a DNA polymerizing enzyme. Non-limiting examples of DNA polymerizing enzymes include DNA polymerases derived from various archaeal genera, such as Aeropyrum, Archaeglobus, Desulflococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, or variants thereof, such as 9°N polymerase (e.g., SEQ ID NOs: 280-281), Vent® DNA polymerase (e.g., SEQ ID NO: 283), Deep Vent® (e.g., SEQ ID NO: 284), Therminator™ (e.g., SEQ ID NO: 282), Pyrococcus furiosus, and the like. furiosus DNA polymerase (Pfu polymerase) (e.g., SEQ ID NO: 285), RB69 (e.g., SEQ ID NO: 287), KOD (Sigma-Aldrich), Pfx, and Tgo polymerase.In some embodiments, the polymerase is selected from the group consisting of Candidatus Altiarchaeales archaea (e.g., SEQ ID NOs: 1-268, 288-381), RLF 89458.1 (e.g., SEQ ID NOs: 391-413), RLF 60390.1 (e.g., SEQ ID NOs: 414-430), WP 175059460.1 (e.g., SEQ ID NOs: 431-445), ADK 47977.1 (e.g., SEQ ID NOs: 446-447), GBE 17769.1 (e.g., SEQ ID NOs: 448-449), GBE 55812.1 (e.g., SEQ ID NOs: 450-451), KUO 42443.1 (e.g., SEQ ID NOs: 452-453), KXB 02540.1 (e.g., SEQ ID NOs: 454-455), MBC The polymerase may comprise a wild-type or mutant backbone sequence of a polymerase from WP 7218772.1 (e.g., SEQ ID NOS: 456-457), RMF 90817.1 (e.g., SEQ ID NOS: 458-459), WP 058946753.1 (e.g., SEQ ID NOS: 460-461), or WP 167886206.1 (e.g., SEQ ID NOS: 462-463). In some embodiments, the polymerase may comprise an amino acid sequence having a level of identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or greater to any of SEQ ID NOS: 1-390, 464, 391-463, or 465. The sequences of the wild-type and mutant polymerases are listed and described in Table 1A, Table 1B, Table 2, Table 3, Table 4, and Table 5, and Figures 17-52. Table 1A lists wild-type polymerases having the backbone sequence of RLI 89578.1 (SEQ ID NO: 1) and mutational variants (SEQ ID NOs: 2-268, 288-381) containing amino acid substitutions relative to wild-type RLI 89578.1. Any of the amino acid sequences listed in Table 1A can contain the substitutions D141A and / or E143A. Table 1B lists various polymerase backbones and their variants (SEQ ID NOs: 269-287). Table 2 lists wild-type polymerases having the backbone sequence of RLF 89458.1 (SEQ ID NO: 391) and mutational variants (SEQ ID NOs: 392-413) containing amino acid substitutions relative to wild-type RLF 89458.1.Table 3 lists wild-type polymerases having the backbone sequence of RLF 60390.1 (SEQ ID NO: 414) and mutant variants (SEQ ID NOs: 415-430) containing amino acid substitutions relative to wild-type RLF 60390.1. Table 4 lists wild-type polymerases having the backbone sequence of WP 175059460.1 (SEQ ID NO: 431) and mutant variants (SEQ ID NOs: 431-445) containing amino acid substitutions relative to wild-type WP 175059460.1. Table 5 lists various wild-type and mutant polymerases with backbone sequences from ADK 47977.1 (SEQ ID NOs: 446-447), GBE 17769.1 (SEQ ID NOs: 448-449), GBE 55812.1 (SEQ ID NOs: 450-451), KUO 42443.1 (SEQ ID NOs: 452-453), KXB 02540.1 (SEQ ID NOs: 454-455), MBC 7218772.1 (SEQ ID NOs: 456-457), RMF 90817.1 (SEQ ID NOs: 458-459), WP 058946753.1 (SEQ ID NOs: 460-461), or WP 167886206.1 (SEQ ID NOs: 462-463). In Table 1A, Table 1B, Table 2, Table 3, Table 4, and Table 5, an underscore ("_") indicates a space between two or more amino acid substitutions.

[0129] [Table 2-1]

[0130] [Table 2-2]

[0131] [Table 2-3]

[0132] [Table 2-4]

[0133] [Table 2-5]

[0134]

Table 2-6

[0135]

Table 2-7

[0136]

Table 2-8

[0137]

Table 2-9

[0138]

Table 2-10

[0139]

Table 2-11

[0140]

Table 2-12

[0141]

Table 3

[0142]

Table 4

[0143]

Table 5

[0144]

Table 6

[0145]

Table 7

[0146] Some embodiments disclosed herein include compositions comprising nucleotide conjugates. The nucleotide conjugates of the present disclosure can be useful for forming the binding complexes disclosed herein, such as in capture reactions, nucleotide binding reactions, or nucleic acid sequencing reactions. In some embodiments, the nucleotide conjugates can comprise a nucleotide and a moiety. In some embodiments, the moiety can be tethered, bonded, attached, or linked to the nucleotide via a covalent or non-covalent bond. In some embodiments, the moiety can comprise a chemical moiety or a biochemical moiety. In some embodiments, the nucleotide conjugates can comprise multivalent nucleotide conjugates. In some embodiments, the multivalent nucleotide conjugates can be capable of forming binding complexes. In some embodiments, the multivalent nucleotide conjugates can form multivalent binding complexes. In some embodiments, the nucleotide conjugates can comprise a core, a core-attachment moiety, a spacer, a linker, a nucleotide unit, or any combination thereof. In some embodiments, the core can comprise a single molecule, a monomer, a single molecular unit, or a polymer. In some embodiments, the polymer can comprise a polypeptide, a protein, a peptide, or a derivative thereof. In some embodiments, the polymer may comprise a modified polypeptide. In some embodiments, the modification of the polypeptide may include any chemical, biological, or physical modification that can enable the polypeptide to form the nucleotide conjugate disclosed herein. In some embodiments, the modification may include modification with polyethylene glycol (PEG), or PEGylation.In some embodiments, polypeptides containing modifications may include, but are not limited to, those disclosed in the following references, which are incorporated herein by reference in their entirety: Zuma LK, Gasa NL, Makhoba XH, Pooe OJ. Protein PEGylation: Navigating Recombinant Protein Stability, Aggregation, and Bioactivity. Biomed Research International. (2022) Jul 25; 2022:8929715. doi:10.1155 / 2022 / 8929715. PMID:35924267; PMCID:PMC9343206; Suk JS, Xu Q, Kim N, Hanes J, Ensign LM. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Advanced Drug Delivery Reviews. (2016) April 1;99(Pt A):28-51.doi:10.1016 / j.addr.2015.09.012.Epub 2015 Oct 9.PMID:26456916;PMCID:PMC4798869.

[0147] In some embodiments, the nucleotide conjugate comprises a core tethered to multiple "nucleotide arms," ​​for example, as shown in Figures 1-3. In some embodiments, the nucleotide arms may be modular. In some embodiments, the nucleotide arms may comprise a core attachment moiety. In some embodiments, the nucleotide arms may comprise a spacer. In some embodiments, the nucleotide arms may comprise a linker. In some embodiments, the nucleotide arms may comprise a nucleotide unit. In some embodiments, the nucleotide arms may comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit. Thus, the nucleotide conjugate may comprise multiple nucleotide units (see, for example, Figures 1-3). Alternatively, the nucleotide conjugate may comprise a single nucleotide unit.

[0148] In some embodiments, the nucleotide conjugates of the present disclosure increase binding of nucleotide units to a polymerizing enzyme or complexed polymerizing enzyme by increasing the effective concentration of at least the nucleotide units with the enzyme. Such an increase is observed by increasing the nucleotide concentration in solution or by increasing the amount of nucleotides in proximity to the relevant binding or incorporation site of the polymerizing enzyme. This increase can also be achieved by physically restricting the number of nucleotides to a limited amount, resulting in a localized increase in nucleotide concentration. Nucleotide units of nucleotide arms (e.g., attached to a core) can bind to the polymerizing enzyme binding site with a high apparent avidity that can be observed with individual nucleotides that are unconjugated, untethered, or otherwise unconstrained. One way to achieve such a restriction can be by providing a nucleotide conjugate in which multiple nucleotide units are tethered to the core. In some embodiments, the core can be a particle, such as a polymer, branched polymer, dendrimer, micelle, liposome, microparticle, nanoparticle, quantum dot, or other suitable particle.

[0149] The present disclosure provides various embodiments of nucleotides, each configured to include a core tethered (e.g., attached) to multiple nucleotide arms. In some embodiments, the nucleotide arms can include a core attachment moiety. In some embodiments, the nucleotide arms can include a spacer. In some embodiments, the nucleotide arms can include a linker. In some embodiments, the nucleotide arms can include a nucleotide unit. In some embodiments, the nucleotide arms can include a core attachment moiety, a spacer, a linker, and a nucleotide unit. Each nucleotide arm can include (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit (see, e.g., Figure 4). In some embodiments, the nucleotide arms can include a nucleoside unit instead of a nucleotide unit. The nucleotide arms can include biotin. In some embodiments, the nucleotide conjugate can include multiple copies of the same type of nucleotide attached to the core via multiple nucleotide arms (see, e.g., Figures 1, 2, and 3). In the nucleotide conjugate, the tethered nucleotide is referred to as a nucleotide unit. In some embodiments, multiple copies of the nucleotide unit can be covalently or non-covalently bound to the core. The nucleotide arms are designed so that the nucleotide units of the nucleotide arms can interact with one or more polymerases in the same way as free nucleotides. The nucleotide units of each nucleotide arm can bind to a polymerase complexed with a nucleic acid template and a nucleic acid primer (e.g., nucleotide-associated). The nucleotide units can also separate from the complexed polymerase to re-bind the same complexed polymerase or bind another complexed polymerase in the vicinity of the nucleotide conjugate. Because a nucleotide conjugate can contain multiple nucleotide arms, the nucleotide units of a single nucleotide conjugate can simultaneously bind multiple complexed polymerases.The level of valency in the nucleotide units of a given nucleotide conjugate can correspond to the number of nucleotide arms linked to the core. In some embodiments, a single nucleotide conjugate can essentially simultaneously bind multiple complexed polymerases bound to the same template molecule (e.g., a concatemer). The nucleotide conjugate can effectively increase the local concentration of nucleotides, which can enhance the signal in the nucleotide binding reaction.

[0150] The complexed polymerase can comprise a synthetic polypeptide disclosed herein bound to a nucleic acid duplex, which can comprise a nucleic acid template molecule hybridized to a nucleic acid primer, where the primer can comprise an extendable or non-extendable 3' end. The template molecule can be a single-stranded or double-stranded linear or circular nucleic acid. The template molecule can be a clonally amplified nucleic acid molecule. The template molecule can be a nucleic acid concatemer. In some embodiments, the concatemer can comprise two or more tandem copies of a sequence of interest. The template and primer can be fully or partially complementary along the hybridized region.

[0151] Alternatively, the complexed polymerase can comprise a polymerase bound to a self-priming nucleic acid template molecule, where the self-priming portion can comprise an extendable or non-extendable 3' end. The template molecule can comprise a single-stranded or double-stranded region. The template molecule can be a clonally amplified nucleic acid molecule. The template molecule can be, for example, a nucleic acid concatemer containing two or more tandem copies of a sequence of interest. The template and the self-priming portion can be fully or partially complementary along the hybridized region.

[0152] A single nucleotide conjugate can bind multiple complexed polymerases that form multivalent binding complexes by binding to the same template molecule (e.g., concatemers having repeats of the same target nucleic acid sequence). For example, (i) a first binding complex can include a first nucleic acid primer, a first polymerase, and a first nucleotide conjugate that forms a first binding complex by binding to a first portion of the concatemer template molecule, where the first nucleotide unit of the first nucleotide conjugate can bind to the first polymerase; (ii) a second binding complex can include a second nucleic acid primer, a second polymerase, and the same first nucleotide conjugate that can form a second binding complex by binding to a second portion of the concatemer template molecule, where the second nucleotide unit of the first nucleotide conjugate can bind to the second polymerase; and the first and second binding complexes containing the same nucleotide conjugates can form a multivalent binding complex. Thus, in some embodiments, the multivalent binding complex of the present disclosure comprises at least two copies of a target nucleic acid sequence bound to a single nucleotide conjugate disclosed herein. In some embodiments, the nucleotide units of the nucleotide conjugate are complementary to nucleotides of at least two copies of the target nucleic acid sequence. In some embodiments, the multivalent binding complex comprises one or more polymerase enzymes (e.g., synthetic polypeptides) disclosed herein.

[0153] The nucleotide unit of the nucleotide conjugate can bind to a complexed polymerase (e.g., a synthetic polypeptide disclosed herein) by binding to the 3' end or self-priming portion of a primer (or initial extension primer) without undergoing polymerase-catalyzed incorporation. The nucleotide unit bound to the complexed polymerase can form a bound complex. The bound complex can be stable such that the nucleotide unit exhibits a low dissociation rate, with a duration indicating the stability of the bound complex and the strength of the binding interaction. Conditions suitable for the nucleotide unit to bind to the 3' end of the primer at a position opposite the complementary nucleotide in the template strand (where the nucleotide unit does not undergo polymerase-catalyzed incorporation) can include the presence of at least one non-catalytic divalent cation, including strontium, barium, calcium, or a combination thereof.

[0154] In some embodiments, the nucleotide conjugate can include at least one nucleotide unit having a sugar moiety with a 3' OH group or a 3' blocking group. The nucleotide unit with the sugar 3' OH group or a 3' blocking group can bind to a complexed polymerase to interrogate a complementary nucleotide in the template strand, and the nucleotide unit does not undergo nucleotide incorporation. Conditions suitable for binding the nucleotide unit to a complexed polymerase (where the nucleotide unit is attached to the 3' end of the primer at a position opposite the complementary nucleotide in the template strand to interrogate a complementary nucleotide in the template strand, and the nucleotide unit does not undergo polymerase-catalyzed incorporation) can include the presence of at least one non-catalytic divalent cation, including strontium, barium, calcium, or a combination thereof.

[0155] Alternatively, a nucleotide unit can bind to a complexed polymerase by binding to the 3' end or self-priming portion of a primer (or initial extension primer) and undergo polymerase-catalyzed incorporation into the 3' end or self-priming portion of the extendable primer, resulting in primer extension. If the nucleotide unit contains a hydroxyl group at the 3' sugar position, the nucleotide can subsequently be incorporated into the initial extension primer. If the nucleotide unit contains a blocking group at the 3' sugar position, it can prevent subsequent nucleotides from being incorporated into the initial extension primer strand. Conditions suitable for the nucleotide unit to bind to the 3' end of the primer at a position opposite the complementary nucleotide in the template strand (in which case the nucleotide unit undergoes polymerase-catalyzed incorporation) can include the presence of at least one non-catalytic divalent cation, including magnesium, manganese, or a combination of magnesium and manganese.

[0156] In some embodiments, a nucleotide conjugate can include at least one nucleotide unit having a sugar moiety with a 3' OH group or a 3' blocking group. The nucleotide unit with the sugar 3' OH group can bind to a complexed polymerase to interrogate a complementary nucleotide in a template strand, allowing the nucleotide unit to undergo polymerase-catalyzed incorporation. The nucleotide unit with the sugar 3' blocking group can bind to a complexed polymerase to interrogate a complementary nucleotide in a template strand, allowing the nucleotide unit to undergo polymerase-catalyzed incorporation, while the 3' blocking group can inhibit / prevent incorporation of a subsequent nucleotide (or the next nucleotide unit of a nucleotide conjugate). The 3' blocking group can be removed to facilitate incorporation of a subsequent nucleotide. Conditions suitable for binding of a nucleotide unit to a complexed polymerase (wherein the nucleotide unit binds to the 3' end of the primer at a position opposite the complementary nucleotide in the template strand to interrogate the complementary nucleotide in the template strand, and the nucleotide unit undergoes polymerase-catalyzed incorporation) can include the presence of at least one non-catalytic divalent cation, including magnesium, manganese, or a combination of magnesium and manganese.

[0157] In some embodiments, the linker unit of the nucleotide arm can contribute to the binding of polymerase, the stabilization of the polymerase-nucleotide triple complex, or a combination of the binding of polymerase and the stabilization of the polymerase-nucleotide triple complex. The present inventors have found that both the linker and the nucleotide can be recognized and discriminated by DNA polymerase. The present inventors have found that the proximal portion of the nucleotide unit can play an important role in facilitating the heavy enzyme-nucleotide binding interaction. Optimizing the linker region can improve nucleotide conjugates useful for specific applications, such as nucleic acid sequencing.

[0158] Crystal structures of sequencing polymerases co-crystallized with primed template molecules and nucleotide conjugates show that the carbonyl oxygen of the nucleotide arm interacts with the lysine residue of the polymerase (see, e.g., Figure 16 and Example 8). In the nucleotide arm, the carbonyl oxygen is located near the nucleotide unit. The polymerase used in Example 8 has a backbone sequence based on the amino acid sequence of SEQ ID NO: 1, with the lysine residue located at position 495. Binding studies comparing various types of nucleotide conjugates with nucleotide arms bearing various linkers revealed varying levels of binding. Binding data suggest that the presence of an aromatic moiety (e.g., an aryl group) on the linker can improve binding of the nucleotide unit to the polymerase (see, e.g., Figures 11 and 2, and Example 5).

[0159] Nucleotide conjugates can be labeled with a detectable reporter moiety. In some embodiments, the core of a nucleotide conjugate can be labeled with a detectable reporter moiety (e.g., a fluorophore) to enable differentiation between various nucleotide conjugates possessing different types of nucleotide units. For example, the core unit of a first nucleotide conjugate can be labeled with a first fluorophore, in which case the first nucleotide conjugate can contain multiple nucleotide arms having the same type of nucleotide unit, e.g., dGTP nucleotide units. The core unit of a second nucleotide conjugate can be labeled with a second fluorophore (different from the first fluorophore), in which case the second nucleotide conjugate can contain multiple nucleotide arms having the same type of nucleotide unit, e.g., dATP nucleotide units. A mixture of labeled nucleotide conjugates can contain any combination of two or more subpopulations of nucleotide conjugates, in which each subpopulation contains nucleotide conjugates having a reporter-labeled core corresponding to one type of nucleotide unit and a specific type of nucleotide unit. The mixture of labeled and unlabeled nucleotide conjugates can include any combination of at least one subpopulation of nucleotide conjugates containing a plurality of nucleotide conjugates having a reporter-labeled core corresponding to one type of nucleotide unit and a particular type of nucleotide unit, and at least one subpopulation of nucleotide conjugates containing a plurality of nucleotide conjugates having a different type of nucleotide unit and an unlabeled core corresponding to another type of nucleotide unit. A single population or a mixture of various subpopulations of labeled nucleotide conjugates can be used in nucleotide binding assays, nucleotide incorporation assays, nucleic acid sequencing methods, or a combination thereof. The nucleotide conjugates can be useful for massively parallel nucleic acid sequencing.

[0160] Disclosed herein are formulations comprising a first nucleotide conjugate and a second nucleotide conjugate. In some embodiments, the formulation comprises a first nucleotide conjugate, a second nucleotide conjugate, and a third nucleotide conjugate. In some embodiments, the formulation comprises a first nucleotide conjugate, a second nucleotide conjugate, a third nucleotide conjugate, and a fourth nucleotide conjugate. In some embodiments, the first nucleotide conjugate comprises a first label. In some embodiments, the second nucleotide conjugate comprises a second label. In some embodiments, the third nucleotide conjugate comprises a third label. In some embodiments, the fourth nucleotide conjugate comprises a fourth label. In some embodiments, the first label comprises a first fluorophore. In some embodiments, the second label comprises a second fluorophore. In some embodiments, the third label comprises a third fluorophore. In some embodiments, the fourth label comprises a fourth fluorophore. In some embodiments, the first fluorophore emits light at a first wavelength. In some embodiments, the second fluorophore emits light at a second wavelength. In some embodiments, the third fluorophore emits light at a third wavelength. In some embodiments, the fourth fluorophore emits light at a fourth wavelength. In some embodiments, the first wavelength is different from the second wavelength. In some embodiments, at least two of the first wavelength, second wavelength, and third wavelength are different from each other. In some embodiments, each of the first wavelength, second wavelength, and third wavelength are different from each other. In some embodiments, all of the first wavelength, second wavelength, and third wavelength are different from each other. In some embodiments, at least two of the first wavelength, second wavelength, third wavelength, and fourth wavelength are different from each other. In some embodiments, at least three of the first wavelength, second wavelength, third wavelength, and fourth wavelength are all different from each other. In some embodiments, each of the first wavelength, the second wavelength, the third wavelength, and the fourth wavelength are different from each other, hi some embodiments, all of the first wavelength, the second wavelength, the third wavelength, and the fourth wavelength are different from each other.The mixture of different subpopulations of labeled nucleotide conjugates can include a first subpopulation of nucleotide conjugates that include a first fluorophore and a second subpopulation of nucleotide conjugates that include a second fluorophore.

[0161] In some embodiments, at least one nucleotide unit of a nucleotide conjugate can be labeled with a detectable reporter moiety (e.g., a fluorophore) to enable differentiation between various nucleotide conjugates possessing different types of nucleotide units. For example, the base of a nucleotide unit of a first nucleotide conjugate can be labeled with a first fluorophore, in which case the first nucleotide conjugate can contain multiple nucleotide arms having the same type of nucleotide unit, for example, dGTP nucleotide units. The base of a nucleotide unit of a second nucleotide conjugate can be labeled with a second fluorophore (different from the first fluorophore), in which case the second nucleotide conjugate can contain multiple nucleotide arms having the same type of nucleotide unit, for example, dATP nucleotide units. A mixture of labeled nucleotide conjugates can contain any combination of two or more subpopulations of nucleotide conjugates, in which each subpopulation contains nucleotide conjugates having one type of nucleotide unit and a reporter-labeled nucleic acid base corresponding to a particular type of nucleotide unit. The mixture of labeled and unlabeled nucleotide conjugates can include any combination of at least one subpopulation of nucleotide conjugates comprising a plurality of nucleotide conjugates having one type of nucleotide unit and a reporter-labeled nucleic acid base corresponding to a particular type of nucleotide unit, and at least one subpopulation of nucleotide conjugates comprising a plurality of nucleotide conjugates having another type of nucleotide unit and an unlabeled nucleic acid base corresponding to another type of nucleotide unit. A single population or a mixture of various subpopulations of labeled nucleotide conjugates can be used in nucleotide binding assays, nucleotide incorporation assays, nucleic acid sequencing methods, or a combination thereof. The nucleotide conjugates can be useful for massively parallel nucleic acid sequencing.

[0162] Nucleotide conjugates can be used to localize detectable signals at active regions of biochemical interactions, such as protein-nucleic acid interactions, nucleic acid hybridization reactions, or enzymatic reactions, such as polymerase-mediated reactions. For example, the nucleotide conjugates described herein can be used to identify the binding or incorporation sites of nucleic acid bases during polymerase-catalyzed reactions and to distinguish bases for sequencing and array-based applications. Increased binding or incorporation between template nucleic acids and nucleotide units can provide enhanced signals, greatly improving base calling accuracy and reducing imaging time, when the nucleotide units are complementary to the "N" base in the template nucleic acid.

[0163] Furthermore, labeled nucleotide conjugates can form multivalent binding complexes that increase the base call signal from a given polony containing multiple copies of a template nucleic acid strand (e.g., a concatemer). Sequencing workflows that involve generating polonies with clonally amplified copies of a template strand can have the advantage of amplifying the signal due to the presence of multiple simultaneous sequences within a given region, each resulting in a unique signal. The presence of multiple signals within a given region can also reduce the impact of any incorrectly advanced or skipped cycles due to the fact that the signal from the large number of correct base calls can overwhelm the signal from the small number of incorrectly advanced or skipped base calls, thereby providing a method for reducing pre-phasing or phasing errors, improving the read length of sequencing reactions, or combining reduced phasing errors with improved read length of sequencing reactions.

[0164] The nucleotide conjugates disclosed herein and their use can result in one or more of: (i) a stronger signal for better base calling accuracy compared to nucleic acid amplification and sequencing methods; (ii) a greater discrimination of sequence-specific signals from background signals; (iii) a reduced requirement for the amount of starting material; (iv) increased sequencing speed and reduced sequencing time; (v) reduced phasing errors; and (vi) improved read length of sequencing reactions.

[0165] The present disclosure provides nucleotide conjugates comprising a core attached to at least one nucleotide arm. In some embodiments, at least one nucleotide arm can comprise a core attachment moiety. In some embodiments, at least one nucleotide arm can comprise a spacer. In some embodiments, at least one nucleotide arm can comprise a linker. In some embodiments, at least one nucleotide arm can comprise a nucleotide unit. In some embodiments, at least one nucleotide arm can comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit. In some embodiments, the core can comprise a bead, particle, or nanoparticle. In some embodiments, the core can comprise an alkyl, alkenyl, or alkynyl core, such as may be present in a branched polymer or dendrimer. In some embodiments, the core can comprise a moiety that mediates conjugation of the core to the nucleotide arm. In some embodiments, the core can be attached to multiple nucleotide arms. Optionally, the core can be attached to between about 1 and about 50 nucleotide arms. Optionally, the core is attached to between about 2 and about 20 nucleotide arms. Optionally, the core is attached to between about 2 and about 4 nucleotide arms. Optionally, the core is attached to between about 4 and about 10 nucleotide arms. Optionally, the core is attached to between about 10 and about 15 nucleotide arms. Optionally, the core is attached to between about 15 and about 20 nucleotide arms. Figures 1, 2, and 3 show the overall structure of the nucleotide conjugate.

[0166] The present disclosure provides a nucleotide conjugate comprising a core attached to at least one biotinylated nucleotide arm. In some embodiments, at least one biotinylated nucleotide arm can comprise a core attachment moiety. In some embodiments, at least one biotinylated nucleotide arm can comprise a spacer. In some embodiments, at least one biotinylated nucleotide arm can comprise a linker. In some embodiments, at least one biotinylated nucleotide arm can comprise a nucleotide unit. In some embodiments, at least one biotinylated nucleotide arm can comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit. In some embodiments, the core can comprise a streptavidin- or avidin-type moiety, and the biotin unit of the biotinylated nucleotide arm can mediate conjugation of the core to the biotinylated nucleotide arm (Figure 2). The streptavidin- or avidin-type core can be a tetrameric biotin-binding protein capable of binding one, two, three, or up to four biotinylated nucleotide arms.

[0167] In some embodiments, the nucleotide conjugate comprises a core. In some embodiments, the core is a particle. In some embodiments, the particle is a nanoparticle or a microparticle. In some embodiments, the material of the particle comprises a polymer or a metal. In some embodiments, the polymer is synthetic. In some embodiments, the polymer is natural. In some embodiments, the polymer comprises a plastic or a protein. In some embodiments, the protein comprises streptavidin, or avidin, or derivatives, analogs, and other non-natural forms thereof capable of binding to at least one biotin moiety. In some embodiments, the plastic is or comprises polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), or polyethylene glycol (PEG), or any combination thereof. In some embodiments, the core may comprise polyethylene glycol (PEG), polypropylene glycol (PPG), polyvinyl acetate (PVA), polylactic acid (PLA), or polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), chlorinated or fluorinated derivatives thereof, or combinations thereof.

[0168] In some embodiments, the core may comprise avidin, streptavidin, or the like, or derivatives thereof; branched polymers; dendrimers; crosslinked polymer particles, such as agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, or methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dots; liposomes; emulsion particles, or other suitable particles (e.g., nanoparticles, microparticles, etc.). In some embodiments, the core may comprise streptavidin-type or avidin-type moieties, including streptavidin or avidin proteins, as well as any derivatives, analogs, and other non-natural forms of streptavidin or avidin that can bind to at least one biotin moiety. Streptavidin or avidin moieties may include native or recombinant forms, as well as mutant and derivatized molecules. Streptavidin and avidin mutants may include any one or a combination of two or more amino acid insertions, deletions, substitutions, or truncations. Mutants may also include fusion polypeptides.

[0169] Nucleotide conjugates can be configured using streptavidin or avidin cores that have high affinity for the biotin moiety on biotinylated nucleotide arms, thereby reducing the dissociation of nucleotide arms from the cores.A mixture of nucleotide conjugates can be prepared, in which the mixture contains two or more subpopulations of nucleotide conjugates, each subpopulation containing nucleotide conjugates with one type of nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP).Nucleotide conjugates configured to have high affinity between the core and the nucleotide arm can reduce the undesired dissociation of nucleotide arms from the cores and the exchange of nucleotide arms between different cores.The exchange of nucleotide arms during sequencing reactions can cause inaccurate base calling and reduced sequencing accuracy. In some embodiments, a nucleotide conjugate with increased stability (e.g., reduced dissociation of the biotinylated nucleotide arm) can include dye-labeled streptavidin, where the streptavidin subunit carries a Lys121Arg mutation, which can exhibit reduced dissociation of the biotinylated nucleotide arm from the streptavidin core.

[0170] The streptavidin moiety can include full-length or truncated forms that have high affinity for binding to biotin. For example, the streptavidin moiety can be about 10 -14 mol / L, or approximately 10 -15 Dissociation constant (K in mol / L d) can be expressed as follows. In some embodiments, the streptavidin portion can comprise a polypeptide having the backbone sequence of any of SEQ ID NOs: 466-470. The streptavidin portion can comprise an amino acid sequence having a level of identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more to any of SEQ ID NOs: 466-470. The streptavidin portion can comprise a core portion of a streptavidin protein that is truncated at the N-terminus, C-terminus, or a combination of the N-terminus and C-terminus of a streptavidin protein having the amino acid sequence of any of SEQ ID NOs: 466-470. For example, the streptavidin portion can lack the N-terminal portion of any of SEQ ID NOs: 466-468, 470-471 (e.g., the underlined N-terminal portion in Figures 17-19 and 21-22). The streptavidin portion can lack the C-terminal portion of any of SEQ ID NOs: 466-468 (e.g., the underlined C-terminal portion in Figures 17-19). The streptavidin portion can include a core portion comprising the amino acid sequence of SEQ ID NO: 469 or 470.

[0171] In some embodiments, the streptavidin moiety can include any amino acid substitution mutation at the site that can be labeled with a dye. For example, the dye-labeling site can include a lysine at position 121 (see, e.g., SEQ ID NO: 469), which can overlap with the biotin-binding site. In some embodiments, a dye attached to streptavidin at Lys121 can block or inhibit biotin binding to the dye-labeled streptavidin. A nucleotide conjugate containing a dye-labeled streptavidin possessing a lysine at position 121 can exhibit dissociation of the biotinylated nucleotide arm from the streptavidin core. A nucleotide conjugate with increased stability can include a dye-labeled streptavidin possessing a Lys121Arg mutation (see, e.g., SEQ ID NO: 469), which can exhibit reduced dissociation of the biotinylated nucleotide arm from the streptavidin core.

[0172] In some embodiments, the streptavidin moiety increases the affinity for biotin binding (e.g., K d About 10 -16 mol / L), up to about 60° C., about 65° C., about 70° C., or about 80° C., or a combination of increased affinity for biotin binding and improved biotin retention. Amino acid substitutions can include His151Lys or His151Asp of SEQ ID NO: 466, His128Lys or His128Asp of SEQ ID NO: 467, His127Lys or His127Asp of SEQ ID NO: 468, His116Lys or His116Asp of SEQ ID NO: 469, or His135Lys or His135Asp of SEQ ID NO: 470. Histidine residues that can be substituted with lysine or aspartic acid are shown in bold and underlined in Figures 17-21.

[0173] The avidin moiety can include full-length or truncated forms that have high affinity for binding to biotin. For example, the avidin moiety can have a length of about 10 -14 mol / L, or approximately 10 -15 Dissociation constant (K in mol / L d) can exhibit a structural identity similar to that of SEQ ID NO: 471 or 472. In some embodiments, the avidin portion can comprise a polypeptide having the backbone sequence of SEQ ID NO: 471 or 472. The avidin portion can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or greater identity level to SEQ ID NO: 471 or 472. The avidin portion can comprise a core portion of an avidin protein truncated at the N-terminus, C-terminus, or a combination of the N-terminus and C-terminus of an avidin protein having the amino acid sequence of SEQ ID NO: 471. For example, the avidin portion can lack the N-terminal portion of SEQ ID NO: 471 (e.g., the underlined N-terminal portion in Figures 17-19 and 22). In some embodiments, the avidin can comprise a substitution of any one of eight arginine residues, or any combination thereof (e.g., those underlined and bolded in Figure 22 or 23). The amino acid substitutions can include replacing five of the eight arginine residues with neutral amino acids at positions 26, 50, 83, 111, 124, 138, 146, 148 of SEQ ID NO:471, or a combination thereof, or replacing five of the eight arginine residues with neutral amino acids at positions 2, 26, 59, 87, 100, 114, 122, 124 of SEQ ID NO:472, or a combination thereof.

[0174] Avidin can include partially deglycosylated and non-glycosylated forms. Avidin moieties can include derivatized forms, such as N-acylated avidins, e.g., N-acetyl, N-phthalyl, and N-succinyl avidins, as well as commercially available products, including ExtrAvidin®, CaptAvidin™ (selective nitration of tyrosine residues at the four biotin-binding sites to produce avidin that reversibly binds biotin), NeutrAvidin™ (chemically deglycosylated and containing modified arginine residues), and Neutralite Avidin™ (five of the eight arginine residues are replaced with neutral amino acids, two of the lysine residues are replaced with glutamic acid, and Asp17 is replaced with isoleucine). Amino acids with neutral, nonpolar side chains include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Amino acids having neutral polar side chains include asparagine, cysteine, glutamine, serine, threonine, and tyrosine.

[0175] In some embodiments, the core can be labeled with a detectable reporter moiety. Non-limiting examples of detectable moieties include fluorescently detectable moieties, bioluminescently detectable moieties, chemiluminescently detectable moieties, and radiologically detectable moieties. In some embodiments, the nucleotide conjugates can be unlabeled. The core can be a streptavidin or avidin homotetramer. Each subunit in the homotetramer can contain at least one lysine residue that can be conjugated to a fluorophore. N-hydroxysuccinimide (NHS) ester-conjugated fluorophores can be used in labeling reactions. The maximum number of fluorophores that can be attached to a streptavidin or avidin subunit can be determined by the number of lysine residues in the subunit. The amino acid sequences of various streptavidin and avidin subunits are shown in Figures 17-23. For example, a streptavidin subunit can include four lysines (e.g., SEQ ID NO: 469), eight lysines (e.g., SEQ ID NOs: 467, 468, 470), or nine lysines (e.g., SEQ ID NO: 466). An avidin subunit can include nine lysines (e.g., SEQ ID NOs: 471 and 472).

[0176] When preparing labeled streptavidin or avidin cores, the labeling reaction can be optimized to achieve a predetermined degree of labeling (sometimes abbreviated as DoL). The degree of labeling can be expressed as a molar ratio in the form of label / protein. The lower the degree of labeling of the dye core conjugate, the weaker the fluorescence intensity. Dye core conjugates with a very high degree of labeling (e.g., DoL greater than 6) may exhibit reduced fluorescence due to self-quenching from the conjugated fluorophore. In some embodiments, the predetermined degree of labeling for the streptavidin or avidin core may be dye-dependent. Fluorescent dyes include, but are not limited to, CF647, CF680, CF570, and CF532 dyes manufactured by Biotium, AF647, AF680, AF568, and AF532 manufactured by Thermo Fisher Scientific, IFluor 647, IFluor 680, IFlour 568, and IFlour 532 manufactured by AATBio, DY648P1, DY679P1, DY585, and DY530 manufactured by Dyomics, and AFDy 647, IFlour 680LT, AFDye 568, and AFDye 532 manufactured by Fluoroprobes. The predetermined degree of labeling can be about 1-10, about 3-8, about 3.5-7, or about 1.6-4.

[0177] Because red fluorophores are brighter (more intense) than green dyes, color bleeding can occur when imaging both red- and green-labeled nucleotide conjugates on the same support (e.g., a flow cell). To achieve improved signal balance from a mixture of labeled nucleotide conjugates, the degree of labeling of a subpopulation of nucleotide conjugates can be increased or decreased. For example, the degree of labeling of a subpopulation of nucleotide conjugates labeled with a red fluorophore can be decreased compared to the degree of labeling of a subpopulation of nucleotide conjugates labeled with a green fluorophore. In some embodiments, the degree of labeling of a subpopulation of nucleotide conjugates labeled with a red fluorophore can be about 1-3, about 2-3, or about 3-6. In some embodiments, the degree of labeling of a subpopulation of nucleotide conjugates labeled with a green fluorophore can be about 4-7.

[0178] Solution fluorescence measurements can be used to determine the relative brightness of the labeled streptavidin or avidin cores. Alternatively, the degree of labeling can be determined by utilizing a functional assay (e.g., a flow cell capture assay) in which clonal amplified template molecules immobilized on a flow cell are contacted with a primer, a polymerase, and a fluorescently labeled nucleotide conjugate under conditions suitable for binding the nucleotide conjugate to the complexed polymerase without incorporating a nucleotide unit into the primer, and the signal intensity can be detected.

[0179] In some embodiments, the nucleotide conjugate comprises at least two nucleotide arms. In some embodiments, one of the at least two nucleotide arms comprises a core attachment moiety attached to a core. In some embodiments, one of the at least two nucleotide arms comprises a spacer attached to a core attachment moiety. In some embodiments, one of the at least two nucleotide arms comprises a linker attached to a spacer. In some embodiments, one of the at least two nucleotide arms comprises a nucleotide unit attached to a linker. In some embodiments, the nucleotide conjugate comprises (a) a core and (b) at least two nucleotide arms. In some embodiments, one of the at least two nucleotide arms comprises (i) a core attachment moiety attached to a core, (ii) a spacer attached to a core attachment moiety, (iii) a linker attached to the spacer, and (iv) a nucleotide unit attached to the linker. In some embodiments, the linker is

[0180] [ka]

[0181] [ka] wherein n is 1 to 6 and m is 0 to 10.

[0182] In some embodiments, the linker is

[0183] [ka] Includes.

[0184] In some embodiments, the linker is

[0185] [ka] Includes.

[0186] In some embodiments, the linker is

[0187] [ka] Includes.

[0188] In some embodiments, the linker is

[0189] [ka] Includes.

[0190] In some embodiments, the linker is

[0191] [ka] wherein n is 1 to 6 and m is 0 to 10.

[0192] In some embodiments, the linker is

[0193] [ka] wherein n is 1 to 6 and m is 0 to 10.

[0194] In some embodiments, the linker is

[0195] [ka] wherein n is 1 to 6 and m is 0 to 10.

[0196] In some embodiments, the linker is

[0197] [ka] wherein n is 1 to 6 and m is 0 to 10.

[0198] In some embodiments, the linker is

[0199] [ka] Includes.

[0200] In some embodiments, the linker is

[0201] [ka] Includes.

[0202] In some embodiments, the linker is

[0203] [ka] Includes.

[0204] In some embodiments, the linker is

[0205] [ka] Includes.

[0206] In some embodiments, the linker is

[0207] [ka] Includes.

[0208] In some embodiments, the at least two nucleotide arms comprise 3 to 20 nucleotide arms, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide arms. In some embodiments, the at least two nucleotide arms comprise 4 to 19, 5 to 18, 6 to 17, 7 to 16, 8 to 15, 9 to 14, 10 to 13, or 12 nucleotide arms. In some embodiments, the core comprises a polypeptide. In some embodiments, the polypeptide comprises streptavidin or avidin. In some embodiments, the polypeptide comprises streptavidin. In some embodiments, the polypeptide comprises avidin. In some embodiments, the core-attached moiety comprises biotin.

[0209] In some embodiments, the nucleotide conjugate comprises a label, such as a detectable label. In some embodiments, the label is attached to the core or the nucleotide arm. In some embodiments, the label is attached to the core. In some embodiments, the label is attached to the nucleotide arm. In some embodiments, the label is or comprises a fluorescent label. In some embodiments, the composition further comprises a fluorescent label attached to the core. In some embodiments, the composition further comprises a fluorescent label attached to the nucleotide arm.

[0210] In some embodiments, the spacer comprises:

[0211] [ka] In the formula, m is 20 to 500, and o is 1 to 10.

[0212] In some embodiments, the nucleotide arm further comprises a reactive group. In some embodiments, the reactive group is attached to the nucleotide unit. In some embodiments, the reactive group is configured to react with a drug. In some embodiments, the nucleotide arm further comprises a reactive group attached to the nucleotide unit, the reactive group being configured to react with a drug. In some embodiments, the reactive group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group. In some embodiments, the alkyl, alkenyl, alkynyl, or allyl group of the reactive group reacts with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ). In some embodiments, the aryl or benzyl group of the reactive group reacts with H2 and palladium on carbon (Pd / C). In some embodiments, the amine, amide, keto, isocyanate, phosphate, thiol, or disulfide group of the reactive group reacts with a phosphine or a thiol group containing beta-mercaptoethanol or dithiothreitol (DTT). In some embodiments, the carbonate group of the reactive group reacts with potassium carbonate (K2CO3) in methanol (MeOH), triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the urea or silyl group of the reactive group reacts with tetrabutylammonium fluoride, hydrogen fluoride-pyridine (HF-pyridine), ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, the azido group of the reactive group comprises an azide, azide, or azidomethyl group. In some embodiments, the agent comprises a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety, hi some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).In some embodiments, the nucleotide units of at least two nucleotide arms comprise the same nucleobase type.

[0213] In some embodiments, the nucleotide unit comprises a blocking group. In some embodiments, the blocking group is linked to the 3' carbon of the sugar moiety of the nucleotide unit. In some embodiments, the blocking group reacts with a chemical compound to remove the blocking group from the nucleotide unit. In some embodiments, the blocking group reacts with a chemical compound to remove the blocking group from the nucleotide unit and generate a nucleotide unit comprising an OH moiety. In some embodiments, the nucleotide unit comprises a blocking group linked to the 3' carbon of the sugar moiety of the nucleotide unit, and the blocking group reacts with a chemical compound to remove the blocking group from the nucleotide unit and generate a nucleotide unit comprising an OH moiety on the 3' carbon of the sugar moiety of the nucleotide unit. In some embodiments, the blocking group reacts with a chemical compound to remove the blocking group from the nucleotide unit and generate a nucleotide unit comprising an OH moiety on the 3' carbon of the sugar moiety of the nucleotide unit. In some embodiments, the blocking group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group.

[0214] In some embodiments, the alkyl, alkenyl, alkynyl, or aryl group of the blocking group reacts with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl or benzyl group of the blocking group reacts with H and palladium on carbon (Pd / C). In some embodiments, the amine, amide, keto, isocyanate, phosphate, thiol, or disulfide group of the blocking group reacts with a phosphine or thiol group with beta-mercaptoethanol or dithiothreitol (DTT). In some embodiments, the carbonate group of the blocking group reacts with potassium carbonate (KCO) in methanol (MeOH), triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the urea or silyl group of the blocking group is reacted with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride, or triethylamine trihydrofluoride.

[0215] In some embodiments, the azide of the blocking group comprises an azide, azide, or azidomethyl group. In some embodiments, the azide, azide, or azidomethyl group reacts with a chemical. In some embodiments, the chemical comprises a phosphine compound. In some embodiments, the azide, azide, or azidomethyl group reacts with a chemical comprising a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

[0216] Nucleotide Arm The present disclosure provides a nucleotide conjugate comprising one or more nucleotide arms. In some embodiments, the "nucleotide arm" may be modular. In some embodiments, the nucleotide arm may comprise a core attachment moiety. In some embodiments, the nucleotide arm may comprise a spacer. In some embodiments, the nucleotide arm may comprise a linker. In some embodiments, the nucleotide arm may comprise a nucleotide unit. In some embodiments, the nucleotide arm may comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit. In some embodiments, the nucleotide arm may be a nucleoside unit instead of a nucleotide unit. In some embodiments, the nucleotide or nucleoside may comprise an analog thereof.

[0217] In some embodiments, a nucleotide arm may be attached to a core. In some embodiments, two or more nucleotide arms can be attached to a core to form a nucleotide conjugate. A nucleotide conjugate can include multiple nucleotide arms (e.g., Figures 1-3), where each nucleotide arm includes a nucleotide unit that can bind to different complexed polymerases to form a multivalent binding complex.

[0218] Spacer In some embodiments, the composition (e.g., nucleotide conjugate) comprises a spacer. In some embodiments, the nucleotide arm can comprise a spacer. In some embodiments, the spacer can be attached to any of the other components of the nucleotide arm, including, but not limited to, the core, the nucleotide unit, the linker, or other components disclosed herein.

[0219] The spacer can physically separate the nucleotide or nucleoside unit from the core. A spacer is shown in Figure 5A. The spacer can be any length, for example, the value of m can be 1 or at least 2, at least 5, at least 10, at least 20, at least 25, at least 50, at least 75, at least 100, at least 200, at least 500, at least 750, at least 1000, or 2000 or more. In some embodiments, the value of m can be about 20-500, or about 100-110 (e.g., 5,000 g / mol PEG). In some embodiments, in the spacer shown in Figure 5A, the value of o can be 1-50. In some embodiments, the value of o can be about 1-10, or the value of o is about 4. The spacer can be a linear or branched molecule.

[0220] In some embodiments, the spacer can include polyethylene glycol, polypropylene glycol, vinyl acetate, polylactic acid, or polyglycolic acid.

[0221] In some embodiments, the spacer can comprise polyethylene glycol (PEG) with a PEG molecular weight of about 100-200 Da, 200-300 Da, 300-400 Da, 400-500 Da, 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 40K Da, 50K Da, or more.

[0222] In some embodiments, the spacer unit of the nucleotide arm can be attached to a biotin moiety, thereby forming a biotinylated nucleotide arm. In some embodiments, the biotinylated nucleotide arm can comprise a core attachment moiety. In some embodiments, the biotinylated nucleotide arm can comprise a spacer. In some embodiments, the biotinylated nucleotide arm can comprise a linker. In some embodiments, the biotinylated nucleotide arm can comprise a nucleotide unit. In some embodiments, the biotinylated nucleotide arm can comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit. In some embodiments, the biotinylated nucleotide arm can comprise (i) biotin, (ii) a spacer, (iii) a linker, and (iv) a nucleotide (or nucleoside) (see, e.g., Figures 7A-7D).

[0223] Linker In some embodiments, the nucleotide arm may include a linker. In some embodiments, the linker may be attached to any of the other components of the nucleotide arm, including, but not limited to, the core, the nucleotide unit, the spacer, or other components disclosed herein. In some embodiments, the nucleotide arm may include a linker having any one or any combination of two or more moieties, including an amide, a carbonyl oxygen, an aromatic moiety, a polyether moiety, or a combination thereof. The aromatic moiety may include a 6-carbon ring structure such as a benzene ring. The polyether moiety may be polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid. The linker may include a linear or branched molecule. The linker may include or lack a reactive moiety (e.g., a cleavable moiety). The linker moiety of the nucleotide arm can interact with a polymerase. The type and position of the moiety in the linker can be selected to optimize interaction with the polymerase binding pocket. For example, an amide moiety in the linker can interact with the polymerase through hydrogen bonding. An aromatic moiety in the linker can interact with the polymerase through hydrophobic interactions. The nucleotide arm can include a linker 6 that includes a carbonyl oxygen adjacent to the nucleotide unit and an aryl moiety. Crystallographic data indicate that the carbonyl oxygen interacts with a lysine residue in the polymerase binding pocket. Capture assays indicate that linkers with an aromatic moiety can exhibit improved binding to polymerases compared to linkers lacking the aromatic moiety (see, e.g., Figures 11 and 12, and Example 5).

[0224] In some embodiments, the nucleotide arm can comprise a linker that can include any of the linker structures shown in Figures 5A-5F. In some embodiments, R1 of the linker can comprise any group, e.g., a nucleotide, a nucleoside, or an analog thereof. In some embodiments, R2 of the linker can comprise any group, e.g., a spacer (see, e.g., the top of Figure 5A). In some embodiments, in the linker, the value of m can be 0-10. In some embodiments, in the linker, the value of n can be 1-6.

[0225] In some embodiments, the linker can comprise an aliphatic chain having 2 to 8 units. In some embodiments, the linker can comprise an oligoethylene glycol moiety having 2 to 8 units. In some embodiments, the linker can comprise an aromatic group. In some embodiments, the linker can comprise an aromatic group or an oligoethylene glycol moiety having 2 to 8 units. In some embodiments, the linker can comprise an aliphatic chain having 2 to 6 subunits. In some embodiments, the linker can comprise an oligoethylene glycol moiety having 2 to 6 subunits. In some embodiments, the aromatic group can comprise an aryl group (see, e.g., N3 linker and linkers 1-7 in Figures 5A-5C). In some embodiments, the aromatic group can comprise a 6-carbon ring group. In some embodiments, the aromatic group can comprise a meta-aminomethylbenzoic acid (also known as 3-aminomethylbenzoic acid) group (mAMBA) (e.g., linkers 8 and 9 in Figure 5C). In some embodiments, the linker can comprise a fluorenylmethoxycarbonyl protecting group (Fmoc), which can be removed when the linker is attached to the spacer. In some embodiments, the linker can include an NHS ester group (N-hydroxysuccinimide) that can be removed when the linker is attached to a nucleotide unit.

[0226] Nucleotide Arm: Spacer-Linker-Nucleotide In some embodiments, the nucleotide arm can include a spacer connected to any of the linkers shown in Figures 5A-5F, and the linker can be connected to any nucleotide or nucleoside. The nucleotide arm is shown in Figures 6A and 6B.

[0227] In some embodiments, the nucleotide arm can comprise a spacer having the structure shown in Figure 5A (top). The spacer can be any length, for example, the value of m can be 1 or at least 2, at least 5, at least 10, at least 20, at least 25, at least 50, at least 75, at least 100, at least 200, at least 500, at least 750, at least 1000, or 2000 or more. In some embodiments, the value of m can be about 20-500, or about 100-110 (e.g., 5,000 g / mol PEG). In some embodiments, in the spacer shown in Figure 5A, the value of o can be 1-50. In some embodiments, the value of o can be about 1-10, or the value of o can be about 4. In some embodiments, the spacer can comprise polyethylene glycol, polypropylene glycol, vinyl acetate, polylactic acid, or polyglycolic acid.

[0228] In some embodiments, the nucleotide arm can comprise any type of nucleotide or nucleoside connected to a linker. Nucleotides include, but are not limited to, dATP, dGTP, dCTP, dTTP, or dUTP. The nucleotide unit can be connected to the linker via propargylamine attachment at the 5-position of the pyrimidine base, the 7-position of the purine base, the 1-position of the pyrimidine base, or the 9-position of the purine base.

[0229] In some embodiments, certain combinations of linkers and nucleotides can exhibit improved nucleotide discrimination by the polymerase, as indicated by brighter fluorescent signals in nucleotide capture assays (see, e.g., Figures 9, 10, 11, and Example 4). Note that the "N3" linker is photosensitive. The "N3" moiety can be useful for reducing residual fluorescent signals seen in circulation assays.

[0230] Linker-nucleotide unit In some embodiments, any of the linkers described herein can be attached to a nucleotide to form a nucleotide-linker molecule. Non-limiting examples of nucleotide-linker configurations are shown in Figures 7A-7G. For example, a nucleotide unit can be attached to the linker at the 5-position of the pyrimidine base, or the 7-position of the purine base, or the 1-position of the pyrimidine base, or the 9-position of the purine base, via a propargylamine attachment.

[0231] Biotinylated nucleotide arms In some embodiments, the nucleotide arm can be a biotinylated nucleotide arm. In some embodiments, the biotinylated nucleotide arm comprises (i) biotin, (ii) a spacer, (iii) a linker, and (iv) a nucleotide (or nucleoside). Non-limiting examples of biotinylated nucleotide arms are shown in Figures 8A and 8B.

[0232] In some embodiments, the nucleotide conjugate can comprise a core attached to multiple biotinylated nucleotide arms. In some embodiments, the biotinylated nucleotide arms can comprise a core attachment moiety. In some embodiments, the biotinylated nucleotide arms can comprise a spacer. In some embodiments, the biotinylated nucleotide arms can comprise a linker. In some embodiments, the biotinylated nucleotide arms can comprise a nucleotide unit. In some embodiments, the biotinylated nucleotide arms can comprise a core attachment moiety, a spacer, a linker, and a nucleotide unit. In some embodiments, each biotinylated nucleotide arm comprises (i) a biotin moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit or nucleoside unit.

[0233] Nucleotide Unit In some embodiments, any of the nucleotide arms and biotinylated nucleotide arms described herein can comprise a nucleotide unit. In some embodiments, the nucleotide unit can bind to a polymerase complexed with a nucleic acid template and a nucleic acid primer (e.g., nucleotide-associated). The nucleotide unit can also be separated from the complexed polymerase and re-associated with the same complexed polymerase or another complexed polymerase in the vicinity of the nucleotide conjugate.

[0234] The nucleotide arm and the biotinylated nucleotide arm can comprise a nucleotide unit, where the nucleotide unit can comprise a heterocyclic base, a sugar, and at least one phosphate group. In some embodiments, the nucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate groups.

[0235] The nucleotide arm and biotinylated nucleotide arm can include a nucleotide unit (or nucleoside unit) containing a heterocyclic base, including a purine base or a pyrimidine base or an analog thereof.

[0236] In some embodiments, the 5-position of the pyrimidine base can be joined to a linker, the 7-position of the purine base can be joined to a linker, the 1-position of the pyrimidine base can be joined to a linker, or the 9-position of the purine base can be joined to a linker.

[0237] In some embodiments, the nucleotide arm and biotinylated nucleotide arm comprise a nucleotide arm unit that is a propargylamine group-modified nucleotide, in which the 5-position of the pyrimidine base or the 7-position of the purine base can be connected to the linker via a propargylamine group, or the 1-position of the pyrimidine base or the 9-position of the purine base can be connected to the linker via a propargylamine group.

[0238] In some embodiments, the nucleotide arm and the biotinylated nucleotide arm can comprise a nucleotide unit having a ribose or deoxyribose sugar moiety. In some embodiments, the nucleotide unit can be adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), and deoxyadenosine monophosphate (dAMP), thymidine triphosphate (TTP), thymidine diphosphate (TDP), thymidine monophosphate (TMP), deoxythymidine triphosphate (dTTP), deoxythymidine diphosphate (dTDP), deoxythymidine monophosphate (dTMP), uridine triphosphate (UTP), uridine diphosphate (UDP), uridine monophosphate (UMP), deoxyuridine triphosphate (UTP), deoxyuridine diphosphate (UDP), deoxyuridine monophosphate (UMP), deoxyuridine triphosphate (UTP), deoxyuridine diphosphate (UDP), deoxyuridine monophosphate (UDP), deoxyuridine monophosphate (UDP), deoxyuridine triphosphate (UTP), deoxyuridine di ...UTP), deoxyuridine diphosphate (UDP), deoxyuridine monophosphate (UDP), deoxyuridine triphosphate (UTP), deoxy It may be selected from the group consisting of lysine triphosphate (dUTP), deoxyuridine diphosphate (dUDP), deoxyuridine monophosphate (dUMP), cytidine triphosphate (CTP), cytidine diphosphate (CDP), cytidine monophosphate (CMP), deoxycytidine triphosphate (dCTP), deoxycytidine diphosphate (dCDP), deoxycytidine monophosphate (dCMP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), deoxyguanosine triphosphate (dGTP), deoxyguanosine diphosphate (dGDP), and deoxyguanosine monophosphate (dGMP).

[0239] In some embodiments, the nucleotide arm and the biotinylated nucleotide arm can comprise a nucleotide unit (or nucleoside unit) having a sugar moiety comprising ribose, deoxyribose, or an analog thereof. In some embodiments, the sugar moiety can comprise a 3' OH group. In some embodiments, the nucleotide unit having a sugar 3' OH group can bind to a complexed polymerase, including a polymerase bound to a nucleic acid template (or a self-priming template / primer nucleic acid molecule) that can hybridize to a nucleic acid primer. For example, the nucleotide unit can bind to a polymerase and be attached to the 3' end of the primer at a position opposite the complementary nucleotide in the template strand. The nucleotide unit having a sugar 3' OH group can undergo nucleotide incorporation in a polymerase-catalyzed reaction. The sugar 3' OH group on the incorporated nucleotide unit can mediate subsequent polymerase-catalyzed incorporation of a nucleotide, for example, in a primer extension reaction.

[0240] In some embodiments, the nucleotide arm and biotinylated nucleotide arm can comprise a nucleotide unit (or nucleoside unit) having a sugar moiety in which the 3'OH group is replaced with a blocking group. In some embodiments, the nucleotide unit having a 3' blocking group can bind to a complexed polymerase, including a polymerase bound to a nucleic acid template (or a self-priming template / primer nucleic acid molecule) hybridized to a nucleic acid primer. For example, the nucleotide unit can bind to a polymerase and can be attached to the 3' end of the primer at a position opposite the complementary nucleotide in the template strand. The nucleotide unit having a 3' blocking group can undergo nucleotide incorporation in a polymerase-catalyzed reaction. The 3' blocking group on the incorporated nucleotide unit can inhibit / prevent subsequent polymerase-catalyzed incorporation of a nucleotide, for example, in a primer extension reaction.

[0241] Modified nucleotide arms In some embodiments, any of the nucleotide arms and biotinylated nucleotide arms described herein can include a linker having a reactive group at any position along the linker. For example, the azide moiety in the N3 linker (FIG. 5A) can be replaced with a reactive group. In some embodiments, any of the nucleotide arms and biotinylated nucleotide arms described herein can include a linker having a reactive group selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0242] In some embodiments, the reactive groups in the linker can react with chemical reagents. For example, alkyl, alkenyl, alkynyl, and allyl reactive groups react with tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Aryl and benzyl reactive groups react with H and palladium on carbon (Pd / C). Amine, amide, keto, isocyanate, phosphate, thiol, and disulfide reactive groups react with phosphines or thiol groups containing β-mercaptoethanol or dithiothritol (DTT). Carbonate reactive groups react with potassium carbonate (KCO) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The reactive groups urea and silyl react with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0243] In some embodiments, the reactive group in the linker can include an azide, azide, or azidomethyl group. In some embodiments, the azide, azide, or azidomethyl group in the linker can react with a chemical. In some embodiments, the chemical can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0244] In some embodiments, any of the nucleotide arms and biotinylated nucleotide arms described herein can comprise a nucleotide unit having a sugar moiety in which the 3' OH group is replaced with a chain-terminating moiety (blocking group), wherein the sugar 3' blocking group is selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0245] In some embodiments, the sugar 3' blocking group can comprise a 3'-O-azidomethyl group, a 3'-O-methyl group, a 3'-O-alkylhydroxylamino group, a 3'-phosphorothioate group, a 3'-O-malonyl group, or a 3'-O-benzyl group.

[0246] In some embodiments, the nucleotide arm and biotinylated nucleotide arm can include nucleotide units with sugar 3' blocking groups that react with chemical reagents. For example, the sugar 3' blocking groups alkyl, alkenyl, alkynyl, and allyl can react with piperidine and tetrakis(triphenylphosphine)palladium(0) (Pd(P(CH))) or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The aryl and benzyl blocking groups can react with H and palladium on carbon (Pd / C). The amine, amide, keto, isocyanate, phosphate, thiol, and disulfide blocking groups can react with phosphines or thiol groups containing β-mercaptoethanol or dithiothritol (DTT). The carbonate blocking group can react with potassium carbonate (KCO) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). The blocking groups urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0247] In some embodiments, the nucleotide arms and biotinylated nucleotide arms can include nucleotide units having sugar 3' blocking groups that include azide, azido, or azidomethyl groups.

[0248] In some embodiments, the azide, azide, or azidomethyl 3' blocking group can be reacted with a chemical reagent. In some embodiments, the chemical can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bis-sulfotriphenylphosphine (BS-TPP), or tri(hydroxyproyl)phosphine (THPP).

[0249] In some embodiments, the nucleotide arms and biotinylated nucleotide arms can include nucleotide units with a chain of one, two, or three phosphorus atoms, where the chain is attached to the 5' carbon of the sugar moiety via an ester or phosphoramidate linkage. In some embodiments, the nucleotide units can be analogs with phosphorus chains in which the phosphorus atoms are linked with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain can include substituted side chain groups including O, S, or BH3. In some embodiments, the chain can include phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite groups.

[0250] Combinations of Nucleotide Conjugates The present disclosure provides compositions, systems, and kits comprising a plurality of nucleoside conjugates, which may comprise a mixture of at least two subpopulations of nucleoside conjugates labeled with different reporter moieties. In some embodiments, at least a first subpopulation of nucleoside conjugates may be labeled with a first reporter moiety corresponding to a first nucleotide unit on the nucleotide arm. In some embodiments, at least a second subpopulation of nucleoside conjugates may be labeled with a second reporter moiety corresponding to a second nucleotide unit on the nucleotide arm. In some embodiments, the first and second reporter moieties may be different from each other. In some embodiments, the plurality of nucleoside conjugates may further comprise at least a third subpopulation of nucleoside conjugates labeled with a third reporter moiety, and the first, second, and third reporter moieties may be different from each other. In some embodiments, the plurality of nucleoside conjugates can further include at least a fourth subpopulation of nucleoside conjugates labeled with a fourth reporter moiety, where the first, second, third, and fourth reporter moieties can be different from one another. In some embodiments, additional subpopulations of labeled nucleoside conjugates (e.g., fifth, sixth, seventh, eighth, ninth, tenth, or more) can be added to the mixture. In some embodiments, the reporter moiety can be a fluorophore. In some embodiments, a first subpopulation of nucleoside conjugates can be labeled with a first fluorophore, and a second fluorophore of the nucleoside conjugates can be labeled with a second fluorophore. In some cases, the first fluorophore and the second fluorophore can be different.

[0251] The present disclosure provides compositions, systems, and kits comprising a plurality of nucleoside conjugates, which may comprise a mixture of at least two subpopulations of nucleoside conjugates labeled with different reporter moieties. In some embodiments, at least a first subpopulation of nucleoside conjugates can be labeled with a first reporter moiety corresponding to a first nucleotide unit on a nucleotide arm. In some embodiments, at least a second subpopulation of nucleoside conjugates can be unlabeled (e.g., dark nucleoside conjugates).

[0252] The present disclosure provides compositions, systems, and kits comprising a plurality of nucleoside conjugates, including a mixture of at least three subpopulations of nucleoside conjugates labeled with different reporter moieties. In some embodiments, at least a first subpopulation of nucleoside conjugates can be labeled with a first reporter moiety corresponding to a first nucleotide unit on a nucleotide arm. In some embodiments, at least a second subpopulation of nucleoside conjugates can be labeled with a second reporter moiety corresponding to a second nucleotide unit on a nucleotide arm. In some embodiments, at least a third subpopulation of nucleoside conjugates can be unlabeled (e.g., dark nucleoside conjugates). In some embodiments, the first and second reporter moieties can be different from each other.

[0253] The present disclosure provides compositions, systems, and kits comprising a plurality of nucleoside conjugates, including a mixture of at least four subpopulations of nucleoside conjugates labeled with different reporter moieties. In some embodiments, the mixture of nucleoside conjugates can have at least a first subpopulation of nucleoside conjugates, where the first subpopulation can be labeled with a first reporter moiety corresponding to a first nucleotide unit on the nucleotide arm. In some embodiments, the mixture of nucleoside conjugates can have at least a second subpopulation of nucleoside conjugates, where the second subpopulation can be labeled with a first reporter moiety corresponding to a second nucleotide unit on the nucleotide arm. In some embodiments, the mixture of nucleoside conjugates can have at least a second subpopulation of nucleoside conjugates, where a third subpopulation is labeled with a third reporter moiety. In some embodiments, the mixture of nucleoside conjugates can have at least a fourth subpopulation of nucleoside conjugates, and the fourth subpopulation can be unlabeled (e.g., dark nucleoside conjugates). Optionally, the first, second, and third reporter moieties can be different from one another.

[0254] Disclosed herein are formulations comprising a first nucleotide conjugate and a second nucleotide conjugate. In some embodiments, the formulation comprises a first nucleotide conjugate, a second nucleotide conjugate, and a third nucleotide conjugate. In some embodiments, the formulation comprises a first nucleotide conjugate, a second nucleotide conjugate, a third nucleotide conjugate, and a fourth nucleotide conjugate. In some embodiments, the first nucleotide conjugate comprises a first label. In some embodiments, the second nucleotide conjugate comprises a second label. In some embodiments, the third nucleotide conjugate comprises a third label. In some embodiments, the fourth nucleotide conjugate comprises a fourth label. In some embodiments, the first label comprises a first fluorophore. In some embodiments, the second label comprises a second fluorophore. In som...

Claims

1. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:391, wherein said amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO:

391.

2. 2. The synthetic polypeptide of claim 1, wherein the amino acid sequence comprises the D141A mutation and the D143A mutation.

3. 3. The synthetic polypeptide of claim 1 or 2, wherein the amino acid sequence further comprises a Y410A mutation, a L409S mutation, a Y261A mutation, a P411G mutation, a F406I mutation, a P411A mutation, a Y7A mutation, a Y493I mutation, a Y493T mutation, a V513I mutation, a L409A mutation, a A485S mutation, a Y410G mutation, a I521H mutation, or a K507L mutation, or any combination thereof, with reference to SEQ ID NO:

391.

4. The amino acid sequence (a) the Y410A mutation with reference to SEQ ID NO: 391; (b) the L409S mutation, the Y410A mutation, and the Y261A mutation with reference to SEQ ID NO: 391; (c) the L409S mutation, the Y410A mutation, the P411G mutation, and the Y261A mutation with reference to SEQ ID NO: 391; (d) the Y261A mutation, the F406I mutation, the L409S mutation, the Y410A mutation, and the P411A mutation with reference to SEQ ID NO: 391; (e) the Y7A mutation, the Y261A mutation, and the Y410A mutation with reference to SEQ ID NO: 391; (f) the Y261A mutation, the Y410A mutation, and the Y493I mutation with reference to SEQ ID NO: 391; (g) the Y261A mutation, the Y410A mutation, and the Y493T mutation with reference to SEQ ID NO: 391; (h) the Y261A mutation, the Y410A mutation, and the V513I mutation with reference to SEQ ID NO: 391; (i) the Y7A mutation, the Y261A mutation, the L409S mutation, and the Y410A mutation with reference to SEQ ID NO: 391; (j) the Y261A mutation, the L409A mutation, the Y410A mutation, and the P411A mutation with reference to SEQ ID NO: 391; (k) the Y261A mutation, the L409S mutation, the Y410A mutation, and the P411G mutation with reference to SEQ ID NO: 391; (l) the Y261A mutation, the L409S mutation, and the Y410A mutation with reference to SEQ ID NO: 391; (m) the Y261A mutation, the L409S mutation, and the Y410G mutation with reference to SEQ ID NO: 391; (n) the Y261A mutation, the L409A mutation, the Y410A mutation, the P411A mutation, and the A485S mutation with reference to SEQ ID NO: 391; (O) the Y261 mutation, the L409S mutation, the Y410A mutation, the P411G mutation, and the A485S mutation with reference to SEQ ID NO: 391; (p) the Y261A mutation, the L409S mutation, the Y410A mutation, and the A485S mutation with reference to SEQ ID NO: 391; (q) the Y261A mutation, the L409S mutation, the Y410G mutation, and the A485S mutation with reference to SEQ ID NO: 391; (r) the Y261A mutation, the L409A mutation, the Y410A mutation, the P411A mutation, and the I521H mutation with reference to SEQ ID NO: 391; (s) the Y261A mutation, the L409S mutation, the Y410A mutation, the P411G mutation, and the I521H mutation with reference to SEQ ID NO: 391; (T) the Y261A mutation, the L409S mutation, the Y410A mutation, and the I521H mutation with reference to SEQ ID NO: 391; (u) the Y261A mutation, the L409S mutation, the Y410G mutation, and the I521H mutation with reference to SEQ ID NO: 391; or (v) the Y261A mutation, the L409S mutation, the Y410A mutation, the A485S mutation, the K507L mutation, and the I521H mutation with reference to SEQ ID NO:

391. The synthetic polypeptide of claim 3, further comprising:

5. 2. The synthetic polypeptide of claim 1, wherein the amino acid sequence further comprises any one of SEQ ID NOs: 392-413.

6. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:414, wherein said amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO:

414.

7. 7. The synthetic polypeptide of claim 6, wherein the amino acid sequence comprises the D158A mutation and the E160A mutation.

8. 8. The synthetic polypeptide of claim 6 or 7, wherein the amino acid sequence further comprises a L431A mutation, a Y432A mutation, a P433I mutation, an A507S mutation, a K506Q mutation, a P433A mutation, an I543H mutation, a L431S mutation, a P433G mutation, a K529L mutation, or a Y432G mutation, or any combination thereof, with reference to SEQ ID NO:

414.

9. The amino acid sequence (a) the L431A mutation, the Y432A mutation, the P433I mutation, and the A507S mutation with reference to SEQ ID NO: 414; (b) the L431A mutation, the Y432A mutation, the P433I mutation, and the K506Q mutation with reference to SEQ ID NO: 414; (c) the L431A mutation, the Y432A mutation, the P433I mutation, the K506Q mutation, and the A507S mutation with reference to SEQ ID NO: 414; (d) the L431A mutation, the Y432A mutation, and the P433A mutation with reference to SEQ ID NO: 414; (e) the L431A mutation, the Y432A mutation, the P433A mutation, and the A507S mutation with reference to SEQ ID NO: 414; (f) the L431A mutation, the Y432A mutation, the P433A mutation, and the I543H mutation with reference to SEQ ID NO: 414; (g) the L431S mutation, the Y432A mutation, and the P433G mutation with reference to SEQ ID NO: 414; (h) the L431S mutation, the Y432A mutation, the P433G mutation, and the A507S mutation with reference to SEQ ID NO: 414; (i) the L431S mutation, the Y432A mutation, the P433G mutation, and the I543H mutation with reference to SEQ ID NO: 414; (j) the L431S mutation and the Y432A mutation with reference to SEQ ID NO: 414; (l) the L431S mutation, the Y432A mutation, and the A507S mutation with reference to SEQ ID NO: 414; (m) the L431S mutation, the Y432A mutation, the A507S mutation, the K529L mutation, and the I543H mutation with reference to SEQ ID NO: 414; (n) the L431S mutation, the Y432A mutation, and the I543H mutation with reference to SEQ ID NO: 414; (o) the L431S mutation and the Y432G mutation with reference to SEQ ID NO: 414; (p) the L431S mutation, the Y432G mutation, and the A507S mutation with reference to SEQ ID NO: 414; or (q) the L431S mutation, the Y432G mutation, and the I543H mutation with reference to SEQ ID NO:

414. The synthetic polypeptide of claim 8, further comprising:

10. 2. The synthetic polypeptide of claim 1, wherein the amino acid sequence further comprises any one of SEQ ID NOs:415-430.

11. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:431, wherein said amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO:

431.

12. 12. The synthetic polypeptide of claim 11, wherein the amino acid sequence comprises the D141A mutation and the E143A mutation.

13. 13. The synthetic polypeptide of claim 11 or 12, wherein the amino acid sequence further comprises a Y412A mutation, a L411A mutation, a P413A mutation, an A488S mutation, a I524H mutation, a L411S mutation, a P413G mutation, a K510L mutation, or a Y412G mutation, or any combination thereof, with reference to SEQ ID NO:

431.

14. The amino acid sequence (a) the Y412A mutation with reference to SEQ ID NO: 431; (b) the L411A mutation, the Y412A mutation, and the P413A mutation with reference to SEQ ID NO: 431; (c) the L411A mutation, the Y412A mutation, the P413A mutation, and the A488S mutation with reference to SEQ ID NO: 431; (d) the L411A mutation, the Y412A mutation, the P413A mutation, and the I524H mutation with reference to SEQ ID NO: 431; (e) the L411S mutation, the Y412A mutation, and the P413G mutation with reference to SEQ ID NO: 431; (f) the L411S mutation, the Y412A mutation, the P413G mutation, and the A488S mutation with reference to SEQ ID NO: 431; (g) the L411S mutation, the Y412A mutation, the P413G mutation, and the I524H mutation with reference to SEQ ID NO: 431; (h) the L411S mutation and the Y412A mutation with reference to SEQ ID NO: 431; (i) the L411S mutation, the Y412A mutation, and the A488S mutation with reference to SEQ ID NO: 431; (j) the L411S mutation, the Y412A mutation, the A488S mutation, the K510L mutation, and the I524H mutation with reference to SEQ ID NO: 431; (k) the L411S mutation, the Y412A mutation, and the I524H mutation with reference to SEQ ID NO: 431; (l) the L411S mutation and the Y412G mutation with reference to SEQ ID NO: 431; (m) the L411S mutation, the Y412G mutation, and the A488S mutation with reference to SEQ ID NO: 431; or (n) the L411S mutation, the Y412G mutation, and the I524H mutation with reference to SEQ ID NO: 431 14. The synthetic polypeptide of claim 13, further comprising:

15. 12. The synthetic polypeptide of claim 11, wherein the amino acid sequence further comprises any one of SEQ ID NOs: 432-445.

16. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:446, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO:

446.

17. 17. The synthetic polypeptide of claim 16, wherein the amino acid sequence comprises two or more of the D141A mutation, the E143A mutation, and the Y412A mutation with reference to SEQ ID NO:

446.

18. 18. The synthetic polypeptide of claim 16 or 17, wherein the amino acid sequence further comprises SEQ ID NO:

447.

19. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:448, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO:

448.

20. 20. The synthetic polypeptide of claim 19, wherein the amino acid sequence further comprises two or more of the D149A mutation, the E151A mutation, the Y272A mutation, and the Y422A mutation, with reference to SEQ ID NO:

448.

21. 21. The synthetic polypeptide of claim 19 or 20, wherein the amino acid sequence further comprises SEQ ID NO:

449.

22. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:450, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation, with reference to SEQ ID NO:

450.

23. 23. The synthetic polypeptide of claim 22, wherein the amino acid sequence comprises two or more of the D149A mutation, the E151A mutation, the Y272A mutation, and the Y422A mutation, with reference to SEQ ID NO:

450.

24. 24. The synthetic polypeptide of claim 22 or 23, wherein the amino acid sequence further comprises SEQ ID NO:

451.

25. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:452, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO:

452.

26. 26. The synthetic polypeptide of claim 25, wherein the amino acid sequence comprises two or more of the D170A mutation, the E172A mutation, and the Y449A mutation, with reference to SEQ ID NO:

452.

27. 27. The synthetic polypeptide of claim 25 or 26, wherein the amino acid sequence further comprises SEQ ID NO:

453.

28. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:454, wherein the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO:

454.

29. 29. The synthetic polypeptide of claim 28, wherein the amino acid sequence comprises two or more of the D170A mutation, the E172A mutation, and the Y449A mutation, with reference to SEQ ID NO:

454.

30. 30. The synthetic polypeptide of claim 28 or 29, wherein the amino acid sequence further comprises SEQ ID NO:

455.

31. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:456, wherein the amino acid sequence comprises a D173A mutation, an E175A mutation, a Y452A mutation, or a C468A mutation with reference to SEQ ID NO:

456.

32. 32. The synthetic polypeptide of claim 31 , wherein the amino acid sequence comprises two or more of the D173A mutation, the E175A mutation, the Y452A mutation, and the C468A mutation, with reference to SEQ ID NO:

456.

33. 33. The synthetic polypeptide of claim 31 or 32, wherein the amino acid sequence further comprises SEQ ID NO:

457.

34. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:458, wherein the amino acid sequence comprises a D149A mutation, an E151A mutation, a Y272A mutation, or a Y422A mutation with reference to SEQ ID NO:

458.

35. 35. The synthetic polypeptide of claim 34, wherein the amino acid sequence comprises two or more of the D149A mutation, the E151A mutation, the Y272A mutation, and the Y422A mutation, with reference to SEQ ID NO:

458.

36. 36. The synthetic polypeptide of claim 34 or 35, wherein the amino acid sequence further comprises SEQ ID NO:

459.

37. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:460, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO:

460.

38. 38. The synthetic polypeptide of claim 37, wherein the amino acid sequence comprises two or more of the D141A mutation, the E143A mutation, and the Y412A mutation with reference to SEQ ID NO:

460.

39. 39. The synthetic polypeptide of claim 37 or 38, wherein the amino acid sequence further comprises SEQ ID NO:

461.

40. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:462, wherein the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO:

462.

41. 41. The synthetic polypeptide of claim 40, wherein the amino acid sequence further comprises an amino acid deletion from amino acid position I496 to amino acid position S1033, or any portion of that amino acid sequence, with reference to SEQ ID NO:

462.

42. 42. The synthetic polypeptide of claim 40 or 41, wherein the amino acid sequence comprises two or more of the D141A mutation, the E143A mutation, and the Y412A mutation with reference to SEQ ID NO:

462.

43. 43. The synthetic polypeptide of any one of claims 40-42, wherein the amino acid sequence further comprises SEQ ID NO:

463.

44. 44. A synthetic polypeptide according to any one of claims 1 to 43, which is purified or isolated.

45. 45. The synthetic polypeptide of any one of claims 1 to 44, which is a polymerase.

46. 46. ​​The synthetic polypeptide of claim 45, wherein the polymerizing enzyme comprises a DNA polymerase or a polymerizing portion thereof.

47. 11. A method of nucleic acid analysis comprising the steps of providing a formulation comprising: (i) a synthetic polypeptide according to any one of claims 1 to 47; (ii) a primed nucleic acid sequence; and (iii) a nucleotide unit complementary to a nucleotide of the primed nucleic acid, wherein the formulation is provided under conditions sufficient to form a binding complex comprising the synthetic polypeptide, the nucleotide unit, and the primed nucleic acid sequence.

48. 48. The method of claim 47, comprising a nucleic acid sequencing method.

49. 48. The method of claim 47, comprising sequencing by synthesis methods.

50. The formulation further comprises one or more compositions, wherein one of the one or more compositions comprises (a) a core; (b) at least two nucleotide arms, wherein one of the at least two nucleotide arms comprises (i) a core attachment moiety attached to the core; (ii) a spacer attached to the core attachment portion; (iii) a linker attached to the spacer; and (iv) the nucleotide unit bound to the linker and at least two nucleotide arms comprising:

51. The linker is 【Chemistry 1】 【Chemistry 2】 wherein n is 1 to 6 and m is 0 to 10.

52. One or more containers, A synthetic polypeptide according to any one of claims 1 to 46, and Detectable Nucleotide Units A container containing instructions for introducing said synthetic polypeptide and said nucleotide unit into said primed nucleic acid sequence that contains a nucleotide complementary to said nucleotide unit under conditions sufficient to form a binding complex comprising said synthetic polypeptide, said nucleotide unit, and said primed nucleic acid sequence; Including the kit.

53. The method further comprises the steps of: A core, at least two nucleotide arms, wherein one of the at least two nucleotide arms comprises a core attachment portion attached to the core; A spacer coupled to the core attachment portion; a linker attached to the spacer, and The nucleotide unit attached to the linker and at least two nucleotide arms comprising:

54. The linker is 【Chemistry 3】 【Chemistry 4】 wherein n is 1 to 6 and m is 0 to 10.

55. A synthetic polypeptide according to any one of claims 1 to 46, a primed nucleic acid sequence; a nucleotide unit that is detectable and complementary to a nucleotide in the primed nucleic acid sequence; a system configured to form a binding complex comprising the primed nucleic acid sequence, the synthetic polypeptide, and the nucleotide unit.

56. The method further comprises the steps of: A core, at least two nucleotide arms, wherein one of the at least two nucleotide arms comprises a core attachment portion attached to the core; A spacer coupled to the core attachment portion; a linker attached to the spacer, and The nucleotide unit attached to the linker and at least two nucleotide arms comprising:

56. The system of claim 55, wherein the binding complex comprises the primed nucleic acid sequence, the synthetic polypeptide, and the one composition.

57. two or more copies of the primed nucleic acid sequence; two or more of the synthetic polypeptides, wherein the composition is configured to form a multivalent binding complex comprising two or more nucleotide units of one of the compositions, the two or more copies of the primed nucleic acid sequence, and two or more of the synthetic polypeptides; 56. The system of claim 55, further comprising:

58. The linker is 【Chemistry 5】 【Chemistry 6】 wherein n is 1 to 6 and m is 0 to 10.

59. 50. A system comprising: (i) a synthetic polypeptide according to any one of claims 1 to 46; (ii) a primed nucleic acid sequence; and (iii) a nucleotide unit complementary to a nucleotide of the primed nucleic acid, provided under conditions sufficient to form a binding complex comprising said synthetic polypeptide, said nucleotide unit, and said primed nucleic acid sequence.

60. A system comprising (i) a synthetic polypeptide according to any one of claims 1 to 46, and (ii) a nucleotide.

61. 61. The system of claim 60, wherein the nucleotide comprises a blocking group.

62. 61. The system of claim 60, wherein the nucleotide does not contain a blocking group.

63. 61. The system of claim 60, wherein the nucleotide comprises a label.

64. 61. The system of claim 60, wherein the nucleotide is unlabeled.

65. 62. The system of claim 61, wherein the blocking group is linked to a 3' carbon of a sugar moiety of the nucleotide, and the blocking group reacts with a chemical compound to remove the blocking group from the nucleotide and generate a nucleotide that includes an OH moiety at the 3' carbon of the sugar moiety of the nucleotide.

66. 66. The system of claim 65, wherein the blocking group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group.

67. a) the alkyl, alkenyl, alkynyl, or aryl group of the blocking group is tetrakis(triphenylphosphine)palladium(0) (Pd(P(C ６ H ５ ) ３ ) ４ ), or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ), b) the aryl or benzyl group of the blocking group is H ２ and palladium on carbon (Pd / C), c) reacting an amine, amide, keto, isocyanate, phosphate, thiol, or disulfide group of the blocking group with a phosphine or thiol group with beta-mercaptoethanol or dithiothreitol (DTT); d) The carbonate group of the blocking group is dissolved in potassium carbonate (KCO 3 ) in methanol (MeOH). ２ CO ３ ), triethylamine in pyridine, or Zn in acetic acid (AcOH), 67. The system of claim 66, wherein e) the urea or silyl group of the blocking group reacts with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride, or triethylamine trihydrofluoride.

68. 67. The system of claim 66, wherein the azide of the blocking group comprises an azide, azido, or azidomethyl group.

69. 69. The system of claim 68, wherein the azide, azide, or azidomethyl group reacts with a chemical comprising a phosphine compound.

70. 70. The system of claim 69, wherein the phosphine compound comprises a derivatized tri-alkylphosphine moiety or a derivatized tri-arylphosphine moiety.

71. 70. The system of claim 69, wherein the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

72. (a) a core; (b) at least two nucleotide arms, wherein one of the at least two nucleotide arms comprises (i) a core attachment moiety attached to the core; (ii) a spacer attached to the core attachment portion; (iii) a linker attached to the spacer; and (iv) a nucleotide unit bound to the linker and at least two nucleotide arms comprising: The linker is 【Chemistry 7】 【Chemistry 8】 wherein n is 1-6 and m is 0-10.

73. The linker is 【Chemistry 9】 73. The composition of claim 72, comprising:

74. The linker is 【Chemistry 10】 73. The composition of claim 72, comprising:

75. The linker is 【Chemistry 11】 73. The composition of claim 72, comprising:

76. The linker is 【Chemistry 12】 73. The composition of claim 72, comprising:

77. The linker is 【Chemistry 13】 wherein n is 1 to 6 and m is 0 to 10.

78. The linker is 【Chemistry 14】 wherein n is 1 to 6 and m is 0 to 10.

79. The linker is 【Chemistry 15】 wherein n is 1 to 6 and m is 0 to 10.

80. The linker is 【Chemistry 16】 wherein n is 1 to 6 and m is 0 to 10.

81. The linker is 【Chemistry 17】 73. The composition of claim 72, comprising:

82. The linker is 【Chemistry 18】 73. The composition of claim 72, comprising:

83. The linker is 【Chemistry 19】 73. The composition of claim 72, comprising:

84. The linker is 【Chemistry 20】 73. The composition of claim 72, comprising:

85. The linker is 【Chemistry 21】 73. The composition of claim 72, comprising:

86. 73. The composition of claim 72, wherein the at least two nucleotide arms comprise between 3 and 20 nucleotide arms.

87. 73. The composition of claim 72, wherein the core comprises a polypeptide.

88. 88. The composition of claim 87, wherein the polypeptide comprises streptavidin or avidin.

89. 73. The composition of claim 72, wherein the core attachment moiety comprises biotin.

90. 73. The composition of claim 72, further comprising a fluorescent label attached to said core or said one nucleotide arm.

91. The spacer is 【Chemical 22】 wherein m is 20 to 500 and o is 1 to 10.

92. 73. The composition of claim 72, wherein the nucleotide arm further comprises a reactive group attached to the nucleotide unit, the reactive group configured to react with a drug.

93. 93. The composition of claim 92, wherein the reactive group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group.

94. (a) The alkyl, alkenyl, alkynyl, or aryl group of the reactive group is tetrakis(triphenylphosphine)palladium(0) (Pd(P(C ６ H ５ ) ３ ) ４ ), or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), (b) the aryl or benzyl group of the reactive group is H ２ and palladium on carbon (Pd / C), (c) reacting an amine, amide, keto, isocyanate, phosphate, thiol, or disulfide group of the reactive group with a phosphine or thiol group with beta-mercaptoethanol or dithiothreitol (DTT); (d) The carbonate group of the reactive group is reacted with potassium carbonate (KCO3) in methanol (MeOH). ２ CO ３ ), triethylamine in pyridine, or Zn in acetic acid (AcOH), 94. The composition of claim 93, wherein (e) the urea or silyl group of the reactive group reacts with tetrabutylammonium fluoride, hydrogen fluoride pyridine (HF-pyridine), ammonium fluoride, or triethylamine trihydrofluoride.

95. 94. The composition of claim 93, wherein the azide group of the reactive group comprises an azide, azido, or azidomethyl group.

96. 96. The composition of claim 95, wherein the agent comprises a phosphine compound.

97. 97. The composition of claim 96, wherein the phosphine compound comprises a derivatized tri-alkyl or tri-aryl phosphine moiety.

98. 97. The composition of claim 96, wherein the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

99. 73. The composition of claim 72, wherein said one nucleotide unit of said at least two nucleotide arms comprises the same nucleobase type.

100. 73. The composition of claim 72, wherein the nucleotide unit comprises a blocking group linked to the 3' carbon of a sugar moiety of the nucleotide unit, and the blocking group reacts with a chemical compound to remove the blocking group from the nucleotide unit and generate a nucleotide unit comprising an OH moiety at the 3' carbon of the sugar moiety of the nucleotide unit.

101. 101. The composition of claim 100, wherein the blocking group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group.

102. a) the alkyl, alkenyl, alkynyl, or aryl group of the blocking group is tetrakis(triphenylphosphine)palladium(0) (Pd(P(C ６ H ５ ) ３ ) ４ ), or 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ), b) the aryl or benzyl group of the blocking group is H ２ and palladium on carbon (Pd / C), c) reacting an amine, amide, keto, isocyanate, phosphate, thiol, or disulfide group of the blocking group with a phosphine or thiol group with beta-mercaptoethanol or dithiothreitol (DTT); d) The carbonate group of the blocking group is substituted with potassium carbonate (K ２ CO ３ ), triethylamine in pyridine, or Zn in acetic acid (AcOH), The composition of claim 101, wherein e) the urea or silyl group of the blocking group is reacted with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride, or triethylamine trihydrofluoride.

103. 102. The composition of claim 101, wherein the azide of the blocking group comprises an azide, azido, or azidomethyl group.

104. 104. The composition of claim 103, wherein the azide, azide, or azidomethyl group is reacted with a chemical that comprises a phosphine compound.

105. The composition of claim 104, wherein the phosphine compound comprises a derivatized tri-alkyl or tri-aryl phosphine moiety.

106. 105. The composition of claim 104, wherein the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfotriphenylphosphine (BS-TPP).

107. 73. A formulation comprising at least two compositions according to claim 72, wherein the at least two compositions comprise a first composition and a second composition, and wherein a nucleotide unit of the first composition comprises a different nucleobase than a nucleotide unit of the second composition.

108. 73. A formulation comprising at least two compositions according to claim 72, wherein the at least two compositions comprise a first composition and a second composition; (a) a nucleotide unit of the first composition comprises a first blocking group linked to a 3' carbon of a sugar moiety, and the first blocking group is reacted with a chemical compound to remove the first blocking group and generate an OH moiety at the 3' carbon of the sugar moiety of the nucleotide unit of the first composition; (b) a nucleotide unit of the second composition comprises a second blocking group linked to the 3' carbon of a sugar moiety, the second blocking group reacts with a chemical compound to remove the second blocking group and generate an OH moiety at the 3' carbon of the sugar moiety of the nucleotide unit of the second composition, and the nucleotide unit of the first composition is different from the nucleotide unit of the second composition.

109. 73. A formulation comprising at least two compositions according to claim 72, wherein at least two of said compositions comprise a first composition and a second composition, and wherein the linker of said first composition is different from the linker of said second composition.

110. 73. A formulation comprising at least two compositions according to claim 72, wherein the at least two compositions comprise a first composition and a second composition, wherein a linker of the first composition comprises a first reactive group and a linker of the second composition comprises a second reactive group, the second reactive group being different from the first reactive group.

111. 73. A formulation comprising at least two compositions according to claim 72, wherein the at least two compositions comprise a first composition and a second composition, the first composition comprising a first fluorophore and the second composition comprising a second fluorophore, the first fluorophore emitting light at a wavelength different from the wavelength of light emitted from the second fluorophore.

112. 73. A formulation comprising at least two compositions according to claim 72, wherein the at least two compositions comprise a first composition and a second composition, the first composition comprising a fluorescent label and the second composition being unlabeled.

113. A kit for processing nucleic acid molecules, comprising: (a) a composition according to any one of claims 72 to 106 or a formulation according to any one of claims 107 to 112; (b) instructions for using the composition in a nucleotide identification reaction; and Kit including:

114. The kit of claim 113, further comprising an agent that reacts with a reactive group in a linker of the composition.

115. 114. The kit of claim 113, further comprising an agent that reacts with a reactive group at the 3' carbon of a sugar moiety in a nucleotide unit of the composition.

116. 114. The kit of claim 113, further comprising reagents for use in nucleotide coupling reactions.

117. 117. The kit of claim 116, wherein the reagent comprises a cation.

118. (i) a solution containing a cation; (ii) one or more polymerases; (iii) one or more primer sequences; (iv) one or more unlabeled nucleotides, or (v) any combination of (i) to (iv). The kit of claim 113, further comprising:

119. (a) a composition according to any one of claims 72 to 106; (b) two or more copies of a primed nucleic acid sequence, the two or more copies of the primed nucleic acid sequence comprising nucleotides complementary to the nucleotide units of the composition; and (c) two or more polymerases; wherein the system is configured to form a multivalent binding complex comprising two or more of said primed nucleic acid sequences, said two or more polymerases, and said composition.

120. The system of claim 119, wherein the multivalent binding complex is formed under conditions such that the nucleotide units of the composition are not incorporated into the two or more copies of the primed nucleic acid sequence.

121. The system of claim 120, wherein the two or more copies of the nucleic acid sequence and the two or more copies of the nucleic acid sequence primer molecule are immobilized to the support under conditions sufficient to immobilize the multivalent binding complex to the support.

122. A plurality of the multivalent binding complexes are immobilized on the support, and the density of the plurality of the multivalent binding complexes immobilized on the support is expressed as a density per square millimeter (mm ２ ) per 10 ２ ~10 ９ The system of claim 120,

123. The system of claim 122, wherein a plurality of said multivalent binding complexes on said support are in fluid communication with each other and with a solution of a reagent under conditions sufficient for a plurality of said multivalent binding complexes to react with the solution of a reagent in a massively parallel manner.

124. 107. A method of nucleic acid analysis comprising the step of introducing a composition according to any one of claims 72 to 106 to a primed nucleic acid sequence under conditions sufficient to form a binding complex comprising (i) a nucleotide unit of the composition and (ii) a nucleotide in the primed nucleic acid sequence, wherein the nucleotide is complementary to the nucleotide unit of the composition.

125. 107. A method of nucleic acid analysis comprising introducing a composition according to any one of claims 72 to 106 to two or more copies of a primed nucleic acid sequence under conditions sufficient to form a multivalent binding complex comprising (i) two or more nucleotide units of the composition and (ii) two or more nucleotides in the two or more copies of the primed nucleic acid sequence, wherein the two or more nucleotides are complementary to the two or more nucleotide units of the composition.

126. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

127. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:391, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

128. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:414, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

129. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:431, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

130. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:446, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

131. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:448, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

132. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:450, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

133. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:452, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

134. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:454, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

135. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:456, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

136. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:458, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

137. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:460, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

138. A synthetic polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:462, wherein the amino acid sequence provides a binding constant of at least 3 between (i) the amino acid sequence and a primed nucleic acid sequence, (ii) the amino acid sequence and a nucleotide, or (iii) both (i) and (ii).

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