Multivalent binding compositions with reactive groups

Abstract

The present disclosure provides nucleotide conjugates, each configured to include a core attached to a plurality of nucleotide arms, where the nucleotide arms are modular and include (i) a core attachment moiety, (ii) a spacer region, (iii) a linker, and (iv) a nucleotide unit. The nucleotide unit of each nucleotide arm can bind to a polymerase 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 units of the nucleotide conjugate bind to the primer strand, but are not subjected to polymerase-catalyzed incorporation. A binding event can be detected, and a specific base of a nucleotide unit can be identified. The nucleotide conjugates described herein are useful in nucleic acid sequencing methods, in particular for massively parallel sequencing methods employing next generation sequencing platforms.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 328,663, filed on April 7, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named 52933-750_601_SL.XML, created on April 6, 2023, and 753,818 bytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. Background Art

[0005] Nucleic acid sequencing can be used to obtain information in a variety of biomedical settings, including diagnosis, prognosis, biotechnology, and forensic biology. Various sequencing methods have been developed, including Maxam-Gilbert sequencing and chain termination methods, or de novo sequencing methods, including shotgun sequencing and bridge PCR, or next-generation methods, including polymerase community (polony) sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, HeliScope single molecule sequencing, Sequencing, etc. Despite the continuous progress in DNA sequencing, many challenges remain unsolved. Summary of the invention

[0006] In some embodiments, aspects disclosed herein 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, an A485S mutation, a Y410G mutation, an I521H mutation, or a K507L mutation with reference to SEQ ID No: 391, or any combination thereof. In some embodiments, the amino acid sequence further comprises: (a) with reference to the Y410A mutation of SEQ ID No: 391; (b) with reference to the L409S mutation, Y410A mutation and Y261A mutation of SEQ ID No: 391; (c) with reference to the L409S mutation, Y410A mutation, P411G mutation and Y261A mutation of SEQ ID No: 391; (d) with reference to the Y261A mutation, F406I mutation, L409S mutation, Y410A mutation and P411A mutation of SEQ ID No: 391; (e) with reference to the Y7A mutation, Y261A mutation and Y410A mutation of SEQ ID No: 391; (f) with reference to the Y261A mutation, Y410A mutation and Y493I mutation of SEQ ID No: 391; (g) with reference to the Y261A mutation, Y410A mutation and Y493I mutation of SEQ ID No: 391; No: 391 of the Y261A mutation, Y410A mutation and Y493T mutation; (h) with reference to SEQ ID No: 391 of the Y261A mutation, Y410A mutation and V513I mutation; (i) with reference to SEQ ID No: 391 of the Y7A mutation, Y261A mutation, L409S mutation and Y410A mutation; (j) with reference to SEQ ID No: 391 of the Y261A mutation, L409A mutation, Y410A mutation and P411A mutation; (k) with reference to SEQ ID No: 391 of the Y261A mutation, L409S mutation, Y410A mutation and P411G mutation; (l) with reference to SEQ ID No: 391 of the Y261A mutation, L409S mutation and Y410A mutation; (m) with reference to SEQ ID No: Y261A mutation, L409S mutation and Y410G mutation of 391; (n) with reference to Y261A mutation, L409A mutation, Y410A mutation, P411A mutation and A485S mutation of SEQ ID No: 391;(o) with reference to the Y261A mutation, L409S mutation, Y410A mutation, P411G mutation and A485S mutation of SEQ ID No: 391; (p) with reference to the Y261A mutation, L409S mutation, Y410A mutation and A485S mutation of SEQ ID No: 391; (q) with reference to the Y261A mutation, L409S mutation, Y410G mutation and A485S mutation of SEQ ID No: 391; (r) with reference to the Y261A mutation, L409A mutation, Y410A mutation, P411A mutation and I521H mutation of SEQ ID No: 391; (s) with reference to the Y261A mutation, L409S mutation, Y410A mutation, P411G mutation and I521H mutation of SEQ ID No: 391; (t) with reference to the Y261A mutation, L409S mutation, Y410A mutation, P411G mutation and I521H mutation of SEQ ID No: 391. No: 391 of Y261A mutation, L409S mutation, Y410A mutation and I521H mutation; (u) with reference to SEQ ID No: 391 of Y261A mutation, L409S mutation, Y410G mutation and I521H mutation; or (v) with reference to SEQ ID No: 391 of Y261A mutation, L409S mutation, Y410A mutation, A485S mutation, K507L mutation and I521H mutation. In some embodiments, the amino acid sequence further comprises any one of SEQ ID NO: 392-413. In some embodiments, the amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NO: 392-413. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof. ;

[0007] In some embodiments, aspects disclosed herein 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 an 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 with reference to SEQ ID No: 414, or any combination thereof.In some embodiments, the amino acid sequence further comprises: (a) with reference to the L431A mutation, Y432A mutation, P433I mutation and A507S mutation of SEQ ID No: 414; (b) with reference to the L431A mutation, Y432A mutation, P433I mutation and K506Q mutation of SEQ ID No: 414; (c) with reference to the L431A mutation, Y432A mutation, P433I mutation, K506Q mutation and A507S mutation of SEQ ID No: 414; (d) with reference to the L431A mutation, Y432A mutation and P433A mutation of SEQ ID No: 414; (e) with reference to the L431A mutation, Y432A mutation, P433A mutation and A507S mutation of SEQ ID No: 414; (f) with reference to the L431A mutation, Y432A mutation, P433A mutation and A507S mutation of SEQ ID No: 414. No: 414 of the L431A mutation, Y432A mutation, P433A mutation and I543H mutation; (g) with reference to SEQ ID No: 414 of the L431S mutation, Y432A mutation and P433G mutation; (h) with reference to SEQ ID No: 414 of the L431S mutation, Y432A mutation, P433G mutation and A507S mutation; (i) with reference to SEQ ID No: 414 of the L431S mutation, Y432A mutation, P433G mutation and I543H mutation; (j) with reference to SEQ ID No: 414 of the L431S mutation and Y432A mutation; (l) with reference to SEQ ID No: 414 of the L431S mutation, Y432A mutation and A507S mutation; (m) with reference to SEQ ID No: 414; (n) with reference to the L431S mutation, Y432A mutation, A507S mutation, K529L mutation and I543H mutation of SEQ ID No: 414; (o) with reference to the L431S mutation and Y432G mutation of SEQ ID No: 414; (p) with reference to the L431S mutation, Y432G mutation and A507S mutation of SEQ ID No: 414; or (q) with reference to the L431S mutation, Y432G mutation and I543H mutation of 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 polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0008] In some embodiments, aspects disclosed herein 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 with reference to SEQ ID No: 431, or any combination thereof. In some embodiments, the amino acid sequence further comprises: (a) with reference to the Y412A mutation of SEQ ID No: 431; (b) with reference to the L411A mutation, Y412A mutation and P413A mutation of SEQ ID No: 431; (c) with reference to the L411A mutation, Y412A mutation, P413A mutation and A488S mutation of SEQ ID No: 431; (d) with reference to the L411A mutation, Y412A mutation, P413A mutation and I524H mutation of SEQ ID No: 431; (e) with reference to the L411S mutation, Y412A mutation and P413G mutation of SEQ ID No: 431; (f) with reference to the L411S mutation, Y412A mutation, P413G mutation and A488S mutation of SEQ ID No: 431; (g) with reference to the L411A mutation, Y412A mutation, P413G mutation and A488S mutation of SEQ ID No: 431. No: 431; (h) with reference to the L411S mutation and Y412A mutation of SEQ ID No: 431; (i) with reference to the L411S mutation, Y412A mutation and A488S mutation of SEQ ID No: 431; (j) with reference to the L411S mutation, Y412A mutation, A488S mutation, K510L mutation and I524H mutation of SEQ ID No: 431; (k) with reference to the L411S mutation, Y412A mutation and I524H mutation of SEQ ID No: 431; (l) with reference to the L411S mutation and Y412G mutation of SEQ ID No: 431; (m) with reference to the L411S mutation, Y412G mutation and A488S mutation of SEQ ID No: 431; or (n) with reference to SEQ ID No: L411S mutation, Y412G mutation and I524H mutation of No: 431. In some embodiments, the amino acid sequence further comprises any one of SEQ ID NO: 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 polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0009] In some embodiments, aspects disclosed herein 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 447. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0010] In some embodiments, aspects disclosed herein 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 449. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0011] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity with 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 451. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0012] In some embodiments, aspects disclosed herein 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 453. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0013] In some embodiments, aspects disclosed herein 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 455. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0014] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity with 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 457. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0015] In some embodiments, aspects disclosed herein 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 459. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0016] In some embodiments, aspects disclosed herein 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 461. In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0017] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity with 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 with reference to SEQ ID NO: 462, or any portion of its amino acid sequence. 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with 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 polymerase comprises a DNA polymerase or a polymerizing portion thereof.

[0018] In some embodiments, aspects disclosed herein are methods of nucleic acid analysis, the methods comprising: providing a preparation comprising: (i) a synthetic polypeptide disclosed herein; (ii) a primer nucleic acid sequence; and (ii) a nucleotide unit complementary to a nucleotide of the primer nucleic acid, wherein the preparation is provided under conditions sufficient to form a binding complex comprising the synthetic polypeptide, the nucleotide unit, and the primer nucleic acid sequence. In some embodiments, the method comprises a nucleic acid sequencing method. In some embodiments, the method comprises a sequencing by synthesis method. In some embodiments, the preparation further comprises one or more compositions, wherein the composition in the one or more compositions comprises: (a) a core; and (b) at least two nucleotide arms, wherein the nucleotide arms in the at least two nucleotide arms comprise: (i) a core attachment portion coupled to the core; (ii) a spacer coupled to the core attachment portion; (iii) a linker coupled to the spacer; and (iv) a nucleotide unit coupled to the linker. In some embodiments, the linker comprises:

[0019]

[0020]

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

[0022] In some embodiments, a kit is disclosed herein, comprising: one or more containers comprising: a synthetic polypeptide; and a nucleotide unit, wherein the nucleotide unit is detectable; and instructions for introducing the synthetic polypeptide and the nucleotide unit into a primer nucleic acid sequence comprising nucleotides complementary to the nucleotide unit under conditions sufficient to form a binding complex comprising the synthetic polypeptide, the nucleotide unit, and the primer nucleic acid sequence. In some embodiments, the kit further comprises: one or more compositions, wherein the composition in the one or more compositions comprises: a core; and at least two nucleotide arms, wherein the nucleotide arms in the at least two nucleotide arms comprise: a core attachment portion coupled to the core; a spacer coupled to the core attachment portion; a linker coupled to the spacer; and a nucleotide unit coupled to the linker. In some embodiments, the linker comprises:

[0023]

[0024]

[0025] wherein n is 1 to 6 and m is 0 to 10.

[0026] In some embodiments, aspects disclosed herein are systems comprising: a synthetic polypeptide; a primer nucleic acid sequence; and a nucleotide unit, wherein the nucleotide unit is detectable and complementary to a nucleotide in the primer nucleic acid sequence, wherein the system is configured to form a binding complex comprising the primer nucleic acid sequence, the synthetic polypeptide, and the nucleotide unit. In some embodiments, the system further comprises: one or more compositions, wherein the composition in the one or more compositions comprises: a core; and at least two nucleotide arms, wherein the nucleotide arms in at least two nucleotide arms comprise: a core attachment portion coupled to the core; a spacer coupled to the core attachment portion; a linker coupled to the spacer; and a nucleotide unit coupled to the linker, wherein the binding complex comprises the primer nucleic acid sequence, the synthetic polypeptide, and the composition. In some embodiments, the system further comprises: two or more copies of the primer nucleic acid sequence; and two or more synthetic polypeptides, wherein the composition is configured to form a multivalent binding complex comprising two or more nucleotide units of the composition, two or more copies of the primer nucleic acid sequence, and two or more synthetic polypeptides. In some embodiments, the linker comprises:

[0027]

[0028]

[0029] wherein n is 1 to 6 and m is 0 to 10.

[0030] In some embodiments, aspects disclosed herein are systems comprising: (i) a synthetic polypeptide disclosed herein; (ii) a primer nucleic acid sequence; and (iii) nucleotide units complementary to the nucleotides of the primer nucleic acid, wherein the system is provided under conditions sufficient to form a binding complex comprising the synthetic polypeptide, the nucleotide units, and the primer nucleic acid sequence.

[0031] In some embodiments, aspects disclosed herein are systems comprising: (i) a synthetic polypeptide disclosed herein; (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 attached to the 3' carbon of the sugar portion of the nucleotide, and wherein the blocking group reacts with a compound to remove the blocking group from the nucleotide to generate a nucleotide comprising an OH moiety on the 3' carbon of the sugar portion of the nucleotide. In some embodiments, the blocking group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, ketone, isocyanate, phosphate, thiol, disulfide, carbonate, urea or silyl group. In some embodiments, (a) an alkyl, alkenyl, alkynyl or allyl group in the blocking group reacts with tetrakis(triphenylphosphine)palladium(0) (Pd(P(C6H5)3)4) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ); (b) an aryl or benzyl group in the blocking group reacts with H2 and palladium on carbon (Pd / C); (c) an amine, amide, ketone, isocyanate, phosphate, thiol or disulfide group in the blocking group reacts with a phosphine or a thiol group including β-mercaptoethanol or dithiothreitol (DTT); (d) a carbonate group in the blocking group reacts with potassium carbonate (K2CO3) in methanol (MeOH), triethylamine in pyridine or Zn in acetic acid (AcOH); and (e) a urea or silyl group in the blocking group reacts with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride or triethylamine trihydrofluoride. In some embodiments, the azide in the blocking group includes an azide, an azido or an azidomethyl group. In some embodiments, the azide, an azido or an azidomethyl group reacts with a chemical reagent including a phosphine compound. In some embodiments, the phosphine compound includes a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound includes tris(2-carboxyethyl)phosphine (TCEP) or bissulfotriphenylphosphine (BS-TPP).

[0032] In some embodiments, aspects disclosed herein are compositions comprising: (a) a core; and (b) at least two nucleotide arms, wherein the nucleotide arms in the at least two nucleic acid arms comprise: (i) a core attachment portion coupled to the core; (ii) a spacer coupled to the core attachment portion; (iii) a linker coupled to the spacer; and (iv) a nucleotide unit coupled to the linker, wherein the linker comprises:

[0033]

[0034]

[0035]

[0036] wherein n is 1 to 6 and m is 0 to 10.

[0037] In some embodiments, the linker comprises:

[0038] In some embodiments, the linker comprises:

[0039] In some embodiments, the linker comprises:

[0040] In some embodiments, the linker comprises:

[0041] In some embodiments, the linker comprises:

[0042] wherein n is 1 to 6 and m is 0 to 10.

[0043] In some embodiments, the linker comprises:

[0044] wherein n is 1 to 6 and m is 0 to 10.

[0045] In some embodiments, the linker comprises:

[0046] wherein n is 1 to 6 and m is 0 to 10.

[0047] In some embodiments, the linker comprises:

[0048]

[0049] wherein n is 1 to 6 and m is 0 to 10.

[0050] In some embodiments, the linker comprises:

[0051] In some embodiments, the linker comprises:

[0052] In some embodiments, the linker comprises:

[0053] In some embodiments, the linker comprises:

[0054] In some embodiments, the linker comprises:

[0055]

[0056] In some embodiments, at least two nucleotide arms include 3 to 20 nucleotide arms. In some embodiments, the core includes a polypeptide. In some embodiments, the polypeptide includes streptavidin or avidin. In some embodiments, the core attachment portion includes biotin. In some embodiments, the composition further includes a fluorescent label coupled to the core or the nucleotide arm. In some embodiments, the spacer includes the following structure:

[0057] wherein m is 20 to 500 and o is 1 to 10. In some embodiments, the nucleotide arm further comprises a reactive group coupled to the nucleotide unit, wherein the reactive group is configured to react with a reagent. In some embodiments, the reactive group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, ketone, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group. In some embodiments, the alkyl, alkenyl, alkynyl, or allyl group in (a) the reactive group is substituted with tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H 5 ) 3 ) 4 ) reacts with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ); (b) the aromatic or benzyl group in the reactive group reacts with H 2 (c) the amine, amide, ketone, isocyanate, phosphate, thiol or disulfide group in the reactive group reacts with a phosphine or a thiol group including β-mercaptoethanol or dithiothreitol (DTT); (d) the carbonate group in the reactive group reacts with potassium carbonate (K2CO3) in methanol (MeOH); 2 CO 3), triethylamine in pyridine or Zn in acetic acid (AcOH); and (e) urea or silyl groups in reactive groups react with tetrabutylammonium fluoride, hydrofluoride pyridine (HF-pyridine), ammonium fluoride or triethylamine trihydrofluoride. In some embodiments, the azide group in the reactive group includes an azide, an azido or an azidomethyl group. In some embodiments, the reagent includes a phosphine compound. In some embodiments, the phosphine compound includes a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound includes tri(2-carboxyethyl)phosphine (TCEP) or bissulfotriphenylphosphine (BS-TPP). In some embodiments, the nucleotide units of at least two nucleotide arms include the same nucleobase type. In some embodiments, the nucleotide unit includes a blocking group attached to the 3' carbon of the sugar portion of the nucleotide unit, and wherein the blocking group reacts with a compound to remove the blocking group from the nucleotide unit to generate a nucleotide unit including an OH moiety on the 3' carbon of the sugar portion of the nucleotide unit. In some embodiments, the blocking group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, ketone, isocyanate, phosphate, thiol, disulfide, carbonate, urea, or silyl group. In some embodiments, the alkyl, alkenyl, alkynyl, or allyl group in the blocking group (a) is substituted with tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H 5 ) 3 ) 4 ) reacts with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ); (b) the aromatic or benzyl group in the blocking group reacts with H 2 and palladium on carbon (Pd / C); (c) the amine, amide, ketone, isocyanate, phosphate, thiol or disulfide group in the blocking group reacts with a phosphine or a thiol group including β-mercaptoethanol or dithiothreitol (DTT); (d) the carbonate group in the blocking group reacts with potassium carbonate (K2CO3) in MeOH. 2 CO 3 ), triethylamine in pyridine or Zn in acetic acid (AcOH); and (e) the urea or silyl group in the blocking group reacts with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride or triethylamine trihydrofluoride. In some embodiments, the azide in the blocking group comprises an azide, an azido or an azidomethyl group. In some embodiments, the azide, an azido or an azidomethyl group reacts with a chemical reagent comprising a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine portion or a derivatized triarylphosphine portion. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP) or bissulfotriphenylphosphine (BS-TPP).

[0058] In some embodiments, aspects disclosed herein are formulations comprising: at least two of the compositions disclosed herein, wherein at least two of the compositions include a first composition and a second composition, wherein the nucleotide units of the first composition comprise different nucleobases than the nucleotide units of the second composition.

[0059] In some embodiments, aspects disclosed herein are formulations comprising: at least two of the compositions, wherein at least two of the compositions include a first composition and a second composition, wherein: (a) the nucleotide units of the first composition comprise a first blocking group attached to the 3' carbon of the sugar moiety, wherein the first blocking group reacts with a 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 attached to the 3' carbon of the sugar moiety, wherein the second blocking group reacts with a 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, and wherein the nucleotide units of the first composition are different from the nucleotide units of the second composition.

[0060] In some embodiments, aspects disclosed herein are formulations comprising at least two of the compositions disclosed herein, wherein at least two of the compositions include a first composition and a second composition, wherein the linker of the first composition is different from the linker of the second composition.

[0061] In some embodiments, aspects disclosed herein are formulations comprising: at least two of the compositions disclosed herein, wherein at least two of the compositions include 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, wherein the second reactive group is different from the first reactive group.

[0062] In some embodiments, aspects disclosed herein are formulations comprising: at least two of the compositions disclosed herein, wherein at least two of the compositions include a first composition and a second composition, wherein the first composition comprises a first fluorophore and the second composition comprises a second fluorophore, and wherein the wavelength of light emitted by the first fluorophore is different from the wavelength of light emitted by the second fluorophore.

[0063] In some embodiments, aspects disclosed herein are formulations comprising: at least two of the compositions disclosed herein, wherein at least two of the compositions include a first composition and a second composition, wherein the first composition comprises a fluorescent label and the second composition is unlabeled.

[0064] In some embodiments, aspects disclosed herein are kits for nucleic acid molecule processing, the kit comprising: (a) a composition disclosed herein or a formulation disclosed herein; and (b) instructions for the use of the composition in a nucleotide identification reaction. In some embodiments, the kit further comprises: a reagent that reacts with a reactive group in a joint of the composition. In some embodiments, the kit further comprises: a reagent that reacts with a reactive group at the 3' carbon of a sugar moiety in a nucleotide unit of the composition. In some embodiments, the kit further comprises a reagent for use in a nucleotide binding reaction. In some embodiments, the reagent comprises a cation. In some embodiments, the kit further comprises: a solution comprising a cation; one or more polymerases; one or more primer sequences; one or more unlabeled nucleotides; or any combination of (i) to (iv).

[0065] In some embodiments, aspects disclosed herein are systems comprising: (a) a composition disclosed herein; (b) two or more copies of a primer nucleic acid sequence, wherein the two or more copies of the primer nucleic acid sequence comprise 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 primer nucleic acid sequences, two or more polymerases, and the composition. In some embodiments, the multivalent binding complex is formed under conditions where the nucleotide units of the composition are not incorporated into the two or more copies of the primer nucleic acid sequence. In some embodiments, two or more copies of the nucleic acid sequence and two or more copies of the nucleic acid primer molecule are fixed to a support under conditions sufficient to fix the multivalent binding complex to the support. In some embodiments, a plurality of multivalent binding complexes are fixed to a support, wherein the density of the plurality of multivalent binding complexes fixed to the support is 10 2 –10 9 per square millimeter (mm 2 ). In some embodiments, the plurality of multivalent binding complexes on the support are in fluid communication with each other and the reagent solution under conditions sufficient to allow the plurality of multivalent binding complexes to react with the reagent solution in a massively parallel manner.

[0066] In some embodiments, aspects disclosed herein are methods of nucleic acid analysis, the method comprising: introducing a composition disclosed herein into a primer nucleic acid sequence under conditions sufficient to form a binding complex, the binding complex comprising (i) nucleotide units of the composition and (ii) nucleotides in the primer nucleic acid sequence, wherein the nucleotides are complementary to the nucleotide units of the composition.

[0067] In some embodiments, aspects disclosed herein are methods of nucleic acid analysis, the methods comprising: introducing a composition disclosed herein into two or more copies of a primer nucleic acid sequence under conditions sufficient to form a multivalent binding complex, the multivalent binding complex comprising (i) two or more nucleotide units of the composition and (ii) two or more nucleotides of the two or more copies of the primer nucleic acid sequence, wherein the two or more nucleotides are complementary to the two or more nucleotide units of the composition.

[0068] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0069] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 391, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0070] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:414, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0071] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:431, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0072] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:446, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0073] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:448, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0074] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:450, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0075] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:452, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0076] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:454, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0077] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:456, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0078] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:458, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0079] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:460, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0080] In some embodiments, aspects disclosed herein are synthetic polypeptides comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:462, wherein the amino acid sequence: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both (i) and (ii).

[0081] Incorporation by reference

[0082] All publications, patents, and patent applications mentioned in this specification are incorporated herein 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 that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The novel features of the present method and system are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present method and system will be obtained by reference to the following detailed description of illustrative embodiments in which the principles of the present method and system are utilized, and the accompanying drawings, in which:

[0084] Figure 1 is a schematic diagram of a non-limiting example of a nucleotide conjugate according to some embodiments herein, the nucleotide conjugate comprising a universal core conjugated to a plurality of nucleotide arms. In some embodiments, a single nucleotide arm comprises a spacer, a linker, and a nucleotide unit.

[0085] Figure 2 is a schematic diagram of a non-limiting example of a nucleotide conjugate according to some embodiments herein, the nucleotide conjugate comprising a dendrimer core conjugated to a plurality of nucleotide arms. In some embodiments, a single nucleotide arm comprises a spacer, a linker, and a nucleotide unit.

[0086] Figure 3A schematic diagram of a non-limiting example of a nucleotide conjugate according to some embodiments herein comprising a core attached / bound to a plurality of nucleotide arms, wherein an individual nucleotide arm comprises a core attachment portion, a spacer, a linker, and a nucleotide unit.

[0087] Figure 4 is a schematic diagram of a non-limiting example of a nucleotide arm according to some embodiments herein, the nucleotide arm comprising a core attachment portion, a spacer, a linker and a nucleotide unit.

[0088] Figure 5A Non-limiting exemplary chemical structures of spacers (at the top) and linkers (at the bottom) are shown, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker, according to some embodiments herein.

[0089] Figure 5B Non-limiting exemplary chemical structures of linkers according to some embodiments herein are shown, including Linkers 1-6.

[0090] Figure 5C Shown are chemical structures of non-limiting examples of linkers according to some embodiments herein, including linkers 7-9.

[0091] Figure 5D Non-limiting exemplary chemical structures of linkers according to some embodiments herein are shown, including linkers 10 and 11 joined to nucleotide units.

[0092] Figure 5E Non-limiting exemplary chemical structures of linkers according to some embodiments herein are shown, including linkers 12 and 13 joined to nucleotide units.

[0093] Fig. 5F Non-limiting exemplary chemical structures of linkers according to some embodiments herein are shown, including linkers 14-16 joined to nucleotide units.

[0094] Fig. 6A Shown are non-limiting exemplary chemical structures of nucleotide arms according to some embodiments herein, the nucleotide arms comprising a spacer joined to a linker and a linker joined to a nucleotide unit.

[0095] Figure 6B Shown are non-limiting exemplary chemical structures of nucleotide arms according to some embodiments herein, the nucleotide arms comprising a spacer joined to a linker and a linker joined to a nucleotide unit.

[0096] Fig. 7AShown are non-limiting exemplary chemical structures of linkers joined to nucleotide units via propargylamine attachment according to some embodiments herein.

[0097] Figure 7B Additional chemical structures of non-limiting examples of linkers joined to nucleotide units via propargylamine attachment according to some embodiments herein are shown.

[0098] Figure 7C Additional chemical structures of non-limiting examples of linkers joined to nucleotide units via propargylamine attachment according to some embodiments herein are shown.

[0099] Fig.7D Additional chemical structures of non-limiting examples of linkers joined to nucleotide units via propargylamine attachment according to some embodiments herein are shown.

[0100] Fig. 7E Additional chemical structures of non-limiting examples of linkers joined to nucleotide units via propargylamine attachment according to some embodiments herein are shown.

[0101] Figure 7F Additional chemical structures of non-limiting examples of linkers joined to nucleotide units via propargylamine attachment according to some embodiments herein are shown.

[0102] Figure 7G Additional chemical structures of non-limiting examples of linkers joined to nucleotide units via propargylamine attachment according to some embodiments herein are shown.

[0103] Fig. 8A The chemical structure of a non-limiting example of a biotinylated nucleotide arm according to some embodiments herein is shown, the biotinylated nucleotide arm comprising a biotin moiety, a spacer, a linker (e.g., an 11-atom linker) and a nucleotide unit. In this non-limiting example, the nucleotide unit is attached to the linker via propargylamine at position 5 of a pyrimidine base or position 7 of a purine base, or position 1 of a pyrimidine base or position 9 of a purine base.

[0104] Figure 8B The chemical structure of a non-limiting example of a biotinylated nucleotide arm according to some embodiments herein is shown, the biotinylated nucleotide arm comprising a biotin moiety, a spacer, a linker (e.g., linker 6), and a nucleotide unit. In this non-limiting example, the nucleotide unit is attached to the linker via propargylamine at position 5 of a pyrimidine base or position 7 of a purine base, or position 1 of a pyrimidine base or position 9 of a purine base.

[0105] Fig. 9is a bar graph showing the results of trapping assays performed by reacting various fluorescently labeled nucleotide conjugates with the corresponding correct DNA templates.

[0106] Fig.10 is a bar graph showing the results of a capture assay in which increasing concentrations of various fluorescently labeled nucleotide conjugates reacted with the corresponding correct DNA template.

[0107] Fig.11 Four figures showing the results of capture assays are shown, which compare the signal intensity of fluorescently labeled nucleotide conjugates carrying nucleotide arms containing one of an N3 linker, linker 6, linker 8, or propargyl linker. Nucleotide conjugates were labeled with CF680 or CF532 fluorophores. Two different concentrations of nucleotide conjugates (20 and 80 nM) were tested. The figures show capture time (x-axis) and P90 signal intensity (y-axis) in seconds.

[0108] Fig.12 Four figures showing the results of capture assays are shown, which compare the signal intensity of fluorescently labeled nucleotide conjugates carrying nucleotide arms containing one of an N3 linker, linker 6, linker 8, or propargyl linker. The nucleotide conjugates were labeled with AF647 or CF570 fluorophores. Two different concentrations of nucleotide conjugates (20 and 80 nM) were tested. The figures show the capture time (x-axis) and P90 signal intensity (y-axis) in seconds.

[0109] Fig.13 Three graphs showing the results of real-time imaging capture kinetic assays comparing the signal intensity of fluorescently labeled nucleotide conjugates carrying nucleotide arms comprising one of linkers 6 or 10-16 are shown. Three different concentrations of nucleotide conjugates (15, 7.5, and 2.5 nM) were tested. The graphs show capture time (x-axis) and signal intensity (y-axis) in seconds.

[0110] Fig.14 is a graph showing the results of binding kinetic studies of fluorescently labeled nucleotide conjugates carrying nucleotide arms comprising one of linkers 6 or 10-16. The graph shows nucleotide conjugate concentration (x-axis, nM) and rate (y-axis). Fig.14 The legend shown in also applies to Fig.13 .

[0111] Fig.15 is a bar graph showing the binding constants (K) determined for fluorescently labeled nucleotide conjugates carrying nucleotide arms comprising one of linkers 6 or 10-16.

[0112] Fig.16Shown is a 2.8 angstrom model determined by X-ray crystallography in which a sequencing polymerase is co-crystallized with a template molecule hybridized to a primer having a 3' terminal dideoxynucleotide 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.

[0113] Fig.17 is a non-limiting exemplary amino acid sequence of a streptavidin subunit (SEQ ID NO: 466).

[0114] Fig.18 is a non-limiting exemplary amino acid sequence of a streptavidin subunit (SEQ ID NO: 467).

[0115] Fig.19 is a non-limiting exemplary amino acid sequence of a streptavidin subunit (SEQ ID NO: 468).

[0116] Fig. 20 is a non-limiting exemplary amino acid sequence of a streptavidin subunit (SEQ ID NO: 469).

[0117] Fig.21 is a non-limiting exemplary amino acid sequence of a streptavidin subunit (SEQ ID NO: 470).

[0118] Fig. 22 is a non-limiting exemplary amino acid sequence of an avidin subunit (SEQ ID NO: 471).

[0119] Fig.23 is a non-limiting exemplary amino acid sequence of an avidin subunit (SEQ ID NO: 472).

[0120] Fig.24 is the amino acid sequence (SEQ ID NO: 1) of the wild-type DNA polymerase from the archaeon Candidatus altiarchaeales (GenBank Accession No. RLI89578.1).

[0121] Fig.25 is the amino acid sequence (SEQ ID NO: 464) of the wild-type DNA polymerase from the archaeon Candidatus altiarchaeales (GenBank Accession No. OYT41123.1).

[0122] Fig.26is the amino acid sequence of the N-terminal domain of the DNA polymerase from the archaeon Candidatus altiarchaeales RLI 89578.1 (SEQ ID NO: 269).

[0123] Fig. 27 is the amino acid sequence of the exonuclease domain of the DNA polymerase from the archaeon Candidatus altiarchaeales RLI 89578.1 (SEQ ID NO: 270).

[0124] Fig.28 is the amino acid sequence of the palm (1) domain of the DNA polymerase from the archaeon Candidatus altiarchaeales RLI 89578.1 (SEQ ID NO: 271).

[0125] Fig.29 is the amino acid sequence of the finger domain of the DNA polymerase from Candidatus altiarchaeales archaeon RLI 89578.1 (SEQ ID NO: 272).

[0126] Fig.30 is the amino acid sequence of the palm (2) domain of the DNA polymerase from the archaeon Candidatus altiarchaeales RLI 89578.1 (SEQ ID NO: 273).

[0127] Fig.31 is the amino acid sequence of the thumb domain of the DNA polymerase from the archaeon Candidatus altiarchaeales RLI 89578.1 (SEQ ID NO: 274).

[0128] Fig.32 is the amino acid sequence of 9°N polymerase (SEQ ID NO: 280).

[0129] Fig.33 It is the amino acid sequence of 9°N polymerase UniProtKB-Q56366 (DPOL_THES9) (SEQ ID NO: 281).

[0130] Fig.34 is the amino acid sequence of THERMINATOR polymerase (SEQ ID NO: 282).

[0131] Fig.35is the amino acid sequence of a DNA polymerase from Pyrococcus abyssi (SEQ ID NO: 286).

[0132] Fig.36 is the amino acid sequence of VENT polymerase (SEQ ID NO: 283).

[0133] Fig.37 is the amino acid sequence of DEEP VENT polymerase (SEQ ID NO: 284).

[0134] Fig.38 is the amino acid sequence of Pfu polymerase (SEQ ID NO: 285).

[0135] Fig.39 is the amino acid sequence of the wild-type DNA polymerase from Geobacillus stearothermophilus (SEQ ID NO: 275).

[0136] Fig.40 is the amino acid sequence of RB69 polymerase (SEQ ID NO: 287).

[0137] Fig.41A is the amino acid sequence of a wild-type DNA polymerase from the archaeon of the class Thermococci with a backbone sequence from RLF 89458.1 (SEQ ID NO: 391).

[0138] Fig.41B is the amino acid sequence of a wild-type DNA polymerase from the Pyrococcus archaeon with a backbone sequence from RLF 78286.1 (SEQ ID NO: 465).

[0139] Fig.42 is the amino acid sequence of a wild-type DNA polymerase from the Thermoplasmata archaeon with a backbone sequence from RLF 60390.1 (SEQ ID NO: 414).

[0140] Fig.43 is the amino acid sequence of the wild-type DNA polymerase from Thermococcus sp. 2319x1 with the backbone sequence from WP175059460.1 (SEQ ID NO: 431).

[0141] Fig.44is the amino acid sequence of the wild-type DNA polymerase from Thermococcus litoralis having a backbone sequence from ADK 47977.1 (SEQ ID NO: 446).

[0142] Fig.45 is the amino acid sequence of the wild-type DNA polymerase from the archaea BMS3Abin16 with the backbone sequence from GBE 17769.1 (SEQ ID NO: 448).

[0143] Fig.46 is the amino acid sequence of the wild-type DNA polymerase from the archaea BMS3Bbin16 with the backbone sequence from GBE 55812.1 (SEQ ID NO: 450).

[0144] Fig.47 is the amino acid sequence of the wild-type DNA polymerase from Candidatus Hadarchaeum yellowstonense with the backbone sequence from KUO 42443.1 (SEQ ID NO: 452).

[0145] Fig.48 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from KXB 02540.1 (SEQ ID NO: 454).

[0146] Fig.49 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from MBC 7218772.1 (SEQ ID NO: 456).

[0147] Fig.50 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from RMF 90817.1 (SEQ ID NO: 458).

[0148] Fig.51 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from WP 058946753.1 (SEQ ID NO: 460).

[0149] Fig.52 is the amino acid sequence of a wild-type DNA polymerase having a backbone sequence from WP 167886206.1 (SEQ ID NO: 462).

[0150] Fig.53 is a block diagram depicting an exemplary machine including a computer system 100 for nucleic acid processing.

[0151] Fig.54 It is the implementation scheme of the application providing system.

[0152] Fig.55 It is another implementation scheme of the application providing system. DETAILED DESCRIPTION

[0153] In some embodiments, disclosed herein are compositions, systems and kits for processing and / or analyzing nucleic acid sequences. Also provided are methods for processing and / or analyzing nucleic acid sequences using compositions, systems and kits disclosed herein. In some embodiments, the inventive concepts disclosed herein utilize nucleotide conjugates comprising a core, the core having one or more nucleotide units coupled thereto that are complementary to the nucleotides in the nucleic acid sequence to be processed or analyzed. Such nucleotide conjugates can form a multivalent binding complex comprising a nucleotide conjugate bound to the nucleotides in the nucleic acid sequence, and an optional polymerase. In some embodiments, the inventive concepts disclosed herein utilize a synthetic polymerase having one or more mutations in an amino acid sequence, which imparts an enhanced binding to a nucleotide or nucleotide unit of the present disclosure compared to an otherwise identical polymerase without one or more mutations.

[0154] Composition

[0155] In some embodiments, compositions are disclosed herein. In embodiments, compositions can be used for any method, formulation or system disclosed herein. In some embodiments, compositions can be used for nucleic acid processing, analysis, identification or detection methods disclosed herein. In some embodiments, compositions can be used together with polypeptides disclosed herein. In some embodiments, compositions can be or include nucleotide conjugates disclosed herein. In some embodiments, compositions can have the ability to form binding complexes with primer nucleic acid sequences and polypeptides disclosed herein. In some embodiments, compositions can have the ability to form multivalent binding complexes with two or more primer nucleic acid sequences and two or more polypeptides disclosed herein.

[0156] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, nucleotide conjugates may include a core attached to multiple nucleotide arms. In some embodiments, multiple nucleotide arms may include nucleotide units of the same type. For example, nucleotide conjugates may include a core (e.g., streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, wherein all attachment arms have nucleotide units selected from dATP, dGTP, dCTP, dTTP and dUTP.

[0157] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, nucleotide conjugates may include a core attached to a plurality of nucleotide arms. In some embodiments, a plurality of nucleotide arms may include different types of nucleotide units. For example, nucleotide conjugates may include a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein at least the first attachment arm may have a first nucleotide unit selected from dATP, dGTP, dCTP, dTTP and dUTP, and the second attachment arm may have a second nucleotide unit selected from dATP, dGTP, dCTP, dTTP and dUTP, wherein the first and second nucleotide units are different.

[0158] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates may comprise a core attached to a plurality of nucleotide arms. In some embodiments, a plurality of nucleotide arms may comprise a spacer of the same type. For example, a nucleotide conjugate may comprise a core (e.g., a streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein all attached arms have the same spacer. In some embodiments, the spacer may be selected from any spacer described herein (e.g., Figure 5A (top)).

[0159] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugate may comprise a core attached to a plurality of nucleotide arms. In some embodiments, a plurality of nucleotide arms may comprise different types of spacers. For example, a nucleotide conjugate may comprise a core (e.g., a streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein at least the first attachment arm may have a first type of spacer, and the second attachment arm may have a second type of spacer, wherein the first and second spacer units are different. In some embodiments, the first and second types of linkers may be selected from any spacer described herein (e.g., Figure 5A (top)).

[0160] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates may comprise a core attached to a plurality of nucleotide arms. In some embodiments, a plurality of nucleotide arms may comprise a linker of the same type. For example, a nucleotide conjugate may comprise a core (e.g., a streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein all attached arms have the same linker. In some embodiments, the linker may be selected from any linker described herein (e.g., Figure 5A (bottom) and Figure 5B -F).

[0161] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugate may comprise a core attached to a plurality of nucleotide arms. In some embodiments, a plurality of nucleotide arms may comprise different types of linkers. For example, a nucleotide conjugate may comprise a core (e.g., a streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein at least the first attachment arm may have a first type of linker, and the second attachment arm may have a second type of linker, wherein the first and second linker units are different. In some embodiments, the first and second types of linkers may be selected from any linker described herein (e.g., Figure 5A (bottom) and Figure 5B -F).

[0162] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, nucleotide conjugates may include a core attached to multiple nucleotide arms. In some embodiments, multiple nucleotide arms may include spacers and joints of the same type. For example, nucleotide conjugates may include a core (e.g., streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, wherein all attachment arms have identical spacers and joints. In some embodiments, spacers and joints may be selected from any spacers and joints described herein.

[0163] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, nucleotide conjugates may include a core attached to multiple nucleotide arms. In some embodiments, multiple nucleotide arms may include reactive groups of the same type. For example, nucleotide conjugates may include a core (e.g., streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, wherein all attachment arms have the same reactive groups. In some embodiments, reactive groups may include alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azide groups, amine groups, amide groups, ketone groups, isocyanate groups, phosphate groups, sulphur groups, disulfide groups, carbonate groups, urea groups or silyl groups.

[0164] In some embodiments, the reactive groups in the linker can react with chemical reagents. For example, the reactive groups alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H 5 ) 3 )4 ) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Reactive groups aryl and benzyl can react with H 2 and palladium on carbon (Pd / C). Reactive groups amines, amides, ketones, isocyanates, phosphates, thiols, disulfides can react with phosphines or with thiol groups including β-mercaptoethanol or dithiothreitol (DTT). Reactive groups carbonates can react with potassium carbonate (KCO) in methanol (MeOH). 2 CO 3 ), with triethylamine in pyridine or with Zn in acetic acid (AcOH). The reactive groups urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride or with triethylamine trihydrofluoride.

[0165] In some embodiments, the nucleotide arm can have the same type of reactive groups in the joint, wherein the reactive groups can include azide, azido or azidomethyl groups. In some embodiments, the azide, azido or azidomethyl groups in the joint can react with chemical reagents. In some embodiments, the chemical reagent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkyl phosphine part or a derivatized triaryl phosphine part. In some embodiments, the phosphine compound can include tris (2-carboxyethyl) phosphine (TCEP), bissulfo triphenylphosphine (BS-TPP) or tris (hydroxypropyl) phosphine (THPP).

[0166] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, nucleotide conjugates may include a core attached to a plurality of nucleotide arms. In some embodiments, a plurality of nucleotide arms may include different types of reactive groups in a joint. For example, a nucleotide conjugate may include a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein at least the first attachment arm may have a reactive group of the first type in the first joint unit, and the second attachment arm may have a reactive group of the second type in the second joint unit, wherein the first and second reactive groups are different.

[0167] 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 alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azide groups, amine groups, amide groups, ketone groups, isocyanate groups, phosphate groups, sulfur groups, disulfide groups, carbonate groups, urea groups and silyl groups in any combination.

[0168] In some embodiments, the first and second reactive groups can react with a chemical reagent. For example, the reactive groups alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H 5 ) 3 ) 4 ) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Reactive groups aryl and benzyl can be reacted with H2 and palladium on carbon (Pd / C). Reactive groups amine, amide, ketone, isocyanate, phosphate, thiol, disulfide can be reacted with phosphine or with thiol groups including β-mercaptoethanol or dithiothreitol (DTT). Reactive groups carbonate can be reacted with potassium carbonate (KCO) in MeOH. 2 CO 3 ), with triethylamine in pyridine or with Zn in acetic acid (AcOH). The reactive groups urea and silyl can react with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride or with triethylamine trihydrofluoride.

[0169] In some embodiments, the nucleotide arm can have different types of reactive groups in the joint, wherein the reactive group can include azide, azido or azidomethyl groups. In some embodiments, the azide, azido or azidomethyl groups in the joint can react with chemical reagents. In some embodiments, the chemical reagent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkyl phosphine part or a derivatized triaryl phosphine part. In some embodiments, the phosphine compound can include tris (2-carboxyethyl) phosphine (TCEP), bissulfo triphenylphosphine (BS-TPP) or tris (hydroxypropyl) phosphine (THPP).

[0170] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates may include a core attached to a plurality of nucleotide arms. In some embodiments, a plurality of nucleotide arms may include nucleotide units with sugar 3'OH groups of the same type. For example, the nucleotide conjugates may include a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein all attachment arms have nucleotide units with sugar 3'OH groups of the same type.

[0171] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, nucleotide conjugates may include a core attached to multiple nucleotide arms. In some embodiments, multiple nucleotide arms may include nucleotide units with the same type of sugar 3' blocking groups (e.g., chain termination moieties). For example, nucleotide conjugates may include a core attached to multiple nucleotide arms or biotinylated nucleotide arms (e.g., streptavidin or avidin core), wherein all attachment arms have nucleotide units, and the nucleotide units have the same type of sugar 3' blocking groups. In some embodiments, sugar 3' blocking groups may include alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azide groups, amine groups, amide groups, ketone groups, isocyanate groups, phosphate groups, sulphur groups, disulfide groups, carbonate groups, urea groups or silyl groups. In some embodiments, the sugar 3' blocking group may include 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 may include an azide, an azido, or an azidomethyl group.

[0172] In some embodiments, the sugar 3' blocking group can be reacted with a chemical reagent. For example, the sugar 3' blocking groups alkyl, alkenyl, alkynyl, and allyl can be reacted with tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H 5 ) 3 ) 4 ) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Sugar 3' blocking group aryl and benzyl groups can be reacted with H2 and palladium on carbon (Pd / C). Sugar 3' blocking group amines, amides, ketones, isocyanates, phosphates, thiols, disulfides can be reacted with phosphines or with thiol groups including β-mercaptoethanol or dithiothreitol (DTT). Sugar 3' blocking group carbonates can be reacted with potassium carbonate (KCO) in MeOH. 2 CO 3 ), with triethylamine in pyridine or with Zn in acetic acid (AcOH). Sugar 3' blocking groups urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride or with triethylamine hydrofluoride.

[0173] In some embodiments, the sugar 3' blocking group (e.g., azide, azido, and azidomethyl) can react with a chemical reagent. In some embodiments, the chemical reagent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0174] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates may include a core attached to a plurality of nucleotide arms. In some embodiments, a plurality of nucleotide arms may include nucleotide units with different sugar 3' blocking groups. For example, the nucleotide conjugates may include a core (e.g., streptavidin or avidin core) attached to a plurality of nucleotide arms or biotinylated nucleotide arms, wherein at least the first attachment arm may have a first nucleotide unit, the first nucleotide unit having a first 3' blocking group, and the second attachment arm may have a second nucleotide unit, the second nucleotide unit having a second 3' blocking group, wherein the first and second 3' blocking groups are different.

[0175] 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 alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azide groups, amine groups, amide groups, ketone groups, isocyanate groups, phosphate groups, thiol groups, disulfide groups, carbonate groups, urea groups 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 3'-O-alkylhydroxylamino groups, 3'-phosphorothioate groups, 3'-O-malonyl groups 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 azide, azido or azidomethyl groups in any combination.

[0176] In some embodiments, the first and second 3' blocking groups can react with chemical reagents. For example, the 3' blocking groups alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H 5 ) 3 ) 4) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). 3' blocking groups aryl and benzyl can be reacted with H2 and palladium on carbon (Pd / C). 3' blocking groups amines, amides, ketones, isocyanates, phosphates, thiols, disulfides can be reacted with phosphines or with thiol groups including β-mercaptoethanol or dithiothreitol (DTT). 3' blocking groups carbonates can be reacted with potassium carbonate (KCO) in MeOH. 2 CO 3 ), with triethylamine in pyridine or with Zn in acetic acid (AcOH). 3' blocking groups urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride or with triethylamine trihydrofluoride.

[0177] In some embodiments, the first and second 3' blocking groups (e.g., azide, azido, and azidomethyl) can react with a chemical reagent. In some embodiments, the chemical reagent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0178] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, nucleotide conjugates may include a core attached to multiple nucleotide arms. In some embodiments, multiple nucleotide arms may include a nucleotide unit with a first sugar 3'OH blocking group. In some embodiments, multiple nucleotide arms may include a nucleotide unit with a second 3'OH blocking group. In some cases, the first and second 3'OH blocking groups may be different. For example, a nucleotide conjugate may include a core (e.g., streptavidin or avidin core) attached to multiple nucleotide arms or biotinylated nucleotide arms, wherein (a) at least the first arm may include a first nucleotide unit with a sugar moiety including a 3'OH group, (b) at least the second arm may include a second nucleotide unit with a first 3' blocking group, and (c) at least the third arm may include a third nucleotide unit with a second blocking group, wherein the first and second 3' blocking groups are different from each other.

[0179] 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 alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azide groups, amine groups, amide groups, ketone groups, isocyanate groups, phosphate groups, thiol groups, disulfide groups, carbonate groups, urea groups 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 3'-O-alkylhydroxylamino groups, 3'-phosphorothioate groups, 3'-O-malonyl groups 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 azide, azido or azidomethyl groups in any combination.

[0180] In some embodiments, the first and second 3' blocking groups can react with chemical reagents. For example, the 3' blocking groups alkyl, alkenyl, alkynyl, and allyl can react with tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H 5 ) 3 ) 4 ) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). 3' blocking groups aryl and benzyl can be reacted with H2 and palladium on carbon (Pd / C). 3' blocking groups amines, amides, ketones, isocyanates, phosphates, thiols, disulfides can be reacted with phosphines or with thiol groups including β-mercaptoethanol or dithiothreitol (DTT). 3' blocking groups carbonates can be reacted with potassium carbonate (KCO) in MeOH. 2 CO 3 ), with triethylamine in pyridine or with Zn in acetic acid (AcOH). 3' blocking groups urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride or with triethylamine trihydrofluoride.

[0181] In some embodiments, the first and second 3' blocking groups (e.g., azide, azido, and azidomethyl) can react with a chemical reagent. In some embodiments, the chemical reagent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bissulfotriphenylphosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP).

[0182] The present disclosure provides compositions, systems, methods and kits comprising nucleotide conjugates. In some embodiments, the nucleotide conjugates may have a core. In some embodiments, the core may 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 may include a labeled nucleotide conjugate. In some embodiments, streptavidin or avidin cores may be labeled with 1-6 or more reporter moieties. In some embodiments, the reporter moiety may include a fluorophore.

[0183] In a mixture of nucleotide conjugates with different units in its nucleotide arms, the distinction between different nucleotide conjugates can be achieved. In some embodiments, the core of the first nucleotide conjugate can be labeled with a reporter moiety to distinguish it from the second labeled (or unlabeled) nucleotide conjugate. For example, the unit in the nucleotide arm of the labeled first nucleotide conjugate can be different from the unit in the nucleotide arm of the labeled second nucleotide conjugate. Any unit in the first nucleotide conjugate (for example, a spacer, a joint, 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, wherein the first and second reporting moieties correspond to the units of distinction. In some embodiments, the first and second reporting moieties can be spectrally distinguishable from each other.

[0184] In some embodiments, the core of the first nucleotide conjugate can be labeled with a first reporter portion corresponding to a 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 portion corresponding to a base in the attached nucleotide arm (e.g., dATP, dGTP, dCTP, dTTP or dUTP), wherein the base in the first nucleotide conjugate is different from the base in the second nucleotide conjugate. In some embodiments, the first and second reporter portions are spectrally distinguishable from each other. In some embodiments, the detection of the first reporter portion indicates a binding event, an incorporation event, or a combination of binding and incorporation events of a first nucleotide conjugate with a first base, and the detection of the second reporter portion indicates a binding event, an incorporation event, or a combination of binding and incorporation events of a second nucleotide conjugate with a second base. The binding event can be a nucleotide conjugate binding to a composite polymerase. The incorporation event can be a nucleotide unit incorporating into the terminal 3' end of an extendable primer in a composite polymerase, wherein the nucleotide unit is a part of a nucleotide conjugate.

[0185] Synthetic peptides

[0186] In some embodiments, synthetic polypeptides are disclosed herein. In some embodiments, the synthetic polypeptides are purified or separated. In some embodiments, the synthetic polypeptides are polymerases. In some embodiments, the polymerases include nucleic acid polymerases or their polymerizing parts. In some embodiments, the polymerases include DNA polymerases or their polymerizing parts. In some embodiments, the polymerases include RNA polymerases or their polymerizing parts. In some embodiments, the synthetic polypeptides can use the preparations, systems or methods disclosed herein. For example, the synthetic polypeptides can be used for methods of nucleic acid sequence analysis, sequencing, identification or treatment, such as methods disclosed herein.

[0187] Synthetic polypeptides disclosed herein include amino acid sequences. In some embodiments, the amino acid sequence has a sequence identity of greater than or equal to 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 100% to any one of SEQ ID NO: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460 and 462. In some embodiments, the amino acid sequence includes one or more mutations. In some embodiments, one or more mutations confer enhanced binding to a nucleotide or nucleotide unit disclosed herein, compared to an otherwise identical synthetic polypeptide without one or more mutations. In some embodiments, binding is enhanced 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 without one or more mutations. In some embodiments, binding is enhanced by at least about 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 without one or more mutations. Affinity (e.g., the strength with which a synthetic polypeptide binds to a nucleotide) can be determined by calculating the dissociation constant (K) of the binding complex formed between the nucleotide and the synthetic polypeptide using a suitable technique. d ) as measured by methods such as those 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.

[0188] 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.Amino acids disclosed herein may be referred to by the single-letter or three-letter codes set forth in Table 6 below.

[0189] Table 6. International Union of Pure and Applied Chemistry (IUPAC) amino acid codes

[0190]

[0191] The missense substitution of amino acid can be indicated by A1#A2, wherein A1 is the amino acid replaced with amino acid A2 at amino acid position #. For example, W26C represents that the amino acid 26 (tryptophan, W) of the reference base sequence becomes cysteine ​​(C). In contrast, nonsense substitution is represented by A1#X, wherein A1 is the amino acid replaced with stop codon X at amino acid position #. Deletion is represented by "del" after the amino acid flanking the deletion site. For example, K29del represents the deletion of lysine (K) at amino acid position 29 of the reference base sequence. Insertion between the amino acid flanking the insertion site is represented by "ins", followed by the amino acid to be inserted. For example, K29_M30insQSK represents the insertion of amino acid sequence QSK between lysine (K) at amino acid position 29 and methionine (M) at amino acid position 30 of the reference base sequence. In some embodiments, the base sequence is any one of SEQ ID NO: 1, 391, 414, 431, 446, 448, 450, 452, 454, 456, 458, 460 and 462.

[0192] In some embodiments, the synthetic polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to 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, and the amino acid sequence comprises one or more mutations listed in Table 1A with reference to SEQ ID NO: 1.

[0193] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 391, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391. In some embodiments, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 391, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and the amino acid sequence comprises a D141A mutation or a D143A mutation with reference to SEQ ID NO: 391.

[0194] 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, an 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, an I521H mutation, or a K507L mutation with reference to SEQ ID No: 391, or any combination thereof. 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 an Y261A mutation, a F406I mutation, a 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 an 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 an 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 an 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, an 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, an 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, an 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, an L409S mutation, a Y410A mutation, and an A485S mutation with reference to SEQ ID No: 391. In some embodiments, the amino acid sequence further comprises a Y261A mutation, an 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 Y410G 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, 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.

[0195] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 414, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414. In some embodiments, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 414, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO: 414.

[0196] 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 an L431A mutation, a Y432A mutation, a P433I mutation, an A507S mutation, a K506Q mutation, a P433A mutation, an I543H mutation, a L431 S mutation, a P433G mutation, a K529L mutation, or a Y432G mutation with reference to SEQ ID No: 414, or any combination thereof. In some embodiments, the amino acid sequence further comprises an L431A mutation, a Y432A mutation, a P433I mutation, and an A507S mutation with reference to SEQ ID No: 414. In some embodiments, the amino acid sequence further comprises an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an L431S mutation and a Y432A mutation with reference to SEQ ID No: 414. In some embodiments, the amino acid sequence further comprises an 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 an 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 an 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 an L431S mutation and a Y432G mutation with reference to SEQ ID No: 414. In some embodiments, the amino acid sequence further comprises an 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 an 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.

[0197] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 431, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431. In some embodiments, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 431, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO: 431.

[0198] 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, 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 with reference to SEQ ID No: 431, or any combination thereof. 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 an 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 an L411A mutation, a Y412A mutation, a P413A mutation, and an A488S mutation with reference to SEQ ID No: 431. In some embodiments, the amino acid sequence further comprises an 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 an 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 an 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 an 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 an L411S mutation and a Y412A mutation with reference to SEQ ID No: 431. In some embodiments, the amino acid sequence further comprises an 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 an 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 an L411S mutation, a Y412A mutation, and an I524H mutation with reference to SEQ ID No: 431. In some embodiments, the amino acid sequence further comprises an L411S mutation and a Y412G mutation with reference to SEQ ID No: 431. In some embodiments, the amino acid sequence further comprises an 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 an 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.

[0199] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 446, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 446, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 446, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 446, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 446, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 446, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 446, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 446, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 446, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 446. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 446, and 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, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 446, and 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 81% sequence identity to SEQ ID NO: 446, and 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 82% sequence identity to SEQ ID NO: 446, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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 the D141A mutation, the E143A mutation, and the Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the amino acid sequence comprises the D141A mutation, the E143A mutation, and the Y412A mutation with reference to SEQ ID NO: 446. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 447.

[0200] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 448, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 448, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 448, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 448, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 448, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 448, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 448, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 448, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 448, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 448, and 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, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 448, and 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 polypeptides comprise an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 448, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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 99% sequence identity to SEQ ID NO: 448, and 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 the D149A mutation, E151A mutation, Y272A mutation, and Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the amino acid sequence comprises three or more of the D149A mutation, E151A mutation, Y272A mutation, and Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the amino acid sequence comprises the D149A mutation, E151A mutation, Y272A mutation, and Y422A mutation with reference to SEQ ID NO: 448. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 449.

[0201] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 450, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 450, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 450, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 450, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 450, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 450, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 450, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 450, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 450, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 450, and 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, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 450, and 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 polypeptides comprise an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 450, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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 the D149A mutation, E151A mutation, Y272A mutation, and Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the amino acid sequence comprises three or more of the D149A mutation, E151A mutation, Y272A mutation, and Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the amino acid sequence comprises the D149A mutation, E151A mutation, Y272A mutation, and Y422A mutation with reference to SEQ ID NO: 450. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 451.

[0202] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 452, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 452, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 452, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 452, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 452, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 452, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 452, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 452, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 452, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 452. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 452, and 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, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 452, and 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 81% sequence identity to SEQ ID NO: 452, and 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 82% sequence identity to SEQ ID NO: 452, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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.

[0203] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 454, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 454, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 454, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 454, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 454, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 454, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 454, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 454, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 454, and the amino acid sequence comprises a D170A mutation, an E172A mutation, or a Y449A mutation with reference to SEQ ID NO: 454. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 454, and 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, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 454, and 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 81% sequence identity to SEQ ID NO: 454, and 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 82% sequence identity to SEQ ID NO: 454, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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 the D170A mutation, the E172A mutation, and the Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the amino acid sequence comprises the D170A mutation, the E172A mutation, and the Y449A mutation with reference to SEQ ID NO: 454. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 455.

[0204] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 456, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 456, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 456, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 456, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 456, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 456, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 456, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 456, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 456, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 456, and 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, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 456, and 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 polypeptides comprise an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 456, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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 the D173A mutation, E175A mutation, Y452A mutation, and C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the amino acid sequence comprises three or more of the D173A mutation, E175A mutation, Y452A mutation, and C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the amino acid sequence comprises the D173A mutation, E175A mutation, Y452A mutation, and C468A mutation with reference to SEQ ID NO: 456. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 457.

[0205] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 458, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 458, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 458, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 458, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 458, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 458, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 458, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 458, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 458, and 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 are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 458, and 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, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 458, and 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 polypeptides comprise an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 458, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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 the D149A mutation, E151A mutation, Y272A mutation, and Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the amino acid sequence comprises three or more of the D149A mutation, E151A mutation, Y272A mutation, and Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the amino acid sequence comprises the D149A mutation, E151A mutation, Y272A mutation, and Y422A mutation with reference to SEQ ID NO: 458. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 459.

[0206] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 460, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 460, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 460, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 460, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 460, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 460, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 460, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 460, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 460, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 460. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 460, and 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, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 460, and 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 81% sequence identity to SEQ ID NO: 460, and 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 82% sequence identity to SEQ ID NO: 460, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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 the D141A mutation, the E143A mutation, and the Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the amino acid sequence comprises the D141A mutation, the E143A mutation, and the Y412A mutation with reference to SEQ ID NO: 460. In some embodiments, the amino acid sequence further comprises SEQ ID NO: 461.

[0207] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 462, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to SEQ ID NO: 462, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to SEQ ID NO: 462, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to SEQ ID NO: 462, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to SEQ ID NO: 462, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 462, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to SEQ ID NO: 462, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to SEQ ID NO: 462, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to SEQ ID NO: 462, and the amino acid sequence comprises a D141A mutation, an E143A mutation, or a Y412A mutation with reference to SEQ ID NO: 462. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to SEQ ID NO: 462, and 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, disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 462, and 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 81% sequence identity to SEQ ID NO: 462, and 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 82% sequence identity to SEQ ID NO: 462, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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, and 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 part of its 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 further comprises (i) an amino acid deletion from amino acid position I496 to amino acid position S1033 or any part of its 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 further comprises (i) an amino acid deletion from amino acid position I496 to amino acid position S1033 with reference to SEQ ID NO: 462, or any portion of its amino acid sequence; 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.

[0208] Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 71% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 72% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 73% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 74% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 75% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 76% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 77% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 78% sequence identity to any one of SEQ ID NOs: 1-472. Disclosed herein are synthetic polypeptides comprising an amino acid sequence having at least 79% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptides comprise an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptides comprise an amino acid sequence having at least 81% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptides comprise an amino acid sequence having at least 82% sequence identity to any one of SEQ ID NOs: 1-472. In some embodiments, the synthetic polypeptides comprise 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 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 an amino acid sequence of any one of SEQ ID NOs: 1-472.

[0209] 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: (i) 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, between the amino acid sequence and the primer nucleic acid sequence; (ii) 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, between the amino acid sequence and the nucleotide sequence; or (iii) both of (i) and (ii).

[0210] 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: (i) 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, between the amino acid sequence and the primer nucleic acid sequence; (ii) 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, between the amino acid sequence and the nucleotide sequence; or (iii) both of (i) and (ii).

[0211] 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: (i) 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, between the amino acid sequence and the primer nucleic acid sequence; (ii) 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, between the amino acid sequence and the nucleotide sequence; or (iii) both of (i) and (ii).

[0212] In some embodiments, the synthetic polypeptide is purified or isolated. In some embodiments, the synthetic polypeptide is a polymerase. In some embodiments, the polymerase includes a nucleic acid polymerase or a polymerizing portion thereof. In some embodiments, the polymerase includes a DNA polymerase or a polymerizing portion thereof.

[0213] Disclosed herein are compositions comprising nucleic acid sequences encoding any synthetic polypeptide disclosed herein.Disclosed herein are compositions comprising DNA sequences encoding any synthetic polypeptide disclosed herein.Disclosed herein are compositions comprising messenger RNA sequences encoding any synthetic polypeptide disclosed herein.Disclosed herein are vectors comprising nucleic acid sequences encoding any synthetic polypeptide 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 nucleic acids encoding any synthetic polypeptide disclosed herein.Disclosed herein are cells comprising vectors comprising nucleic acid sequences encoding any synthetic polypeptide disclosed herein.In some embodiments, cells are transduced with nucleic acids or vectors disclosed herein.In some embodiments, cells express any synthetic polypeptide disclosed herein.In some embodiments, synthetic polypeptides are separated or extracted from cells.

[0214] In some embodiments, the synthetic polypeptide is a polymerase, such as a polymerase. Non-limiting 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 phage DNA polymerases; Bacillus subtilis DNA polymerase III and Escherichia coli (E. coli) DNA polymerase IIIα and ε; reverse transcriptases such as HIV M or O reverse transcriptases, avian myeloblastosis virus reverse transcriptases, or Moloney murine leukemia virus (MMLV) reverse transcriptases or telomerases. In some embodiments, the polymerase may include Klenow polymerase. In some embodiments, the polymerase is a DNA polymerase. Non-limiting examples of DNA polymerases include DNA polymerases derived from various archaea such as Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyroditium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, or variants thereof, including such polymerases as 9-degree N polymerase (e.g., SEQ ID NOs: 280-281), DNA polymerase (e.g., SEQ ID NO: 283), Deep (e.g., SEQ ID NO: 284), Therminator TM(e.g., SEQ ID NO: 282), Pyrococcus furiosus DNA polymerase (Pfu polymerase) (e.g., SEQ ID NO: 285), RB69 (e.g., SEQ ID NO: 287), KOD (Sigma-Aldrich), Pfx and Tgo polymerases. In some embodiments, the polymerase can include a wild-type or mutant backbone sequence of a polymerase from Candidatus Altiarchaeales archaea (e.g., SEQ ID NOs: 1-268, 288-381), from RLF 89458.1 (e.g., SEQ ID NOs: 391-413), from RLF 60390.1 (e.g., SEQ ID NOs: 414-430), from WP 175059460.1 (e.g., SEQ ID NOs: 431-445), from ADK 47977.1 (e.g., SEQ ID NOs: 446-447), from GBE 17769.1 (e.g., SEQ ID NOs: 448-449), from GBE 55812.1 (e.g., SEQ ID NOs: 450-451), from KUO 42443.1 (e.g., SEQ ID NOs: 452-453), from KXB 02540.1 (e.g., SEQ ID NOs: 454-455), from MBC 7218772.1 (e.g., SEQ ID NOs: 456-457), from RMF 90817.1 (e.g., SEQ ID NOs: 458-459), from WP 058946753.1 (e.g., SEQ ID NOs: 460-461), or from WP167886206.1 (e.g., SEQ ID NOs: 462-463). In some embodiments, the polymerase may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or higher identity to any one of SEQ ID NOs: 1-390, 464, 391-463, or 465. The sequences of the wild-type and mutant polymerases are in Tables 1A, 1B, 2, 3, 4, and 5, and Figure 17-52Table 1A lists wild-type polymerases having a backbone sequence from RLI 89578.1 (SEQ ID NO: 1) and mutant variants comprising amino acid substitutions relative to wild-type RLI 89578.1 (SEQ ID NOs: 2-268, 288-381). Any of the amino acid sequences listed in Table 1A may comprise the substitutions D141A and / or E143A. Table 1B lists various polymerase backbones and variants thereof (SEQ ID NOs: 269-287). Table 2 lists wild-type polymerases having a backbone sequence from RLF 89458.1 (SEQ ID NO: 391) and mutant variants comprising amino acid substitutions relative to wild-type RLF 89458.1 (SEQ ID NOs: 392-413). Table 3 lists the wild-type polymerase having a backbone sequence from RLF 60390.1 (SEQ ID NO: 414) and mutant variants comprising amino acid substitutions relative to wild-type RLF 60390.1 (SEQ ID NO: 415-430). Table 4 lists the wild-type polymerase having a backbone sequence from WP 175059460.1 (SEQ ID NO: 431) and mutant variants comprising amino acid substitutions relative to wild-type WP 175059460.1 (SEQ ID NO: 431-445). Table 5 lists various wild-type and mutant polymerases having 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), KXB02540.1 (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 Tables 1A, 1B, 2, 3, 4, and 5, underscores ("_") indicate intervals between two or more amino acid substitutions.

[0215] Table 1A: Mutations in the polymerase backbone of Candidatus Altiarchaeales archaea

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228] * = amino acid deletion starts from the codon marked by an asterisk (*) and ends at the stop codon.

[0229] Table 1B: Various backbone sequences of polymerases

[0230]

[0231] Table 2: Mutations of RLF 89458.1 polymerase backbone

[0232]

[0233] Table 3: Mutations of RLF 60390.1 polymerase backbone

[0234]

[0235] Table 4: Mutations of WP 175059460.1 polymerase backbone

[0236]

[0237] Table 5: Mutations of other polymerase backbones

[0238]

[0239] Nucleotide conjugates

[0240] In some embodiments, compositions comprising nucleotide conjugates are disclosed herein. Nucleotide conjugates disclosed herein can be used to form binding complexes disclosed herein, such as in capture reactions, nucleotide binding reactions, or nucleic acid sequencing reactions disclosed herein. In some embodiments, nucleotide conjugates can include nucleotides and parts. In some embodiments, parts can be coupled, bound, attached, or connected to nucleotides by covalent or non-covalent bonds. In some embodiments, parts can include chemical or biological parts. In some embodiments, nucleotide conjugates can include multivalent nucleotide conjugates. In some embodiments, multivalent nucleotide conjugates can have the ability to form binding complexes. In some embodiments, multivalent nucleotide conjugates can form multivalent binding complexes. In some embodiments, nucleotide conjugates can include cores, core attachment parts, spacers, joints, nucleotide units, or any combination thereof. In some embodiments, cores can include unimolecular, monomeric, single molecular units, or polymers. In some embodiments, polymers can include polypeptides, proteins, peptides, or derivatives thereof. In some embodiments, polymers can include polypeptides comprising modifications. In some embodiments, modifications of polypeptides can include any chemical, biological, or physical modifications that can make polypeptides form nucleotide conjugates disclosed herein. In some embodiments, modification can include modification with polyethylene glycol (PEG), or PEGylation.In some embodiments, the polypeptide comprising the modification may include, but is not limited to, those disclosed in the following references, each of which is incorporated herein by reference in its 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 2015Oct 9.PMID:26456916; PMCID:PMC4798869.

[0241] In some embodiments, a nucleotide conjugate comprises a core coupled to a plurality of "nucleotide arms," ​​e.g. Figure 1-3 In some embodiments, the nucleotide arm may be modular. In some embodiments, the nucleotide arm may include a core attachment portion. In some embodiments, the nucleotide arm may include a spacer. In some embodiments, the nucleotide arm may include a linker. In some embodiments, the nucleotide arm may include a nucleotide unit. In some embodiments, the nucleotide arm may include a core attachment portion, a spacer, a linker, and a nucleotide unit. Thus, the nucleotide conjugate may include multiple nucleotide units (e.g., see Figure 1-3 ). Alternatively, the nucleotide conjugate may comprise a single nucleotide unit.

[0242] In some embodiments, the nucleotide conjugates of the present disclosure provide an increase in the binding of nucleotide units to polymerase or to a composite polymerase at least by increasing the effective concentration of nucleotide units and enzymes. Such an increase is observed due to an increase in nucleotide concentration in solution or by increasing the amount of nucleotides adjacent to the site of polymerase-related binding or incorporation. The increase can also be achieved by physically limiting a certain number of nucleotides in a limited volume, thereby causing a local increase in nucleotide concentration. The nucleotide units of the nucleotide arms (e.g., attached to the core) can be bound to the polymerase binding site with a higher apparent affinity than can be observed for unconjugated, untethered or otherwise unrestricted single nucleotides. A method of realizing such restrictions can be by providing a nucleotide conjugate in which a plurality of nucleotide units are tethered to the core. In some embodiments, the core can be a particle, such as a polymer, a branched polymer, a dendrimer, a micelle, a liposome, a microparticle, a nanoparticle, a quantum dot or other suitable particles.

[0243] The present disclosure provides various embodiments of nucleotide conjugates, each of which is configured to include a core coupled (e.g., attached) to a plurality of nucleotide arms. In some embodiments, the nucleotide arms may include a core attachment portion. In some embodiments, the nucleotide arms may include a spacer. In some embodiments, the nucleotide arms may include a linker. In some embodiments, the nucleotide arms may include a nucleotide unit. In some embodiments, the nucleotide arms may include a core attachment portion, a spacer, a linker, and a nucleotide unit. A single nucleotide arm may include (i) a core attachment portion, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit (e.g., see Figure 4 In some embodiments, the nucleotide arms may comprise nucleoside units rather than nucleotide units. The nucleotide arms may include biotin. In some embodiments, the nucleotide conjugates may comprise multiple copies of the same type of nucleotide attached to the core via multiple nucleotide arms (e.g., see Figure 1 , 2and 3). In a nucleotide conjugate, the tethered nucleotide is referred to as a nucleotide unit. In some embodiments, multiple copies of a nucleotide unit may be covalently or non-covalently bound to a core. The nucleotide arm is designed so that the nucleotide unit of the nucleotide arm can interact with one or more polymerases in a manner similar to free nucleotides. The nucleotide unit of each nucleotide arm can bind to a polymerase (e.g., nucleotide association) that is complexed with a nucleic acid template and a nucleic acid primer. The nucleotide unit can also dissociate from the composite polymerase and re-bind to the same composite polymerase or to a different composite polymerase close to the nucleotide conjugate. Since the nucleotide conjugate can include multiple nucleotide arms, the nucleotide unit of a single nucleotide conjugate can simultaneously bind to multiple composite polymerases. The valence level of the nucleotide unit of a given nucleotide conjugate can correspond to the number of nucleotide arms attached to the core. In some embodiments, a single nucleotide conjugate can substantially simultaneously bind to multiple composite polymerases that are bound to the same template molecule (e.g., concatemer). Nucleotide conjugates can effectively increase the local concentration of nucleotides, which can enhance the signal in the nucleotide binding reaction.

[0244] The composite polymerase can include a synthetic polypeptide disclosed herein that is attached to a nucleic acid duplex, wherein the duplex can include a nucleic acid template molecule hybridized with a nucleic acid primer, wherein the primer can include an extendable or non-extendable terminal 3' end. The template molecule can be a single-stranded or double-stranded linear or circular nucleic acid molecule. The template molecule can be a clonal amplification nucleic acid molecule. The template molecule can be a nucleic acid concatemer. In some embodiments, the concatemer can include two or more tandem copies of a sequence of interest. The template and primer can be completely or partially complementary along the hybridization region.

[0245] Alternatively, the composite polymerase can include a polymerase that is bound to a self-priming nucleic acid template molecule, wherein the self-priming portion can include an extendable or non-extendable terminal 3' end. The template molecule can include a single-stranded or double-stranded region. The template molecule can be a clonal amplification nucleic acid molecule. The template molecule can be a nucleic acid concatemer, for example, comprising 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 hybridization region.

[0246] A single nucleotide conjugate can bind to multiple complex polymerases bound to the same template molecule (e.g., a concatemer with the same target nucleic acid sequence repeated), thereby forming a multivalent binding complex. For example, (i) a first binding complex can include a first nucleic acid primer, a first polymerase, and a first nucleotide conjugate bound to a first portion of a concatemer template molecule, thereby forming a first binding complex, wherein the first nucleotide unit of the first nucleotide conjugate can be bound to the first polymerase, and (ii) a second binding complex can include a second nucleic acid primer, a second polymerase, and the same first nucleotide conjugate can be bound to the second portion of the same concatemer template, thereby forming a second binding complex, wherein the second nucleotide unit of the first nucleotide conjugate can be bound to the second polymerase, wherein the first and second binding complexes including the same nucleotide conjugate can form a multivalent binding complex. Therefore, in some embodiments, the multivalent binding complex of the present disclosure includes at least two copies of a target nucleic acid sequence bound to a single nucleotide conjugate disclosed herein. In some embodiments, the nucleotide unit of the nucleotide conjugate is complementary to the nucleotides in at least two copies of the target nucleic acid sequence. In some embodiments, the multivalent binding complex includes one or more polymerases disclosed herein (e.g., synthetic polypeptides).

[0247] The nucleotide unit of the nucleotide conjugate can be bound to a composite polymerase (e.g., a synthetic polypeptide disclosed herein) by binding to a primer (or a nascent extension primer) or the terminal 3' end of the self-priming portion without undergoing polymerase-catalyzed incorporation. The nucleotide unit bound to the composite polymerase can form a binding complex. The binding complex can be stable, wherein the nucleotide unit exhibits a low dissociation rate, and has a duration indicating the stability of the binding complex and the strength of the binding interaction. Conditions suitable for binding the nucleotide unit to the 3' end of the primer at a position relative to the complementary nucleotide in the template strand, wherein 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.

[0248] In some embodiments, the nucleotide conjugate may include at least one nucleotide unit having a sugar moiety with a 3'OH group or a 3' blocking group. The nucleotide unit having a sugar 3'OH group or a 3' blocking group can bind to a composite polymerase and interrogate a complementary nucleotide in a template strand, and the nucleotide unit does not undergo nucleotide incorporation. Conditions suitable for binding the nucleotide unit to a composite polymerase, wherein the nucleotide unit binds to the 3' end of a primer at a position opposite to a complementary nucleotide in a template strand and interrogates a complementary nucleotide in a template strand, and wherein the nucleotide unit does not undergo polymerase-catalyzed incorporation, may include the presence of at least one non-catalytic divalent cation, including strontium, barium, calcium, or a combination thereof.

[0249] Alternatively, the nucleotide unit can be bound to the composite polymerase by binding to the primer (or nascent extension primer) or the terminal 3' end of the self-priming part, and undergo polymerase-catalyzed incorporation into the 3' end of the extendable primer or the self-priming part, resulting in primer extension. When the nucleotide unit includes a hydroxyl group at the 3' sugar position, the subsequent nucleotide can be incorporated into the nascent extension primer. When the nucleotide unit includes a blocking group at the 3' sugar position, the subsequent nucleotide can be blocked and cannot be incorporated into the nascent extension primer chain. Conditions suitable for binding the nucleotide unit to the 3' end of the primer at a position opposite to the complementary nucleotide in the template chain, wherein the nucleotide unit undergoes polymerase-catalyzed incorporation, can include the presence of at least one catalytic divalent cation, including magnesium, manganese, or a combination of magnesium and manganese.

[0250] In some embodiments, the nucleotide conjugate may include at least one nucleotide unit with a sugar moiety having a 3'OH group or a 3' blocking group. The nucleotide unit with a sugar 3'OH group can bind to a composite polymerase and interrogate a complementary nucleotide in a template strand, and the nucleotide unit can undergo polymerase-catalyzed nucleotide incorporation. The nucleotide unit with a sugar 3' blocking group can bind to a composite polymerase, and can interrogate a complementary nucleotide in a template strand, and the nucleotide unit can undergo polymerase-catalyzed nucleotide incorporation, but the 3' blocking group can inhibit / prevent the incorporation of subsequent nucleotides (or the next nucleotide unit of the nucleotide conjugate). The 3' blocking group can be removed to promote the incorporation of subsequent nucleotides. Conditions suitable for binding a nucleotide unit to a composite polymerase, wherein the nucleotide unit binds to the 3' end of a primer at a position relative to a complementary nucleotide in a template strand and interrogates a complementary nucleotide in a template strand, and wherein the nucleotide unit undergoes polymerase-catalyzed incorporation, may include the presence of at least one catalytic divalent cation, including magnesium, manganese, or a combination of magnesium and manganese.

[0251] In some embodiments, the joint unit of the nucleotide arm can help bind to the polymerase, to stabilize the polymerase-nucleotide ternary complex or to bind to the combination of the polymerase and the stable polymerase-nucleotide ternary complex. We found that the joint and nucleotide can be recognized and distinguished by the DNA polymerase. We found that the proximal part of the joint unit can play a key role in promoting the polymerase-nucleotide binding interaction. The optimization of the joint region can provide a nucleotide conjugate that can be used for specific applications such as improved nucleic acid sequencing.

[0252] The crystal structure of a sequencing polymerase co-crystallized with a primer-template molecule and a nucleotide conjugate shows that the carbonyl oxygen of the nucleotide arm interacts with a lysine residue of the polymerase (see, e.g. Fig.16and Example 8). In the nucleotide arm, the carbonyl oxygen is close to the nucleotide unit. The polymerase used in Example 8 has a backbone sequence based on the amino acid sequence of SEQ ID NO: 1, and the lysine residue is located at position 495. In addition, binding studies comparing different types of nucleotide conjugates having nucleotide arms carrying different linkers showed different levels of binding. Data from the binding studies indicate that the presence of an aromatic moiety (e.g., an aryl group) on the linker can improve the binding of the nucleotide unit to the polymerase (e.g., Fig.11 and 2 and Example 5).

[0253] Nucleotide conjugates can be labeled with detectable reporter moieties. In some embodiments, the core of a nucleotide conjugate can be labeled with detectable reporter moieties (e.g., fluorophores) in a manner that allows for distinguishing different nucleotide conjugates carrying different types of nucleotide units. For example, the core unit of a first nucleotide conjugate can be labeled with a first fluorophore, wherein the first nucleotide conjugate can include multiple nucleotide arms with nucleotide units of the same type, such as dGTP nucleotide units. The core unit of a second nucleotide conjugate can be labeled with a second fluorophore (which is different from the first fluorophore), wherein the second nucleotide conjugate can include multiple nucleotide arms with nucleotide units of the same type, such as dATP nucleotide units. The mixture of labeled nucleotide conjugates can include any combination of two or more subgroups of nucleotide conjugates, wherein each subgroup includes a nucleotide conjugate with a type of nucleotide unit and a nucleotide conjugate corresponding to a specific type of nucleotide unit through a reporter molecule labeled core. The mixture of labeled and unlabeled nucleotide conjugates can include at least one subpopulation of nucleotide conjugates comprising a plurality of nucleotide conjugates having one type of nucleotide unit and a reporter-labeled core corresponding to a particular type of nucleotide unit, and at least one subpopulation of nucleotide conjugates comprising a plurality of nucleotide conjugates having different types of nucleotide units and an unlabeled core (wherein the unlabeled core corresponds to a different type of nucleotide unit). A single population of labeled nucleotide conjugates or a mixture of different subpopulations can be used for nucleotide binding assays, nucleotide incorporation assays, nucleic acid sequencing methods, or a combination thereof. Nucleotide conjugates can be used for massively parallel nucleic acid sequencing.

[0254] Disclosed herein is a formulation 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 of a first wavelength. In some embodiments, the second fluorophore emits light of a second wavelength. In some embodiments, the third fluorophore emits light of a third wavelength. In some embodiments, the fourth fluorophore emits light of 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, the second wavelength, and the third wavelength are different from each other. In some embodiments, each of the first wavelength, the second wavelength, and the third wavelength is different from another. In some embodiments, all of the first wavelength, the second wavelength, and the third wavelength are different from each other. In some embodiments, at least two of the first wavelength, the second wavelength, the third wavelength, and the fourth wavelength are different from each other. In some embodiments, at least three of the first wavelength, the second wavelength, the third wavelength, and the fourth wavelength are different from each other. In some embodiments, each of the first wavelength, the second wavelength, the third wavelength, and the fourth wavelength is different from another. In some embodiments, the first wavelength, the second wavelength, the third wavelength, and the fourth wavelength are all different from each other. The mixture of different subpopulations of labeled nucleotide conjugates can include a first subpopulation of nucleotide conjugates comprising a first fluorophore and a second subpopulation of nucleotide conjugates comprising a second fluorophore.

[0255] In some embodiments, at least one nucleotide unit of a nucleotide conjugate can be labeled with a detectable reporter moiety (e.g., a fluorophore) in a manner that allows for different nucleotide conjugates carrying different types of nucleotide units to be distinguished. For example, the base in the nucleotide unit of a first nucleotide conjugate can be labeled with a first fluorophore, wherein the first nucleotide conjugate can include multiple nucleotide arms with nucleotide units of the same type, such as dGTP nucleotide units. The base in the nucleotide unit of a second nucleotide conjugate can be labeled with a second fluorophore (which is different from the first fluorophore), wherein the second nucleotide conjugate can include multiple nucleotide arms with nucleotide units of the same type, such as dATP nucleotide units. The mixture of labeled nucleotide conjugates can include any combination of two or more subgroups of nucleotide conjugates, wherein each subgroup includes a nucleotide conjugate with a type of nucleotide unit and a reporter-labeled core base corresponding to a particular type of nucleotide unit. The mixture of labeled and unlabeled nucleotide conjugates can include at least one subpopulation of nucleotide conjugates comprising a plurality of nucleotide conjugates having one type of nucleotide unit and a reporter-labeled nucleobase corresponding to a particular type of nucleotide unit, and at least one subpopulation of nucleotide conjugates comprising a plurality of nucleotide conjugates having different types of nucleotide units and unlabeled nucleobases (wherein the unlabeled nucleobases correspond to different types of nucleotide units). A single population of labeled nucleotide conjugates or a mixture of different subpopulations can be used for nucleotide binding assays, nucleotide incorporation assays, nucleic acid sequencing methods, or a combination thereof. Nucleotide conjugates can be used for massively parallel nucleic acid sequencing.

[0256] Nucleotide conjugates can be used to locate detectable signals to active regions of biochemical interactions, such as sites of protein-nucleic acid interactions, nucleic acid hybridization reactions, or enzymatic reactions (such as polymerase-mediated reactions). For example, nucleotide conjugates described herein can be used to identify sites where nucleobases bind or incorporate during polymerase-catalyzed reactions, and provide base discrimination for sequencing and array-based applications. When a nucleotide unit is complementary to an "N" base in a template nucleic acid, the increased binding or incorporation between the template nucleic acid and the nucleotide unit can provide an enhanced signal, which greatly improves base determination accuracy and shortens imaging time.

[0257] In addition, the labeled nucleotide conjugate can form a multivalent binding complex that increases the base determination signal from a given polymerase community containing multiple copies (e.g., concatemers) of a template nucleic acid chain. A sequencing workflow comprising generating a polymerase community with cloned amplified copies of a template chain can have the following advantages: since there are multiple simultaneous sequencing reactions in a defined region, each reaction provides its own signal, so the signal can be amplified. The presence of multiple signals in a defined region can also reduce the impact of any erroneously advanced or skipped cycles, since the signal from a large number of correct base determinations can overwhelm the signal from a smaller number of incorrect base determinations that are advanced or skipped, thus providing a method for reducing pre-phasing or phasing errors, improving the read length in a sequencing reaction, or reducing phasing errors and improving the read length in a sequence reaction.

[0258] The nucleotide conjugates and uses thereof disclosed herein can result in one or more of the following: (i) stronger signals for better base calling accuracy compared to nucleic acid amplification and sequencing methods; (ii) allow better discrimination of sequence-specific signals from background signals; (iii) reduce the requirements for the amount of starting material, (iv) increase sequencing rates and shorten sequencing times; (v) reduce phasing errors and (vi) improve read lengths in sequencing reactions.

[0259] The present disclosure provides a nucleotide conjugate comprising a core attached to at least one nucleotide arm. In some embodiments, at least one nucleotide arm may include a core attachment portion. In some embodiments, at least one nucleotide arm may include a spacer. In some embodiments, at least one nucleotide arm may include a joint. In some embodiments, at least one nucleotide arm may include a nucleotide unit. In some embodiments, at least one nucleotide arm may include a core attachment portion, a spacer, a joint and a nucleotide unit. In some embodiments, the core may include beads, particles or nanoparticles. In some embodiments, the core may include an alkyl, alkenyl or alkynyl core, such as may be present in a branched polymer or dendritic macromolecule. In some embodiments, the core may include a portion that mediates the conjugation of the core to the nucleotide arm. In some embodiments, the core may be attached to a plurality of nucleotide arms. In some cases, the core may be attached to about 1 to about 50 nucleotide arms. In some cases, the core is attached to about 2 to about 20 nucleotide arms. In some cases, the core is attached to about 2 to about 4 nucleotide arms. In some cases, the core is attached to about 4 to about 10 nucleotide arms. In some cases, the core is attached to about 10 to about 15 nucleotide arms. In some cases, the core is attached to about 15 to about 20 nucleotide arms. Figure 1 , 2 and 3 show the general architecture of the nucleotide conjugates.

[0260] 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 may comprise a core attachment portion. In some embodiments, at least one biotinylated nucleotide arm may comprise a spacer. In some embodiments, at least one biotinylated nucleotide arm may comprise a joint. In some embodiments, at least one biotinylated nucleotide arm may comprise a nucleotide unit. In some embodiments, at least one biotinylated nucleotide arm may comprise a core attachment portion, a spacer, a joint, and a nucleotide unit. In some embodiments, the core may comprise a streptavidin-type or avidin-type portion, and the biotin unit of the biotinylated nucleotide arm may mediate the conjugation of the core to the biotinylated nucleotide arm ( Figure 2 ). The streptavidin-type or avidin-type core may be a tetrameric biotin-binding protein that may bind one, two, three or up to four biotinylated nucleotide arms.

[0261] 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 includes a polymer or a metal. In some embodiments, the polymer is synthetic. In some embodiments, the polymer is natural. In some embodiments, the polymer includes plastic or protein. In some embodiments, the protein includes streptavidin or avidin, or a derivative thereof, an analog thereof, and other non-natural forms thereof that can be bound to at least one biotin moiety. In some embodiments, plastic is or includes polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cycloolefin polymer (COP), cycloolefin copolymer (COC), polyethylene terephthalate (PET) or polyethylene glycol (PEG), or any combination thereof. In some embodiments, the core can include 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.

[0262] In some embodiments, the core may include avidin, streptavidin, etc., or derivatives thereof; branched polymers; dendrimers; cross-linked polymer particles, such as agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dots; liposomes; emulsion particles or any other suitable particles (e.g., nanoparticles, microparticles, etc.). In some embodiments, the core may include a streptavidin-type or avidin-type portion, including streptavidin or avidin, and any derivatives, analogs, and other non-natural forms of streptavidin or avidin that can be bound to at least one biotin portion. The streptavidin or avidin portion may include natural or recombinant forms, as well as mutant versions and derivatized molecules. Mutant versions of streptavidin and avidin may include any one or any combination of two or more of amino acid insertions, deletions, substitutions, or truncations. Mutant versions may also include fusion polypeptides.

[0263] Nucleotide conjugates can be configured using streptavidin or avidin cores with high affinity for the biotin moiety on the biotinylated nucleotide arm to reduce the dissociation of the nucleotide arm from the core. A mixture of nucleotide conjugates can be prepared, wherein the mixture contains two or more subgroups of nucleotide conjugates, and each subgroup contains a nucleotide conjugate with a 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 undesirable dissociation of the nucleotide arm from the core, and the exchange of nucleotide arms between different cores. During the sequencing reaction, the exchange of nucleotide arms can lead to incorrect base determination and reduced sequencing accuracy. In some embodiments, the nucleotide conjugates with increased stability (e.g., the dissociation of the biotinylated nucleotide arm is reduced) can include dye-labeled streptavidin, wherein the streptavidin subunit carries a Lys121Arg mutation, which can show that the dissociation of the biotinylated nucleotide arm from the streptavidin core is reduced.

[0264] The streptavidin portion can include full-length or truncated forms that have a high affinity for binding biotin. For example, the streptavidin portion can exhibit a dissociation constant (Kd) of about 10 -14 mol / L or about 10 -15mol / L. In some embodiments, the streptavidin portion may include a polypeptide having a backbone sequence of any one of SEQ ID NOs: 466-470. The streptavidin portion may include an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or higher identity to any one of SEQ ID NOs: 466-470. The streptavidin portion may include a streptavidin having an amino acid sequence of any one of SEQ ID NOs: 466-470 with the core portion of the streptavidin truncated at the N-terminus, C-terminus, or a combination of the N-terminus and C-terminus. For example, the streptavidin portion may lack the N-terminal portion of any one of SEQ ID NOs: 466-468, 470-471 (e.g., Figure 17-19 , 21-22 with an underlined N-terminal portion). The streptavidin portion may lack the C-terminal portion of any one of SEQ ID NOs: 466-468 (e.g., Figure 17-19 The streptavidin portion may include a core portion comprising the amino acid sequence of SEQ ID NO: 469 or 470.

[0265] In some embodiments, the streptavidin moiety may be included in any amino acid substitution mutation at a site that can be labeled with a dye. For example, the dye-labeled site may include a lysine at position 121 (e.g., see SEQ ID NO: 469), which may overlap with a biotin binding site. In some embodiments, a dye attached to streptavidin at Lys121 may block or inhibit biotin from binding to dye-labeled streptavidin. A nucleotide conjugate comprising a dye-labeled streptavidin carrying lysine at position 121 may exhibit dissociation of a biotinylated nucleotide arm from a streptavidin core. A nucleotide conjugate with increased stability may include a dye-labeled streptavidin carrying a Lys121Arg mutation (e.g., SEQ ID NO: 469), which may exhibit reduced dissociation of a biotinylated nucleotide arm from a streptavidin core.

[0266] In some embodiments, the streptavidin portion may comprise any of the following amino acid substitutions: d Increase to about 10 -16mol / L), improving biotin retention at temperatures up to about 60°C, about 65°C, about 70°C, or about 80°C, or a combination of increasing affinity for binding biotin and improving biotin retention. Amino acid substitutions may 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 may be substituted with lysine or aspartic acid are Figure 17-21 Medium bold and underlined.

[0267] The avidin portion can include full-length or truncated forms that have a high affinity for binding biotin. For example, the avidin portion can exhibit a dissociation constant (Kd) of about 10 -14 mol / L or about 10 -15 mol / L. In some embodiments, the avidin portion may include a polypeptide having a backbone sequence of SEQ ID NO: 471 or 472. The avidin portion may include an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO: 471 or 472. The avidin portion may include an avidin having an amino acid sequence of SEQ ID NO: 471 that is truncated at the N-terminus, C-terminus, or a combination of the N-terminus and C-terminus. For example, the avidin portion may lack the N-terminal portion of SEQ ID NO: 471 (e.g., Fig. 22 In some embodiments, avidin can include substitutions of any one or any combination of the eight arginine residues (e.g., Fig. 22 or 23 are underlined and bold). Amino acid substitutions may include replacing five of the eight arginine residues at positions 26, 50, 83, 111, 124, 138, 146, 148, or a combination thereof, of SEQ ID NO: 471 with a neutral amino acid; or replacing five of the eight arginine residues at positions 2, 26, 59, 87, 100, 114, 122, 124, or a combination thereof, of SEQ ID NO: 472 with a neutral amino acid.

[0268] Avidin can include partially deglycosylated forms and non-glycosylated forms. Avidin can include derivatized forms, for example, N-acyl avidin, such as N-acetyl, N-phthalyl and N-succinyl avidin, and commercially available products, including CaptAvidin TM (selectively nitrates tyrosine residues at four biotin-binding sites to generate avidin that reversibly binds biotin), NeutrAvidin TM (chemically deglycosylated and including modified arginine residues) and Neutralite Avidin TM (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 with neutral polar side chains include asparagine, cysteine, glutamine, serine, threonine, and tyrosine.

[0269] In some embodiments, the core can be labeled with a detectable portion. Non-limiting examples of detectable portions include fluorescent, bioluminescent, chemiluminescent, radioactive detectable portions. In some embodiments, the nucleotide conjugate can be unlabeled. The core can be a streptavidin or avidin that is a homotetramer. Each subunit in the homotetramer can include at least one lysine residue that can be conjugated to a fluorophore. The labeling reaction can use a fluorophore conjugated to an N-hydroxysuccinimide (NHS) ester. 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 described in Figure 17-23 For example, the streptavidin subunit may include 4 lysines (e.g., SEQ ID NO: 469), 8 lysines (e.g., SEQ ID NO: 467, 468, 470), or 9 lysines (e.g., SEQ ID NO: 466). The avidin subunit may include 9 lysines (e.g., SEQ ID NO: 471 and 472).

[0270] When preparing a labeled streptavidin or avidin core, 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 of label / protein form. Dye-core conjugates with lower degrees of labeling will exhibit weaker fluorescence intensity. Dye-core conjugates with very high degrees of labeling (e.g., DoL>6) can exhibit reduced fluorescence due to self-quenching of the conjugated fluorophore. In some embodiments, the predetermined degree of labeling of the streptavidin or avidin core can depend on the dye. Fluorescent dyes include, but are not limited to, CF647, CF680, CF570, and CF532 dyes from Biotium; AF647, AF680, AF568, and AF532 from Thermo Fisher Scientific; IFluor 647, IFluor 680, IFlour568, and IFlour 532 from AATBio; DY648P1, DY679P1, DY585, and DY530 from Dyomics; and AFDy 647, IRFlour 680LT, AFDye 568, and AFDye 532 from Fluoroprobes. The predetermined degree of labeling may be about 1-10, or about 3-8, or about 3.5-7, or about 1.6-4.

[0271] Red fluorophores are brighter (higher in intensity) than green dyes, which can cause bleed-through when imaging red-labeled and green-labeled nucleotide conjugates on the same support (e.g., flow cell). The degree of labeling of a subpopulation of nucleotide conjugates can be increased or decreased to achieve an improved signal balance from a mixture of labeled nucleotide conjugates. For example, the degree of labeling of a subpopulation of nucleotide conjugates labeled with a red fluorophore can be reduced 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, or 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.

[0272] Solution fluorescence measurements can be used to determine the relative brightness of the labeled streptavidin or avidin core. Alternatively, the degree of labeling can be determined by employing a functional assay (e.g., a flow cell trap assay) in which clonal amplification template molecules immobilized on a flow cell are contacted with primers, polymerase, and fluorescently labeled nucleotide conjugates under conditions suitable for binding the nucleotide conjugates to the composite polymerase without incorporating nucleotide units into the primers, and the signal intensity can be detected.

[0273] In some embodiments, the nucleotide conjugate comprises at least two nucleotide arms. In some embodiments, the nucleotide arms in at least two nucleotide arms comprise: a core attachment portion coupled to a core. In some embodiments, the nucleotide arms in at least two nucleotide arms comprise a spacer coupled to a core attachment portion. In some embodiments, the nucleotide arms in at least two nucleotide arms comprise a linker coupled to the spacer. In some embodiments, the nucleotide arms in at least two nucleotide arms comprise a nucleotide unit coupled to a linker. In some embodiments, the nucleotide conjugate comprises: (a) a core; and (b) at least two nucleotide arms. In some embodiments, the nucleotide arms in at least two nucleotide arms comprise: (i) a core attachment portion coupled to a core; (ii) a spacer coupled to a core attachment portion; (iii) a linker coupled to the spacer; and (iv) a nucleotide unit coupled to a linker. In some embodiments, the linker comprises:

[0274]

[0275]

[0276] And n is 1-6 and m is 0-10.

[0277] In some embodiments, the linker comprises

[0278]

[0279]

[0280] In some embodiments, the linker comprises

[0281]

[0282] In some embodiments, the linker comprises

[0283]

[0284] In some embodiments, the linker comprises

[0285]

[0286] In some embodiments, the linker comprises

[0287]

[0288] wherein n is 1 to 6 and m is 0 to 10.

[0289] In some embodiments, the linker comprises

[0290] wherein n is 1 to 6 and m is 0 to 10.

[0291] In some embodiments, the linker comprises

[0292] wherein n is 1 to 6 and m is 0 to 10.

[0293] In some embodiments, the linker comprises

[0294]

[0295] wherein n is 1 to 6 and m is 0 to 10.

[0296] In some embodiments, the linker comprises

[0297]

[0298] In some embodiments, the linker comprises

[0299]

[0300] In some embodiments, the linker comprises

[0301]

[0302] In some embodiments, the linker comprises

[0303]

[0304] In some embodiments, the linker comprises

[0305]

[0306] In some embodiments, at least two nucleotide arms include 3 to 20 nucleotide arms, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide arms. In some embodiments, at least two nucleotide arms include 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 includes a polypeptide. In some embodiments, the polypeptide includes streptavidin or avidin. In some embodiments, the polypeptide includes streptavidin. In some embodiments, the polypeptide includes avidin. In some embodiments, the core attachment portion includes biotin.

[0307] In some embodiments, the nucleotide conjugate comprises a label, such as a detectable label. In some embodiments, the label is coupled to the core or the nucleotide arm. In some embodiments, the label is coupled to the core. In some embodiments, the label is coupled to the nucleotide arm. In some embodiments, the label is or includes a fluorescent label. In some embodiments, the composition further comprises a fluorescent label coupled to the core. In some embodiments, the composition further comprises a fluorescent label coupled to the nucleotide arm.

[0308] In some embodiments, the spacer comprises the following structure:

[0309]

[0310] wherein m is 20 to 500 and o is 1 to 10.

[0311] In some embodiments, the nucleotide arm further comprises a reactive group. In some embodiments, the reactive group is coupled to the nucleotide unit. In some embodiments, the reactive group is configured to react with a reagent. In some embodiments, the nucleotide arm further comprises a reactive group coupled to the nucleotide unit, and the reactive group is configured to react with a reagent. In some embodiments, the reactive group comprises an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, ketone, isocyanate, phosphate, thiol, disulfide, carbonate, urea or silyl group. In some embodiments, the alkyl, alkenyl, alkynyl or allyl group in the reactive group is coupled to tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H 5 ) 3 ) 4 ) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl or benzyl group in the reactive group reacts with H 2 In some embodiments, the amine, amide, ketone, isocyanate, phosphate, thiol, or disulfide group in the reactive group reacts with a phosphine or a thiol group including β-mercaptoethanol or dithiothreitol (DTT). In some embodiments, the carbonate group in the reactive group reacts with potassium carbonate (K2CO3) in methanol (MeOH). 2 CO 3In some embodiments, the urea or silyl group in the reactive group reacts with tetrabutylammonium fluoride, hydrofluoric acid pyridine (HF-pyridine), ammonium fluoride or triethylamine trihydrofluoride. In some embodiments, the azide group in the reactive group comprises an azide, an azido or an azidomethyl group. In some embodiments, the reagent 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 tri(2-carboxyethyl)phosphine (TCEP) or bissulfotriphenylphosphine (BS-TPP). In some embodiments, the nucleotide units of at least two nucleotide arms comprise the same nucleobase type.

[0312] In some embodiments, the nucleotide unit comprises a blocking group. In some embodiments, the blocking group is attached to the 3' carbon of the sugar part of the nucleotide unit. In some embodiments, the blocking group reacts with a compound to remove the blocking group from the nucleotide unit. In some embodiments, the blocking group reacts with a compound to remove the blocking group from the nucleotide unit to generate a nucleotide unit comprising an OH portion. In some embodiments, the nucleotide unit comprises a blocking group attached to the 3' carbon of the sugar part of the nucleotide unit, and the blocking group reacts with a compound to remove the blocking group from the nucleotide unit to generate a nucleotide unit comprising an OH portion on the 3' carbon of the sugar part of the nucleotide unit. In some embodiments, the blocking group reacts with a compound to remove the blocking group from the nucleotide unit to generate a nucleotide unit comprising an OH portion on the 3' carbon of the sugar part of the nucleotide unit. In some embodiments, the blocking group includes an alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, ketone, isocyanate, phosphate, thiol, disulfide, carbonate, urea or silyl group.

[0313] In some embodiments, the alkyl, alkenyl, alkynyl, or allyl group in the blocking group is fused to tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H 5 ) 3 ) 4 ) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the aryl or benzyl group in the blocking group is reacted with H 2In some embodiments, the amine, amide, ketone, isocyanate, phosphate, thiol or disulfide group in the blocking group reacts with a phosphine or a thiol group including β-mercaptoethanol or dithiothreitol (DTT). In some embodiments, the carbonate group in the blocking 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 in the blocking group reacts with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride or triethylamine trihydrofluoride.

[0314] In some embodiments, the azide in the blocking group includes an azide, an azido or an azidomethyl group. In some embodiments, an azide, an azido or an azidomethyl group reacts with a chemical reagent. In some embodiments, the chemical reagent includes a phosphine compound. In some embodiments, an azide, an azido or an azidomethyl group reacts with a chemical reagent including a phosphine compound. In some embodiments, the phosphine compound includes a derivatized trialkylphosphine portion or a derivatized triarylphosphine portion. In some embodiments, the phosphine compound includes tris (2-carboxyethyl) phosphine (TCEP) or bissulfotriphenylphosphine (BS-TPP).

[0315] Nucleotide arm

[0316] The present disclosure provides nucleotide conjugates comprising one or more nucleotide arms. In some embodiments, a "nucleotide arm" can be modular. In some embodiments, a nucleotide arm can include a core attachment portion. In some embodiments, a nucleotide arm can include a spacer. In some embodiments, a nucleotide arm can include a joint. In some embodiments, a nucleotide arm can include a nucleotide unit. In some embodiments, a nucleotide arm can include a core attachment portion, a spacer, a joint, and a nucleotide unit. In some embodiments, a nucleotide arm can be a nucleoside instead of a nucleotide. In some embodiments, a nucleotide or a nucleoside can include an analog thereof.

[0317] In some embodiments, a nucleotide arm may be attached to a core. In some embodiments, two or more nucleotide arms may be attached to a core to form a nucleotide conjugate. A nucleotide conjugate may comprise multiple nucleotide arms (e.g., Figure 1-3 ), wherein individual nucleotide arms comprise nucleotide units that can bind to different complex polymerases to form multivalent binding complexes.

[0318] Spacer

[0319] In some embodiments, the composition (e.g., nucleotide conjugate) comprises a spacer. In some embodiments, the nucleotide arm may comprise a spacer. In some embodiments, the spacer may be coupled to any other component of the nucleotide arm, including but not limited to a core, nucleotide unit, joint, or any other component disclosed herein.

[0320] The spacer can physically separate the nucleotide unit or nucleoside unit from the core. Figure 5A The spacer can have 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, Figure 5A In the spacers shown in , the value of o can be 1 to 50. In some embodiments, the value of o can be about 1 to 10 or the value of o is about 4. The spacer can be a linear or branched molecule.

[0321] In some embodiments, the spacer can include polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid.

[0322] In some embodiments, the spacer may include polyethylene glycol (PEG) having a 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 greater molecular weight PEG.

[0323] In some embodiments, the spacer unit of the nucleotide arm can be attached to the biotin portion, thereby forming a biotinylated nucleotide arm. In some embodiments, the biotinylated nucleotide arm can include a core attachment portion. In some embodiments, the biotinylated nucleotide arm can include a spacer. In some embodiments, the biotinylated nucleotide arm can include a linker. In some embodiments, the biotinylated nucleotide arm can include a nucleotide unit. In some embodiments, the biotinylated nucleotide arm can include a core attachment portion, a spacer, a linker, and a nucleotide unit. In some embodiments, the biotinylated nucleotide arm can include (i) biotin, (ii) a spacer, (iii) a linker, and (iv) a nucleotide (or nucleoside) (e.g., see Fig. 7A -D).

[0324] Connectors

[0325] In some embodiments, the nucleotide arm may include a joint. In some embodiments, the joint may be coupled to any other component of the nucleotide arm, including but not limited to the core, nucleotide unit, spacer or any other component disclosed herein. In some embodiments, the nucleotide arm may include a joint having any combination of any one or two or more parts including amide, carbonyl oxygen, aromatic moiety, polyether moiety or a combination thereof. The aromatic moiety may include a six-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 joint may include a linear or branched molecule. The joint may include or lack a reactive moiety (e.g., a cleavable moiety). The joint portion of the nucleotide arm may interact with a polymerase. The type of part and the position of the part in the joint may be selected to optimize the interaction with the polymerase binding pocket. For example, the amide moiety in the joint may interact with a polymerase via a hydrogen bond. The aromatic moiety in the joint may interact with a polymerase via a hydrophobic interaction. The nucleotide arm may include a joint 6, which includes a carbonyl oxygen and an aromatic moiety at the proximal end of the nucleotide unit. Crystallographic data indicate that the carbonyl oxygen interacts with a lysine residue in the polymerase binding pocket. Capture assays have shown that linkers carrying aromatic moieties can exhibit improved binding to polymerase when compared to linkers lacking the aromatic moiety (e.g., see Fig.11 and 12 and Example 5).

[0326] In some embodiments, the nucleotide arm may comprise a linker, which may comprise Figure 5A -F. In some embodiments, R1 of the linker may include any group, such as a nucleotide, a nucleoside, or an analog thereof. In some embodiments, R2 of the linker may include any group, such as a spacer (e.g., see Figure 5A In some embodiments, in the linker, m may have a value of 0-10. In some embodiments, in the linker, n may have a value of 1-6.

[0327] In some embodiments, the joint may include an aliphatic chain having 2-8 units. In some embodiments, the joint may include an ethylene glycol oligomer portion having 2-8 units. In some embodiments, the joint may include an aromatic group. In some embodiments, the joint may include an aromatic group and an ethylene glycol oligomer portion having 2-8 units. In some embodiments, the joint may include an aliphatic chain having 2-6 subunits. In some embodiments, the joint may include an ethylene glycol oligomer chain having 2-6 subunits. In some embodiments, the aromatic group may include an aryl group (e.g., see Figure 5A-C in N3 linker and linker 1-7). In some embodiments, the aromatic group may include a six-carbon ring group. In some embodiments, the aromatic group may include a meta-aminomethylbenzoic acid (also known as 3-aminomethylbenzoic acid) group (mAMBA) (e.g., Figure 5C In some embodiments, the linker may include a fluorenylmethoxycarbonyl protecting group (Fmoc), which may be removed when the linker is attached to the spacer. In some embodiments, the linker may include an NHS ester group (N-hydroxysuccinimide), which may be removed when the linker is attached to the nucleotide unit.

[0328] Nucleotide arm: spacer-linker-nucleotide

[0329] In some embodiments, the nucleotide arm may comprise a Figure 5A -F, and the linker can be attached to any nucleotide or nucleoside. The nucleotide arm is Fig. 6A and 6B Shown in.

[0330] In some embodiments, the nucleotide arm may comprise Figure 5A (top) shows the spacer of the structure. The spacer can have any length, for example, the value of m is 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 Figure 5A In the spacer shown in , 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 include polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid.

[0331] In some embodiments, the nucleotide arm can include any type of nucleotide or nucleoside joined to the joint. The nucleotide can include but is not limited to dATP, dGTP, dCTP, dTTP or dUTP. The nucleotide unit can be joined to the joint via a propargylamine attachment at the 5th position of a pyrimidine base or the 7th position of a purine base, or at the 1st position of a pyrimidine base or the 9th position of a purine base.

[0332] In some embodiments, certain combinations of linkers and nucleotides can exhibit improved nucleotide discrimination by a polymerase, as demonstrated by brighter fluorescent signals in nucleotide capture assays (e.g., see Fig. 9 , 10and 11 and Example 4). It is worth noting that the "N3" linker is photolabile. The "N3" portion can be used to reduce the residual fluorescence signal observed in the cycling assay.

[0333] Linker-nucleotide unit

[0334] In some embodiments, any linker described herein can be joined to a nucleotide to generate a nucleotide-linker molecule. Non-limiting examples of nucleotide-linker configurations are Fig. 7A For example, the nucleotide unit can be joined to the linker via a propargylamine attachment at the 5-position of a pyrimidine base or the 7-position of a purine base, or at the 1-position of a pyrimidine base or the 9-position of a purine base.

[0335] Biotinylated nucleotide arm

[0336] 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 Fig. 8A and 8B Shown in.

[0337] In some embodiments, the nucleotide conjugate may include a core attached to a plurality of biotinylated nucleotide arms. In some embodiments, the biotinylated nucleotide arm may include a core attachment portion. In some embodiments, the biotinylated nucleotide arm may include a spacer. In some embodiments, the biotinylated nucleotide arm may include a joint. In some embodiments, the biotinylated nucleotide arm may include a nucleotide unit. In some embodiments, the biotinylated nucleotide arm may include a core attachment portion, a spacer, a joint, and a nucleotide unit. In some embodiments, a single biotinylated nucleotide arm includes (i) a biotin portion, (ii) a spacer, (iii) a joint, and (iv) a nucleotide unit or a nucleoside unit.

[0338] Nucleotide unit

[0339] In some embodiments, any nucleotide arm and biotinylated nucleotide arm described herein can comprise a nucleotide unit. In some embodiments, a nucleotide unit can bind to a polymerase complexed with a nucleic acid template and a nucleic acid primer (e.g., nucleotide association). A nucleotide unit can also dissociate from a composite polymerase and re-bind to the same composite polymerase or to a different composite polymerase close to a nucleotide conjugate.

[0340] Nucleotide arms and biotinylated nucleotide arms can comprise nucleotide units, wherein the nucleotide units can comprise a heterocyclic base, a sugar and at least one phosphate group. In some embodiments, the nucleotides can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate groups.

[0341] Nucleotide arms and biotinylation Nucleotide arms may comprise nucleotide units (or nucleoside units) including heterocyclic bases including purine or pyrimidine bases, or analogs thereof.

[0342] In some embodiments, the 5-position of a pyrimidine base can be joined to a linker, the 7-position of a purine base can be joined to a linker, the 1-position of a pyrimidine base can be joined to a linker, or the 9-position of a purine base can be joined to a linker.

[0343] In some embodiments, the nucleotide arms and biotinylated nucleotide arms may comprise a nucleotide unit which is a propargylamine (PA) modified nucleotide, wherein the 5-position of the pyrimidine base or the 7-position of the purine base may be joined to a linker via a propargylamine group, or wherein the 1-position of the pyrimidine base or the 9-position of the purine base may be joined to a linker via a propargylamine group.

[0344] In some embodiments, the nucleotide arms and biotinylated nucleotide arms may comprise nucleotide units having ribose or deoxyribose sugar moieties. In some embodiments, the nucleotide units may be selected from 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 ( 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).

[0345] In some embodiments, the nucleotide arm and the biotinylated nucleotide arm can include a nucleotide unit (or nucleoside unit) with a sugar moiety, and the sugar moiety includes ribose, deoxyribose or its analog. In some embodiments, the sugar moiety can include a 3'OH group. In some embodiments, the nucleotide unit with a sugar 3'OH group can be combined with a composite polymerase including a polymerase that is bound to a nucleic acid template, and the nucleic acid template can be hybridized with a nucleic acid primer (or a self-starting template / primer nucleic acid molecule). For example, the nucleotide unit can be combined with a polymerase, and the terminal 3' end of the primer can be combined with the position opposite to the complementary nucleotide in the template chain. The nucleotide unit with 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 the incorporation of subsequent nucleotides catalyzed by a polymerase, such as in a primer extension reaction.

[0346] In some embodiments, the nucleotide arm and the biotinylated nucleotide arm can include a nucleotide unit (or nucleoside unit) with a sugar moiety having a 3'OH group substituted with a blocking group. In some embodiments, the nucleotide unit with a 3' blocking group can be combined with a composite polymerase including a polymerase bound to a nucleic acid template, which is hybridized with a nucleic acid primer (or a self-starting template / primer nucleic acid molecule). For example, the nucleotide unit can be combined with a polymerase, and the terminal 3' end of the primer can be combined with the position opposite to the complementary nucleotide in the template chain. The nucleotide unit with a 3' blocking group can undergo nucleotide incorporation in a polymerase-catalyzed reaction. The 3' blocking group on the incorporated nucleotide unit can inhibit / block the incorporation of subsequent nucleotides catalyzed by the polymerase, such as in a primer extension reaction.

[0347] Modified nucleotide arms

[0348] In some embodiments, any of the nucleotide arms and biotinylated nucleotide arms described herein may also include a linker having a reactive group at any position along the linker. For example, the azide portion ( Figure 5A ) can be replaced with a reactive group. In some embodiments, any nucleotide arm and biotinylated nucleotide arm described herein can comprise a linker having a reactive group selected from 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 ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0349] In some embodiments, the reactive groups in the linker can react with chemical reagents. For example, the reactive groups alkyl, alkenyl, alkynyl, and allyl react with tetrakis(triphenylphosphine)palladium(0) (Pd(P(C6H5)3)4) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The reactive groups aryl and benzyl react with H2 and palladium on carbon (Pd / C). The reactive groups amine, amide, ketone, isocyanate, phosphate, thiol, disulfide react with phosphine or with thiol groups including β-mercaptoethanol or dithiothreitol (DTT). The reactive group carbonate reacts with potassium carbonate (K2CO3) in MeOH. 2 CO 3 ), with triethylamine in pyridine or with Zn in acetic acid (AcOH). The reactive groups urea and silyl react with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride or with triethylamine trihydrofluoride.

[0350] In some embodiments, the reactive group in the joint can include azide, azido or azidomethyl groups. In some embodiments, the azide, azido or azidomethyl groups in the joint can react with chemical reagents. In some embodiments, the chemical reagent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine part or a derivatized triarylphosphine part. In some embodiments, the phosphine compound can include tris (2-carboxyethyl) phosphine (TCEP), bissulfotriphenylphosphine (BS-TPP) or tris (hydroxypropyl) phosphine (THPP).

[0351] In some embodiments, any of the nucleotide arms and biotinylated nucleotide arms described herein may further comprise a nucleotide unit having a sugar moiety having a 3' OH group substituted with a chain terminating moiety (blocking group), wherein the sugar 3' blocking group is selected from 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 ketone group, an isocyanate group, a phosphate group, a thiol group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0352] In some embodiments, the sugar 3' blocking group can include 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.

[0353] In some embodiments, the nucleotide arms and biotinylated nucleotide arms can include nucleotide units with sugar 3' blocking groups that react with chemical reagents. For example, sugar 3' blocking groups alkyl, alkenyl, alkynyl, and allyl groups can react with tetrakis(triphenylphosphine)palladium(0)(Pd(P(C 6 H5 ) 3 ) 4 ) with piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Blocking groups aryl and benzyl can be reacted with palladium on carbon (Pd / C). Blocking groups amines, amides, ketones, isocyanates, phosphates, thiols, disulfides can be reacted with phosphines or with thiol groups including β-mercaptoethanol or dithiothreitol (DTT). Blocking groups carbonates can be reacted with potassium carbonate (KCO) in MeOH. 2 CO 3 ), with triethylamine in pyridine or with Zn in acetic acid (AcOH). Blocking groups urea and silyl can be reacted with tetrabutylammonium fluoride, pyridine-HF, with ammonium fluoride or with triethylamine trihydrofluoride.

[0354] In some embodiments, the nucleotide arms and biotinylated nucleotide arms may comprise nucleotide units having a sugar 3' blocking group comprising an azide, an azido, or an azidomethyl group.

[0355] In some embodiments, the azide, azido or azidomethyl 3' blocking group can react with a chemical reagent. In some embodiments, the chemical reagent can include a phosphine compound. In some embodiments, the phosphine compound can include a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound can include tris(2-carboxyethyl)phosphine (TCEP), bissulfotriphenylphosphine (BS-TPP) or tris(hydroxypropyl)phosphine (THPP).

[0356] In some embodiments, the nucleotide arms and biotinylated nucleotide arms can comprise nucleotide units having a chain of one, two or three phosphorus atoms, wherein the chain is attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, the nucleotide units can be analogs having a phosphorus chain in which the phosphorus atoms are linked to an intermediate O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain can include substituted side groups, including O, S or BH 3 In some embodiments, the chain can include phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite groups.

[0357] Nucleotide conjugate combination

[0358] The present disclosure provides compositions, systems and kits comprising a plurality of nucleotide conjugates, which may include a mixture of at least two subgroups of nucleotide conjugates labeled with different reporting moieties. In some embodiments, at least a first subgroup of nucleotide conjugates may be labeled with a first reporting moiety corresponding to a first nucleotide unit on a nucleotide arm. In some embodiments, at least a second subgroup of nucleotide conjugates may be labeled with a second reporting moiety corresponding to a second nucleotide unit on a nucleotide arm. In some cases, the first and second reporting moieties may be different from each other. In some embodiments, a plurality of nucleotide conjugates may further include at least a third subgroup of nucleotide conjugates labeled with a third reporting moiety, wherein the first, second and third reporting moieties may be different from each other. In some embodiments, a plurality of nucleotide conjugates may further include at least a fourth subgroup of nucleotide conjugates labeled with a fourth reporting moiety, wherein the first, second, third and fourth reporting moieties may be different from each other. In some embodiments, other subgroups (e.g., the fifth, sixth, seventh, eighth, ninth, tenth or more) of labeled nucleotide conjugates may be added to the mixture. In some embodiments, the reporting moiety may be a fluorophore. In some embodiments, the first subpopulation of nucleotide conjugates can be labeled with a first fluorophore, and the second fluorophore of the nucleotide conjugates can be labeled with a second fluorophore. In some cases, the first fluorophore and the second fluorophore can be different.

[0359] The present disclosure provides compositions, systems and kits comprising a plurality of nucleotide conjugates, which may comprise a mixture of at least two subpopulations of nucleotide conjugates labeled with different reporter moieties. In some embodiments, at least a first subpopulation of nucleotide conjugates may 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 nucleotide conjugates may be unlabeled (e.g., dark nucleotide conjugates).

[0360] The present disclosure provides compositions, systems and kits comprising a plurality of nucleotide conjugates, which comprise a mixture of at least three subgroups of nucleotide conjugates labeled with different reporter moieties. In some embodiments, at least a first subgroup of nucleotide 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 subgroup of nucleotide 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 subgroup of nucleotide conjugates can be unlabeled (e.g., dark nucleotide conjugates). In some embodiments, the first and second reporter moieties can be different from each other.

[0361] The present disclosure provides compositions, systems and kits comprising a plurality of nucleotide conjugates, which comprise a mixture of at least four subgroups of nucleotide conjugates labeled with different reporting moieties. In some embodiments, the mixture of nucleotide conjugates may have at least a first subgroup of nucleotide conjugates, which may be labeled with a first reporting moiety corresponding to a first nucleotide unit on a nucleotide arm. In some embodiments, the mixture of nucleotide conjugates may have at least a second subgroup of nucleotide conjugates, which may be labeled with a second reporting moiety corresponding to a second nucleotide unit on a nucleotide arm. In some embodiments, the mixture of nucleotide conjugates may have at least a third subgroup of nucleotide conjugates, which may be labeled with a third reporting moiety. In some embodiments, the mixture of nucleotide conjugates may have at least a fourth subgroup of nucleotide conjugates, which may be unlabeled (e.g., dark nucleotide conjugates). In some cases, the first, second and third reporting moieties may be different from each other.

[0362] Disclosed herein is a formulation 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 of a first wavelength. In some embodiments, the second fluorophore emits light of a second wavelength. In some embodiments, the third fluorophore emits light of a third wavelength. In some embodiments, the fourth fluorophore emits light of 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, the second wavelength, and the third wavelength are different from each other. In some embodiments, each of the first wavelength, the second wavelength, and the third wavelength is different from another. In...

Claims

1. A synthetic polypeptide 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.

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

3. The synthetic polypeptide of claim 1 or 2, wherein the amino acid sequence further comprises a Y410A mutation, an 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, an I521H mutation or a K507L mutation with reference to SEQ ID No: 391, or any combination thereof.

4. The synthetic polypeptide of claim 3, wherein the amino acid sequence further comprises: (a) with reference to the Y410A mutation of SEQ ID No: 391; (b) with reference to the L409S mutation, the Y410A mutation and the Y261A mutation of SEQ ID No: 391; (c) with reference to the L409S mutation, the Y410A mutation, the P411G mutation and the Y261A mutation of SEQ ID No: 391; (d) with reference to the Y261A mutation, the F406I mutation, the L409S mutation, the Y410A mutation and the P411A mutation of SEQ ID No: 391; (e) with reference to the Y7A mutation, the Y261A mutation and the Y410A mutation of SEQ ID No: 391; (f) with reference to the Y261A mutation, the Y410A mutation and the Y493I mutation of SEQ ID No: 391; (g) with reference to the Y261A mutation, the Y410A mutation and the Y493T mutation of SEQ ID No: 391; (h) with reference to the Y261A mutation, the Y410A mutation and the V513I mutation of SEQ ID No: 391; (i) with reference to the Y7A mutation, the Y261A mutation, the L409S mutation and the Y410A mutation of SEQ ID No: 391; (j) with reference to the Y261A mutation, the L409A mutation, the Y410A mutation and the P411A mutation of SEQ ID No: 391; (k) with reference to the Y261A mutation, the L409S mutation, the Y410A mutation and the P411G mutation of SEQ ID No: 391; (l) with reference to the Y261A mutation, the L409S mutation and the Y410A mutation of SEQ ID No: 391; (m) with reference to the Y261A mutation, the L409S mutation and the Y410G mutation of SEQ ID No: 391; (n) with reference to the Y261A mutation, the L409A mutation, the Y410A mutation, the P411A mutation and the A485S mutation of SEQ ID No: 391; (o) with reference to the Y261A mutation, the L409S mutation, the Y410A mutation, the P411G mutation and the A485S mutation of SEQ ID No: 391; (p) with reference to the Y261A mutation, the L409S mutation, the Y410A mutation and the A485S mutation of SEQ ID No: 391; (q) with reference to the Y261A mutation, the L409S mutation, the Y410G mutation and the A485S mutation of SEQ ID No: 391; (r) with reference to the Y261A mutation, the L409A mutation, the Y410A mutation, the P411A mutation and the I521H mutation of SEQ ID No: 391; (s) with reference to the Y261A mutation, the L409S mutation, the Y410A mutation, the P411G mutation and the I521H mutation of SEQ ID No: 391; (t) with reference to the Y261A mutation, the L409S mutation, the Y410A mutation and the I521H mutation of SEQ ID No: 391; (u) with reference to the Y261A mutation, the L409S mutation, the Y410G mutation and the I521H mutation of SEQ ID No: 391; or (v) with reference to the Y261A mutation, the L409S mutation, the Y410A mutation, the A485S mutation, the K507L mutation and the I521H mutation of SEQ ID No:

391.

5. 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 the amino acid sequence comprises a D158A mutation or an E160A mutation with reference to SEQ ID NO:

414.

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

8. The synthetic polypeptide of claim 6 or 7, wherein the amino acid sequence further comprises an L431A mutation, a Y432A mutation, a P433I mutation, an A507S mutation, a K506Q mutation, a P433A mutation, an I543H mutation, an L431S mutation, a P433G mutation, a K529L mutation or a Y432G mutation with reference to SEQ ID No: 414, or any combination thereof.

9. The synthetic polypeptide of claim 8, wherein the amino acid sequence further comprises: (a) with reference to the L431A mutation, the Y432A mutation, the P433I mutation and the A507S mutation of SEQ ID No: 414; (b) with reference to the L431A mutation, the Y432A mutation, the P433I mutation and the K506Q mutation of SEQ ID No: 414; (c) with reference to the L431A mutation, the Y432A mutation, the P433I mutation, the K506Q mutation and the A507S mutation of SEQ ID No: 414; (d) with reference to the L431A mutation, the Y432A mutation and the P433A mutation of SEQ ID No: 414; (e) with reference to the L431A mutation, the Y432A mutation, the P433A mutation and the A507S mutation of SEQ ID No: 414; (f) with reference to the L431A mutation, the Y432A mutation, the P433A mutation and the I543H mutation of SEQ ID No: 414; (g) with reference to the L431S mutation, the Y432A mutation and the P433G mutation of SEQ ID No: 414; (h) with reference to the L431S mutation, the Y432A mutation, the P433G mutation and the A507S mutation of SEQ ID No: 414; (i) with reference to the L431S mutation, the Y432A mutation, the P433G mutation and the I543H mutation of SEQ ID No: 414; (j) with reference to the L431S mutation and the Y432A mutation of SEQ ID No: 414; (l) with reference to the L431S mutation, the Y432A mutation and the A507S mutation of SEQ ID No: 414; (m) with reference to the L431S mutation, the Y432A mutation, the A507S mutation, the K529L mutation and the I543H mutation of SEQ ID No: 414; (n) with reference to the L431S mutation, the Y432A mutation and the I543H mutation of SEQ ID No: 414; (o) with reference to the L431S mutation and the Y432G mutation of SEQ ID No: 414; (p) with reference to the L431S mutation, the Y432G mutation and the A507S mutation of SEQ ID No: 414; or (q) Referring to the L431S mutation, the Y432G mutation and the I543H mutation of SEQ ID No:

414.

10. 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 the amino acid sequence comprises a D141A mutation or an E143A mutation with reference to SEQ ID NO:

431.

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

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, a A488S mutation, a I524H mutation, a L411S mutation, a P413G mutation, a K510L mutation or a Y412G mutation with reference to SEQ ID No: 431, or any combination thereof.

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

431.

15. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. The synthetic polypeptide of claim 40, wherein the amino acid sequence further comprises an amino acid deletion from amino acid position 1496 to amino acid position S1033 with reference to SEQ ID NO: 462, or any portion thereof.

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

463.

44. The synthetic polypeptide of any one of claims 1-43, wherein the synthetic polypeptide is purified or isolated.

45. The synthetic polypeptide of any one of claims 1-44, wherein the synthetic polypeptide is a polymerase.

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

47. A method for nucleic acid analysis, the method comprising: A formulation is provided, comprising: (i) a synthetic polypeptide as described in any one of claims 1-47; (ii) primer nucleic acid sequence; and (iii) nucleotide units complementary to nucleotides of the primer nucleic acid, wherein the preparation is provided under conditions sufficient to form a bound complex comprising the synthetic polypeptide, the nucleotide units and the primer nucleic acid sequence.

48. The method of claim 47, wherein the method comprises a nucleic acid sequencing method.

49. The method of claim 47, wherein the method comprises a sequencing-by-synthesis method.

50. The method of claim 49, wherein the formulation further comprises one or more compositions, wherein a composition of the one or more compositions comprises: (a) core; and (b) at least two nucleotide arms, wherein a nucleotide arm of the at least two nucleotide arms comprises: (i) a core attachment moiety coupled to the core; (ii) a spacer coupled to the core attachment moiety; (iii) a linker coupled to the spacer; and (iv) said nucleotide unit coupled to said linker.

51. The method of claim 50, wherein the connector comprises: 11 atomic joints, 16 atomic connectors, 23 atomic joints, N3 connector, Connector 1, Connector 2, Connector 3, Connector 4, Connector 5, Connector 6, Connector 7, Connector 8, or Connector 9, wherein n is 1 to 6 and m is 0 to 10.

52. A kit comprising: One or more containers, the containers comprising: The synthetic polypeptide according to any one of claims 1 to 46; and a nucleotide unit, wherein the nucleotide unit is detectable; and Instructions for introducing the synthetic polypeptide and the nucleotide unit into the primer nucleic acid sequence comprising nucleotides complementary to the nucleotide unit under conditions sufficient to form a binding complex comprising the synthetic polypeptide, the nucleotide unit and the primer nucleic acid sequence.

53. The kit of claim 52, further comprising: One or more compositions, wherein the composition of the one or more compositions comprises: core; and at least two nucleotide arms, wherein a nucleotide arm of the at least two nucleotide arms comprises: a core attachment portion coupled to the core; a spacer coupled to the core attachment moiety; a linker coupled to the spacer; as well as The nucleotide unit is coupled to the linker.

54. The kit of claim 53, wherein the linker comprises: 11 atomic joints, 16 atomic connectors, 23 atomic joints, N3 connector, Connector 1, Connector 2, Connector 3, Connector 4, Connector 5, Connector 6, Connector 7, Connector 8, or Connector 9, wherein n is 1 to 6 and m is 0 to 10.

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

56. The system of claim 55, further comprising: One or more compositions, wherein the composition of the one or more compositions comprises: core; and at least two nucleotide arms, wherein a nucleotide arm of the at least two nucleotide arms comprises: a core attachment portion coupled to the core; a spacer coupled to the core attachment moiety; a linker coupled to the spacer; and said nucleotide unit coupled to said linker, Wherein the binding complex comprises the primer nucleic acid sequence, the synthetic polypeptide and the composition.

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

58. The system of claim 56 or 57, wherein the connector comprises: 11 atomic joints, 16 atomic connectors, 23 atomic joints, N3 connector, Connector 1, Connector 2, Connector 3, Connector 4, Connector 5, Connector 6, Connector 7, Connector 8, or Connector 9, wherein n is 1 to 6 and m is 0 to 10.

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

60. A system comprising: (i) a synthetic polypeptide according to any one of claims 1 to 46; (ii) Nucleotide.

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

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

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

64. The system of claim 60, wherein the nucleotides are unlabeled.

65. A system as described in claim 61, wherein the blocking group is attached to the 3' carbon of the sugar portion of the nucleotide, and wherein the blocking group is reacted with a compound to remove the blocking group from the nucleotide to generate the nucleotide comprising an OH moiety on the 3' carbon of the sugar portion of the nucleotide.

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

67. The system of claim 66, wherein a) reacting the alkyl, alkenyl, alkynyl or allyl group in the blocking group with tetrakis(triphenylphosphine)palladium(0) (Pd(P(C6H5)3)4) and piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ); b) reacting the aryl or benzyl group in the blocking group with H2 and palladium on carbon (Pd / C); c) reacting the amine, amide, ketone, isocyanate, phosphate, thiol or disulfide group in the blocking group with a phosphine or a thiol group including β-mercaptoethanol or dithiothreitol (DTT); d) reacting the carbonate group in the blocking group with potassium carbonate (K2CO3) in methanol (MeOH), triethylamine in pyridine or Zn in acetic acid (AcOH); and e) The urea or silyl group in the blocking group is reacted with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride or triethylamine trihydrofluoride.

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

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

70. The system of claim 69, wherein the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety.

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

72. A composition comprising: (a) core; and (b) at least two nucleotide arms, wherein the nucleotide arms in the at least two nucleic acid arms comprise: (i) a core attachment moiety coupled to the core; (ii) a spacer coupled to the core attachment moiety; (iii) a linker coupled to the spacer; and (iv) a nucleotide unit coupled to said linker, Wherein the connector comprises: 11 atomic joints, 16 atomic connectors, 23 atomic joints, N3 connector, Connector 1, Connector 2, Connector 3, Connector 4, Connector 5, Connector 6, Connector 7, Connector 8, or Connector 9, wherein n is 1 to 6 and m is 0 to 10.

73. The composition of claim 72, wherein the linker comprises 11 Atomic Joints.

74. The composition of claim 72, wherein the linker comprises 16-atom connector.

75. The composition of claim 72, wherein the linker comprises 23 atomic joints.

76. The composition of claim 72, wherein the linker comprises N3 connector.

77. The composition of claim 72, wherein the linker comprises Connector 1, wherein n is 1 to 6 and m is 0 to 10.

78. The composition of claim 72, wherein the linker comprises Connector 2, wherein n is 1 to 6 and m is 0 to 10.

79. The composition of claim 72, wherein the linker comprises Connector 3, wherein n is 1 to 6 and m is 0 to 10.

80. The composition of claim 72, wherein the linker comprises Connector 4, wherein n is 1 to 6 and m is 0 to 10.

81. The composition of claim 72, wherein the linker comprises Connector 5.

82. The composition of claim 72, wherein the linker comprises Connector 6.

83. The composition of claim 72, wherein the linker comprises Connector 7.

84. The composition of claim 72, wherein the linker comprises Connector 8.

85. The composition of claim 72, wherein the linker comprises Connector 9.

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

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

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

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

90. The composition of claim 72, further comprising a fluorescent label coupled to the core or the nucleotide arm.

91. The composition of claim 72, wherein the spacer comprises the following structure: wherein m is 20 to 500 and o is 1 to 10.

92. The composition of claim 72, wherein the nucleotide arm further comprises a reactive group coupled to the nucleotide unit, wherein the reactive group is configured to react with a reagent.

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

94. The composition of claim 93, wherein (a) reacting the alkyl, alkenyl, alkynyl or allyl group in the reactive group with tetrakis(triphenylphosphine)palladium(0) (Pd(P(C6H5)3)4) and piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ); (b) reacting the aryl or benzyl group in the reactive group with H2 and palladium on carbon (Pd / C); (c) the amine, amide, ketone, isocyanate, phosphate, thiol or disulfide group of the reactive group reacts with a phosphine or a thiol group including β-mercaptoethanol or dithiothreitol (DTT); (d) reacting the carbonate group in the reactive group with potassium carbonate (K2CO3) in methanol (MeOH), triethylamine in pyridine or Zn in acetic acid (AcOH); and (e) The urea or silyl group in the reactive group is reacted with tetrabutylammonium fluoride, pyridine hydrogen fluoride (HF-pyridine), ammonium fluoride or triethylamine trihydrogen fluoride.

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

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

97. The composition of claim 96, wherein the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety.

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

99. The composition of claim 72, wherein the nucleotide units of the at least two nucleotide arms comprise the same nucleobase type.

100. A composition as described in claim 72, wherein the nucleotide unit comprises a blocking group attached to the 3' carbon of the sugar portion of the nucleotide unit, and wherein the blocking group is reacted with a compound to remove the blocking group from the nucleotide unit to generate the nucleotide unit comprising an OH moiety on the 3' carbon of the sugar portion of the nucleotide unit.

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

102. The composition of claim 101, wherein a) reacting the alkyl, alkenyl, alkynyl or allyl group in the blocking group with tetrakis(triphenylphosphine)palladium(0) (Pd(P(C6H5)3)4) and piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ); b) reacting the aryl or benzyl group in the blocking group with H2 and palladium on carbon (Pd / C); c) the amine, amide, ketone, isocyanate, phosphate, Thiol or disulfide groups react with phosphines or thiol groups including β-mercaptoethanol or dithiothreitol (DTT); d) reacting the carbonate group in the blocking group with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine or Zn in acetic acid (AcOH); and e) The urea or silyl group in the blocking group is reacted with tetrabutylammonium fluoride, HF-pyridine, ammonium fluoride or triethylamine trihydrofluoride.

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

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

105. The composition of claim 104, wherein the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety.

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

107. A formulation comprising: at least two of the compositions of claim 72, wherein the at least two of the compositions include a first composition and a second composition, wherein the nucleotide units of the first composition comprise different nucleobases than the nucleotide units of the second composition.

108. A formulation comprising: at least two of the compositions of claim 72, wherein the at least two of the compositions comprise a first composition and a second composition, wherein: (a) the nucleotide units of the first composition comprise a first blocking group attached to the 3' carbon of a sugar moiety, wherein the first blocking group reacts with a 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 attached to the 3' carbon of the sugar moiety, wherein the second blocking group reacts with a 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, and wherein the nucleotide units of the first composition are different from the nucleotide units of the second composition.

109. A formulation comprising: at least two of the compositions of claim 72, wherein the at least two of the compositions include a first composition and a second composition, wherein the linker of the first composition is different from the linker of the second composition.

110. A formulation comprising: at least two of the compositions of claim 72, wherein the at least two of the compositions include a first composition and a second composition, wherein the linker of the first composition includes a first reactive group and the linker of the second composition includes a second reactive group, wherein the second reactive group is different from the first reactive group.

111. A formulation comprising: at least two of the compositions of claim 72, wherein the at least two of the compositions include a first composition and a second composition, wherein the first composition comprises a first fluorophore and the second composition comprises a second fluorophore, and wherein the wavelength of light emitted by the first fluorophore is different from the wavelength of light emitted by the second fluorophore.

112. A formulation comprising: at least two of the compositions of claim 72, wherein the at least two of the compositions include a first composition and a second composition, wherein the first composition comprises a fluorescent label and the second composition is unlabeled.

113. A kit for nucleic acid molecule processing, the kit comprising: (a) a composition as described in any one of claims 72 to 106 or a formulation as described in any one of claims 107 to 112; and (b) instructions for use of the composition in a nucleotide identification reaction.

114. The kit of claim 113, further comprising: A reagent that reacts with the reactive group in the linker of the composition.

115. The kit of claim 113, further comprising: A reagent that reacts with the reactive group at the 3' carbon of the sugar moiety in the nucleotide units of the composition.

116. The kit of claim 113, further comprising reagents for use in a nucleotide binding reaction.

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

118. The kit of claim 113, further comprising: (i) a solution comprising cations; (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).

119. A system comprising: (a) a composition as described in any one of claims 72 to 106; (b) two or more copies of a primer nucleic acid sequence, wherein the two or more copies of the primer nucleic acid sequence comprise nucleotides that are 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 the two or more primer nucleic acid sequences, the two or more polymerases, and the composition.

120. The system of claim 119, wherein the multivalent binding complex is formed under conditions where the nucleotide units of the composition are not incorporated into the two or more copies of the primer 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 primer molecule are affixed to the support under conditions sufficient to affix the multivalent binding complex to the support.

122. The system of claim 120, wherein a plurality of said multivalent binding complexes are immobilized on said support, wherein the density of said plurality of said multivalent binding complexes immobilized on said support is 10 2 –10 9 per square millimeter (mm 2 ).

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

124. A method for nucleic acid analysis, the method comprising: The composition described in any one of claims 72-106 is introduced into a primer nucleic acid sequence under conditions sufficient to form a binding complex, wherein the binding complex comprises (i) the nucleotide units of the composition and (ii) nucleotides in the primer nucleic acid sequence, wherein the nucleotides are complementary to the nucleotide units of the composition.

125. A method for nucleic acid analysis, the method comprising: The composition described in any one of claims 72-106 is introduced into two or more copies of a primer nucleic acid sequence under conditions sufficient to form a multivalent binding complex, wherein the multivalent binding complex comprises (i) two or more of the nucleotide units of the composition and (ii) two or more nucleotides in the two or more copies of the primer 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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and a nucleotide sequence; or (iii) both of (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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; 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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; 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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; 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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; 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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; or (iii) both of (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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; 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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; or (iii) both of (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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; or (iii) both of (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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; or (iii) both of (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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; 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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; 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: (i) provides a binding constant of at least 3 between the amino acid sequence and a primer nucleic acid sequence; (ii) provides a binding constant of at least 3 between the amino acid sequence and nucleotides; or (iii) both of (i) and (ii).

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