Methods for Polynucleotide Synthesis

Abstract

Disclosed herein are compositions and methods for de novo enzymatic synthesis of nucleic acid molecules. Specifically, among other things, the disclosed compositions and methods provide for the reduction of non-terminations in nucleic acid molecule products produced by de novo enzymatic synthesis. The disclosed compositions and methods also include the use of phosphatase enzymes to facilitate the reduction of non-terminations.

Description

[Technical field]

[0001] Field The present disclosure relates to technology comprising methods for nucleic acid synthesis.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 290,320, filed December 16, 2021, the entire disclosure of which is incorporated herein by reference for all purposes.

[0003] Reference to sequence listing This application contains a Sequence Listing (XML file named ABB-006WO_SL.xml, generated on Dec. 13, 2022, size 15,606 bytes), which was submitted electronically and is incorporated herein by reference. [Background technology]

[0004] background Enzymatic polynucleotide synthesis can be achieved by a series of repeated steps of a template-independent nucleic acid polymerase (e.g., terminal deoxynucleotidyl transferase) binding to a DNA substrate, incorporation of the nucleotide to be added (e.g., a protected nucleotide), followed by a deprotection step to allow future series of nucleotide incorporation. Enzymatic nucleic acid synthesis can result in undesired further insertion due to non-termination after the addition of a nucleotide. This can result in the addition of two or more nucleotides in a single extension step. Achieving the addition of exactly one nucleotide in each step is essential to generate accurate synthesis of oligonucleotides, and even a small insertion error rate results in a high percentage of incorrect sequences in longer oligonucleotides. Thus, there is a need to develop improved methods and / or techniques of enzymatic nucleic acid synthesis to prevent processes that result in the addition of more than one nucleotide per step. Summary of the Invention

[0005] overview The present disclosure provides, inter alia, methods of polynucleotide synthesis that can be used to improve the precision and accuracy of oligonucleotide synthesis, including, for example, improving the accuracy of single base addition.

[0006] In some aspects, provided herein are methods for reducing non-termination in an oligonucleotide synthesis reaction.

[0007] In some aspects, the disclosure provides a method comprising contacting a conjugate reagent with a phosphatase. For example, in some embodiments, the method comprises contacting a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase, the conjugates comprising nucleotides or modified nucleotides covalently attached to a polymerase via a linker, the phosphatase capable of removing one or more terminal 5' phosphates of unmasked nucleotides.

[0008] In some embodiments, the method of the present disclosure includes contacting a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, the conjugates comprising nucleotides covalently linked to a polymerase via a linker, and contacting a sample comprising a polynucleotide with the conjugate reagent, the polymerase of the conjugate catalyzing the covalent addition of the nucleotide of the conjugate to the 3' hydroxyl of the polynucleotide, the conjugate reagent being incubated or has been incubated with a phosphatase, the phosphatase being capable of removing the terminal 5' phosphate of an unblocked nucleotide. In some embodiments, the covalently added nucleotide is a blocked nucleotide. In some embodiments, the terminal 5' phosphate can be, for example, the alpha phosphate, beta phosphate, chi phosphate, delta phosphate, epsilon phosphate, phi phosphate, or gamma phosphate of an unblocked nucleotide.

[0009] In some embodiments, the methods of the disclosure include contacting the conjugate reagent comprising a polymerase-nucleotide conjugate composition with a phosphatase, wherein the conjugate comprises a nucleotide or modified nucleotide covalently linked to a polymerase via a linker, and the phosphatase is capable of removing the terminal 5' phosphate of an unmasked nucleotide.

[0010] In some embodiments, the disclosure provides a method of nucleic acid synthesis comprising: (i) providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, each of which comprises a nucleotide covalently attached to a polymerase via a linker; and (ii) contacting a sample comprising a polynucleotide with the conjugate reagent, wherein the polymerase of the conjugate catalyzes the covalent addition of a nucleotide of the polymerase-nucleotide conjugate to the 3' hydroxyl of the polynucleotide, and wherein the conjugate reagent is or has been incubated with a phosphatase, the phosphatase being capable of removing the terminal 5' phosphate of an unblocked nucleotide. In some embodiments, the covalently added nucleotide is a blocked nucleotide.

[0011] In some embodiments, the disclosure provides a method comprising: (i) providing a conjugate reagent comprising a polymerase-nucleotide conjugate; and (ii) contacting the conjugate reagent with a phosphatase, wherein the conjugate comprises a nucleotide covalently attached to a polymerase via a linker, and wherein the phosphatase is capable of removing a terminal 5' phosphate of an unmasked nucleotide.

[0012] In some embodiments, the linker is a cleavable linker.

[0013] In some embodiments, the present disclosure provides a method of nucleic acid synthesis comprising: providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, the conjugates comprising a nucleotide or modified nucleotide covalently bound to a polymerase via a linker; and contacting a sample comprising a polynucleotide with the conjugate reagent, the polymerase of the conjugate catalyzing the covalent addition of a blocked nucleotide of the conjugate to the 3' hydroxyl of the polynucleotide, and the conjugate reagent is or has been incubated with a phosphatase, the phosphatase capable of removing one or more terminal 5' phosphates from unblocked nucleotides. In some embodiments, the method provided herein comprises cleaving the linker after addition of the conjugate to remove the polymerase bound to the polynucleotide (e.g., via a linker that tethers the nucleotide to the polymerase). In some embodiments, the method provided herein comprises repeating each step of the method one or more times to synthesize a polynucleotide.

[0014] In some embodiments, the methods provided herein include incubating the conjugate reagent with the phosphatase, which hydrolyzes at least one terminal 5' phosphate of at least one unmasked nucleotide. In some embodiments, the methods provided herein include incubating the conjugate reagent with the phosphatase. In some embodiments, the incubation of the conjugate reagent with the phosphatase occurs before incubating the sample with the conjugate reagent. In some embodiments, the phosphatase is removed from the conjugate reagent before incubating the sample with the conjugate reagent. In some embodiments, the incubation of the conjugate reagent with the phosphatase occurs after contacting the sample with the conjugate reagent.

[0015] In some embodiments, the rate of non-termination in the polynucleotide synthesis is reduced as compared to the same synthesis using an otherwise identical conjugate reagent without contact with a phosphatase.

[0016] In some embodiments, the phosphatase does not remove the terminal 5' phosphate of the blocked nucleotide of the conjugate.

[0017] In some embodiments, the unshielded nucleotide is not bound to a polymerase. In some embodiments, the unshielded nucleotide is part of a conjugate, the polymerase is unfolded or improperly folded, and the nucleotide is bound to the polymerase such that the nucleotide is not shielded from the phosphatase, or multiple nucleotides are bound to the polymerase. In some embodiments, the phosphatase removes the terminal 5' phosphate of the unshielded nucleotide.

[0018] In some embodiments, the polymerase comprises a template-independent polymerase, hi some embodiments, the template-independent polymerase is terminal deoxynucleotidyl transferase (TdT), or a variant thereof.

[0019] In some embodiments, the phosphatase is an alkaline phosphatase or a non-alkaline phosphatase.

[0020] In some embodiments, the polymerase comprises a template-dependent polymerase. In some embodiments, the polymerase comprises a DNA polymerase. In some embodiments, the polymerase comprises an RNA polymerase.

[0021] The present disclosure provides a composition comprising a plurality of conjugates, each conjugate comprising a nucleotide or modified nucleotide bound to a polymerase, wherein the purity of nucleotides shielded by the bound polymerase in the composition is about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, or greater than about 99.9%, or about 99.9%, relative to the total amount of nucleotides (shielded and unshielded) in the composition.

[0022] In some embodiments, the plurality of conjugates comprises less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, less than about 0.05%, or less than about 0.01% unmasked or modified nucleotides.

[0023] In some embodiments, the compositions provided herein comprise at least one phosphatase. In some embodiments, the phosphatase is an alkaline phosphatase or a non-alkaline phosphatase.

[0024] In some embodiments, the multiple conjugates can extend the nucleic acid molecule by one nucleotide. In some embodiments, the multiple conjugates can extend the nucleic acid molecule by no more than one nucleotide.

[0025] In some embodiments, the nucleic acid molecule is single stranded. In some embodiments, the nucleic acid is double stranded.

[0026] In some aspects, the disclosure provides methods of synthesizing a polynucleotide comprising a predetermined sequence, comprising contacting a nucleic acid molecule with a composition provided herein.

[0027] In some embodiments, the method produces a polynucleotide product that comprises a predetermined sequence. In some embodiments, the polynucleotide product has less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, less than about 0.05%, or less than about 0.01% of polynucleotides that comprise a sequence that is not the predetermined sequence, relative to all polynucleotides in the product.

[0028] In some embodiments, the nucleic acid molecule is single-stranded.In some embodiments, the nucleic acid molecule is double-stranded.In some embodiments, the method provided herein comprises treating a composition comprising a polymerase-nucleotide conjugate, comprising contacting the composition with a phosphatase, wherein the phosphatase can remove the terminal 5' phosphate of unblocked nucleotide.

[0029] In some embodiments, the disclosed method is a method of reducing one or more non-terminating reactions in the synthesis of a nucleic acid molecule, wherein the synthesis is performed in an environment comprising one or more unmasked nucleotides, and the reducing comprises contacting a conjugate reagent comprising a polymerase-nucleotide conjugate with a phosphatase, wherein the polymerase-nucleotide conjugate comprises a nucleotide tethered to a polymerase by a linker, each nucleotide comprising one or more phosphates, each nucleotide comprising a terminal 5' phosphate, and wherein the phosphatase is capable of removing the terminal 5' phosphate from the one or more unmasked nucleotides.

[0030] In some embodiments, the number of non-terminations that occur in synthesis reactions performed in the presence of a phosphatase is reduced compared to synthesis performed under the same conditions but in the absence of the phosphatase.

[0031] In some embodiments, the nucleotides are modified nucleotides. [Brief description of the drawings]

[0032] The foregoing and other objects, features and advantages will become apparent from the following description of specific embodiments of the present disclosure as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views, and in which the drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the present disclosure.

[0033] [Figure 1A]

[0023] Figure 1A shows an exemplary template-independent polymerase with an exemplary unshielded nucleotide (e.g., a deoxynucleoside triphosphate or "dNTP") tethered in the wrong position.

[0034] [Figure 1B]1 is a diagram of an exemplary unshielded nucleotide that may be present in a polymerase-nucleotide conjugate reagent. FIG. 2 shows an exemplary unfolded template-independent polymerase with an exemplary tethered nucleotide (e.g., dNTP).

[0035] [Figure 1C] FIG. 1A is a diagram of an exemplary unmasked nucleotide that may be present in a polymerase-nucleotide conjugate reagent. FIG. 1B illustrates an exemplary "free" (or untethered) nucleotide (e.g., dNTP) present in an exemplary conjugate reagent. Such a free dNTP may be present in such a polymerase-nucleotide conjugate reagent due to, for example, cleavage (e.g., due to instability) of a linker between the nucleotide and the polymerase, or, for example, incomplete removal of the free nucleotide from the conjugate after conjugate synthesis. Each nucleotide in FIG. 1A, FIG. 1B, and FIG. 1C has a 5' phosphate group accessible for catalytic removal via a phosphatase.

[0036] [Figure 1D] 1 shows an exemplary polymerase-nucleotide conjugate comprising an exemplary shielded nucleotide (e.g., dNTP), in which the exemplary nucleotide is tethered to the catalytic site of a folded polymerase and sterically hindered by the tethered polymerase from phosphatase cleavage at its 5' phosphate.

[0037] [Figure 2A-1] Figure 1 shows the results of an exemplary single-base addition reaction to an exemplary single-stranded DNA substrate using A, C, T, or G polymerase conjugates in the presence (+Phos) or absence (-Phos) of phosphatase. The resulting synthetic oligonucleotides were analyzed by capillary electrophoresis. The x-axis shows the approximate nucleotide length of the oligonucleotide, and the y-axis shows the relative fluorescence at 517 nm. The reactions were terminated at the indicated time points. [Figure 2A-2]See description of Figure 2A-1.

[0038] [Figure 2B] Shown are expanded views of the results from Figure 2A at 21 minutes 41 seconds in the presence or absence of phosphatase. The specific nucleotides are indicated in each set of panels. The arrow indicates the +2 addition.

[0039] [Figure 3A-1] Figure 1 shows a graphical representation of the results of capillary electrophoresis analysis of single-base addition reactions on single-stranded DNA substrates using T-polymerase conjugates in the presence of the following exemplary phosphatase variants: B. taurus (Quick CIP, NEB), P. borealis (Shrimp alkaline phosphatase, NEB), Antarctic bacterium TAB5 (Antarctic phosphatase, NEB), or E. coli (Takara Bio) phosphatase. Synthesis reactions were performed at room temperature (24°C). Control synthesis reactions were performed without phosphatase. Reactions were terminated at the indicated time points. The x-axis shows the relative electrophoretic migration of oligonucleotides (by approximate nucleotide length) and the y-axis shows the relative fluorescence at 517 nm. [Figure 3A-2] See description of Figure 3A-1.

[0040] [Figure 3B] 1 shows a graphical representation of the results of capillary electrophoresis analysis of an exemplary single-base addition reaction to a single-stranded DNA substrate using a T-polymerase conjugate in the presence of the following exemplary phosphatase variants: bovine (Quick CIP, NEB), Arctic shrimp (shrimp alkaline phosphatase, NEB), Antarctic bacteria TAB5 (Antarctic phosphatase, NEB), or E. coli (Takara Bio) phosphatase. Synthesis reactions were performed at 37° C. (with and without phosphatase) and terminated after 30 minutes. The arrow indicates the expected size of the +2 addition.

[0041] [Figure 4]1 shows a graphical representation of the results of an exemplary conjugate-based polynucleotide synthesis of an exemplary 50-mer polynucleotide performed in the presence or absence of phosphatase using a SeqStudio Genetic Analyzer, with the resulting synthesized polynucleotides distinguished by size along the x-axis. Peaks corresponding to the starter oligo and the correct 50-mer synthesis product are labeled. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description Details of various embodiments of the disclosure are set forth in the description that follows. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

[0043] definition As used herein, the term "nucleotide" refers to a molecule that includes a nucleoside and one or more phosphate groups. A "nucleoside" refers to a molecule that includes a nucleobase (e.g., adenine, thymine, cytosine, guanine, or uracil) and a five-carbon sugar (e.g., ribose or 2'-deoxyribose). Exemplary nucleotides can be or include, but are not limited to, nucleoside monophosphates, nucleoside diphosphates, nucleoside triphosphates, nucleoside tetraphosphates, nucleoside pentaphosphates, or nucleoside hexaphosphates. As provided herein, TdT and TdT variants can incorporate any nucleoside polyphosphate, including nucleotide analogs that, in some embodiments, include modifications to the nucleobase.

[0044] As used herein, a "non-terminating" or "insertion" occurs when two or more nucleotides are added during a single step of cyclic nucleotide extension. This can occur when an unmasked nucleotide with an uncleaved 5' phosphate is added to an oligonucleotide.

[0045] As used herein, the term "phosphatase" refers to an enzyme that can remove the 5' phosphate of a nucleotide, particularly a nucleotide that is not shielded as part of an improperly formed conjugate or tethered to a polymerase. When not referring to a specific phosphatase enzyme as used herein, phosphatase is also meant to include all phosphatase enzymes, engineered enzymes with phosphatase activity, or functional fragments thereof that can remove one or more phosphate group(s) from a nucleotide. Phosphatase can also refer to any biomolecule (e.g., a polypeptide or ribozyme) that can remove one or more phosphate group(s) from a nucleotide, including engineered enzymes with phosphatase activity, or functional fragments thereof.

[0046] As used herein, the term "protected nucleotide" or "shielded nucleotide" refers to a nucleotide that is sterically hindered by a tethered polymerase (or other entity or moiety, such as a blocking group) from a phosphatase that can remove its 5' phosphate. In some embodiments, such a nucleotide is likely to inhibit subsequent nucleotide addition after it has been added to an oligonucleotide and prior to removal of the tethered polymerase.

[0047] As used herein, the term "unprotected nucleotide" or "unshielded nucleotide" refers to a nucleotide that is not sterically hindered by a polymerase (or other entity or moiety, such as a blocking group) tethered from a phosphatase that can remove its 5' phosphate. In some embodiments, the unshielded nucleotide may be tethered to a polymerase, such as a misfolded polymerase, or tethered at an incorrect position. The unshielded nucleotide may be untethered (or free) from a polymerase. An unshielded nucleotide that is not exposed to a phosphatase is more likely to be misadded to a polynucleotide as an insertion after a shielded nucleotide has been properly added.

[0048] Polymerase-Nucleotide Conjugates and Polynucleotide Synthesis Among other things, the present disclosure provides polymerase-nucleotide conjugates. As will be understood by those skilled in the art, there are various challenges associated with high-precision and accurate polynucleotide synthesis. For example, among other things, polymerases can catalyze the covalent addition of nucleotides in error, which can result in the addition of more than one nucleotide per step when using polymerase-nucleotide conjugates in controlled stepwise nucleic acid synthesis (e.g., insertion or non-termination). The techniques provided herein, including combining such conjugates with phosphatases (e.g., providing polymerase-nucleotide conjugates in the presence of phosphatases), overcome such challenges. These techniques help achieve more accurate and precise stepwise addition, which reduces errors compared to the aforementioned synthesis approaches (e.g., those performed in the absence of phosphatases).

[0049] In some embodiments, the conjugate is provided in the presence of a phosphatase. In some embodiments, the disclosure provides a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, wherein the polymerase and the nucleotide are linked via a linker. In some embodiments, the linker is cleavable.

[0050] In some embodiments, the conjugation reagent is in the presence of a phosphatase.

[0051] In some embodiments, the polymerase-nucleotide conjugate is combined with a template (eg, a start oligo or initial oligonucleotide) in the presence of a phosphatase.

[0052] A typical process for stepwise synthesis of a polynucleotide involves the stepwise addition of individual nucleotides to a starter oligo (i.e., an initial oligonucleotide) via cyclic steps. For example, in some embodiments, the steps include the addition of a polymerase-nucleotide conjugate to the oligonucleotide, the covalent addition of a nucleotide to the 3' end of the oligonucleotide catalyzed by a polymerase, and the cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired extended polynucleotide is synthesized, such that the extended polynucleotide has one or more nucleotides longer than the polynucleotide before the steps are repeated one or more times.

[0053] Among other things, provided herein are methods of nucleic acid synthesis. In some embodiments, the methods of nucleic acid synthesis include contacting (e.g., incubating) a conjugate reagent comprising a polymerase-nucleotide conjugate (e.g., a plurality of polymerase-nucleotide conjugates) in the presence of one or more phosphatases. In some embodiments, the plurality of nucleotides are the same nucleotide (e.g., A, G, T, or C, etc.). In some embodiments, the nucleotides are different nucleotides (e.g., A, G, T, and / or C, etc.). In some such embodiments, synthesis performed in the presence of a phosphatase is improved in one or more respects (e.g., more precise, more efficient, more accurate) compared to the same synthesis in the absence of the phosphatase.

[0054] In some embodiments, synthesis performed in the presence of a phosphatase prevents the addition of unmasked nucleotides to the nucleic acid. The methods provided herein include contacting (e.g., incubating) a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase, and when the polymerase-nucleotide conjugates are used for nucleic acid synthesis (e.g., non-termination leading to additional nucleotide insertion), the rate of processes resulting in the addition of more than one nucleotide per step is reduced compared to synthesis without a phosphatase or without treatment of the conjugate reagent with a phosphatase.

[0055] non-terminal Achieving precise single nucleotide addition in each step of stepwise nucleic acid synthesis is essential to generate accurate synthesis of longer oligonucleotides. Performing these additions with accuracy and precision remains a challenge in the industry. For example, stepwise nucleic acid synthesis using polymerase-nucleotide conjugates is susceptible to insertion and / or non-termination, which may result in the addition of two or more nucleotides to a nucleic acid in a single step of cyclic nucleotide elongation.

[0056] Unshielded nucleotides are not sterically hindered or are only partially sterically hindered by tethered polymerase from phosphatase cleavage at their 5' phosphate.During oligonucleotide synthesis, polymerase may catalyze the covalent addition of unshielded nucleotides in error, which may result in the addition of more than one nucleotide per step when using polymerase-nucleotide conjugates in nucleic acid synthesis (e.g., insertion or non-termination).The technology provided herein overcomes this problem and helps achieve accurate and precise stepwise addition with reduced errors compared to the aforementioned synthesis approach.

[0057] In some embodiments, non-termination may occur when an unmasked nucleotide with a non-cleaved 5' phosphate is added to an oligonucleotide. As provided herein, in some embodiments, the phosphatase hydrolyzes the 5' phosphate (e.g., terminal 5' phosphate) of a nucleotide (e.g., nucleotide triphosphate, etc.). In some embodiments, the terminal 5' phosphate can be the alpha phosphate, beta phosphate, chi phosphate, delta phosphate, epsilon phosphate, phi phosphate, or gamma phosphate of the nucleotide. As provided herein, in some embodiments, the phosphatase hydrolyzes the 5' phosphate (e.g., terminal 5' phosphate) of a nucleotide or a free nucleotide of a polymerase-nucleotide conjugate. In some embodiments, the phosphatase disclosed herein hydrolyzes the 5' phosphate of a nucleotide of a plurality of polymerase-nucleotide conjugates, for example, one or more 5' terminal phosphate(s) of a nucleotide, and prevents the hydrolyzed nucleotide from being added to a nucleic acid during oligonucleotide synthesis. In some embodiments, the phosphatase hydrolyzes the 5' phosphate of the unshielded nucleotide of the polymerase-nucleotide conjugate. In some such embodiments, the hydrolysis of the 5' phosphate prevents the unshielded nucleotide from being added to a nucleic acid during oligonucleotide synthesis. In some embodiments, the phosphatase disclosed herein hydrolyzes one or more 5' phosphate(s) of the unshielded nucleotide in a plurality of polymerase-nucleotide conjugates, preventing the unshielded nucleotide from being added to a nucleic acid during oligonucleotide synthesis. In some embodiments, the phosphatase disclosed herein hydrolyzes one or more 5' phosphate(s) of one or more free nucleotides in a composition comprising one or more polymerase-nucleotide conjugates, preventing the free nucleotide from being added to a nucleic acid during oligonucleotide synthesis.In some embodiments, the phosphatase hydrolyzes the 5' phosphate of one or more free nucleotides present in a composition comprising one or more polymerase-nucleotide conjugates. In some such embodiments, hydrolysis of the 5' phosphate prevents one or more free nucleotides from being added to a nucleic acid during oligonucleotide synthesis.

[0058] Unshielded nucleotides The presence of an unmasked nucleotide in a conjugate reagent can result in a non-termination (i.e., insertion) in oligonucleotide synthesis. In some embodiments, an unmasked nucleotide is less likely to inhibit subsequent nucleotide addition after it is added to an oligonucleotide during nucleic acid synthesis.

[0059] In some embodiments, a fraction of the nucleotides in the plurality of polymerase-nucleotide conjugates are not shielded by the polymerase. In some embodiments, the polymerase-nucleotide conjugate comprises an unshielded nucleotide. In some embodiments, the polymerase molecule in the polymerase-nucleotide conjugate does not sterically hinder the phosphatase's access to the 5' phosphate of the tethered nucleotide. In some embodiments, the tethered nucleotide is an unshielded nucleotide. In some embodiments, the unshielded nucleotide in the polymerase-nucleotide conjugate is not sterically hindered by the tethered polymerase from a phosphatase that can remove its 5' phosphate. In some embodiments, removing the 5' phosphate (e.g., terminal 5' phosphate) of the nucleotide in the polymerase-nucleotide conjugate prevents the nucleotide from being added to the nucleic acid during nucleic acid synthesis. In some embodiments, the phosphatase hydrolyzes the 5' phosphate (e.g., terminal 5' phosphate) of the unshielded nucleotide in the polymerase-nucleotide conjugate. In some such embodiments, more than one 5' terminal phosphate is removed, e.g., the 5' terminal phosphate is removed consecutively, i.e., from a first nucleotide, then a second nucleotide, etc. Thus, in some such embodiments, one or more 5' terminal phosphates may be removed, but at a given nucleotide, a single 5' terminal phosphate is present and removed, at which point a different phosphate becomes the 5' terminal phosphate of a nucleotide having at least one 5' terminal phosphate.

[0060] In some embodiments, the unshielded nucleotide is part of an improperly formed conjugate. In some embodiments, the improperly formed conjugate includes a misfolded polymerase, a polymerase with a nucleotide attached at the wrong position, and / or a polymerase with multiple nucleotides attached. In some embodiments, the nucleotide is free or untethered from the polymerase due to instability or incomplete purification. In some embodiments, the free or untethered nucleotide is an unshielded nucleotide.

[0061] Examples of unshielded nucleotides The lack of shielding of a nucleotide in a polymerase-nucleotide conjugate can result from several processes during the preparation of the polymerase-nucleotide conjugate or during the addition reaction itself. A nucleotide that is not bound to a polymerase in a composition containing a polymerase-nucleotide conjugate is considered an unshielded nucleotide. Non-limiting examples of processes that can result in a polymerase-nucleotide conjugate containing an unshielded nucleotide include spontaneous cleavage of the linker between the nucleotide and the polymerase (see, for example, the linkers in Figures 1A-1D), an unfolded polymerase (see, for example, Figure 1B), a polymerase with a nucleotide bound in the wrong position (see, for example, Figure 1A), a polymerase containing multiple bound nucleotides (i.e., on a single polymerase), or an unconstrained free nucleotide (see, for example, Figure 1C). In some embodiments, spontaneous cleavage of the linker between the nucleotide and the polymerase can occur due to instability, resulting in a free nucleotide in the conjugate reagent.

[0062] Shielded Nucleotides In some embodiments, a fraction of the nucleotides in the plurality of polymerase-nucleotide conjugates are shielded by the polymerase. A non-limiting example of a shielded nucleotide includes a nucleotide that is tethered to the catalytic site of a correctly folded polymerase (see, for example, the exemplary schematic diagram in FIG. 1D). In some embodiments, the polymerase-nucleotide conjugate comprises a shielded nucleotide. In some embodiments, the polymerase molecule in the polymerase-nucleotide conjugate sterically prevents the phosphatase from accessing the 5' phosphate (e.g., the 5' terminal phosphate) of the tethered nucleotide. In some embodiments, the 5' phosphate (e.g., the 5' terminal phosphate) can be, for example, an α-phosphate, β-phosphate, χ-phosphate, δ-phosphate, ε-phosphate, φ-phosphate, or γ-phosphate. In some embodiments, the tethered nucleotide is a shielded nucleotide. In some embodiments, the shielded nucleotide in the polymerase-nucleotide conjugate is sterically hindered by the polymerase tethered from a phosphatase capable of removing its 5' phosphate. In some embodiments, the phosphatase is unable to hydrolyze the 5' phosphate (e.g., the 5' terminal phosphate) of the shielded nucleotide in the polymerase-nucleotide conjugate.

[0063] Phosphatase Phosphatases typically use water to cleave phosphate monoesters into phosphate ions and alcohols. Phosphatase enzymes catalyze the hydrolysis of their substrates.

[0064] The 5' phosphate of the nucleotide in the polymerase-nucleotide conjugate is necessary for the addition of the nucleotide to the oligonucleotide by the polymerase. In some embodiments, removal of the 5' phosphate of the nucleotide in the polymerase-nucleotide conjugate prevents the nucleotide from being added to the nucleic acid during oligonucleotide synthesis.

[0065] Disclosed herein is a method comprising adding a phosphatase to a conjugate reagent to hydrolyze the 5' phosphate group of the nucleotide in the polymerase-nucleotide conjugate. In some embodiments, the phosphatase removes a phosphate moiety from the unshielded nucleotide in the polymerase-nucleotide conjugate. In some embodiments, the method comprises adding a phosphatase capable of hydrolyzing the 5' phosphate group of the unshielded nucleotide to the polymerase-nucleotide conjugate.

[0066] Types of phosphatases Any suitable phosphatase, engineered enzyme with phosphatase activity, or functional fragment thereof for the methods described herein is contemplated by the present disclosure. In some embodiments, the phosphatase is a nucleotidase. Enzymes with phosphatase activity are included in the enzyme class EC 3.1.3.-., hydrolases acting on ester bonds, e.g., phosphate monoester hydrolases. However, enzymes with suitable phosphatase activity (such as apyrase) may be found in other enzyme classes. In some embodiments, the phosphatase may optionally be capable of hydrolyzing inorganic phosphate substrates, e.g., pyrophosphate.

[0067] In some embodiments, the phosphatase is immobilized to a solid support. In some embodiments, the phosphatase is a fusion protein. In some embodiments, the phosphatase comprises a detectable label. In some embodiments, the phosphatase is a recombinant polypeptide. In some embodiments, the phosphatase is a wild-type phosphatase. In some embodiments, the wild-type phosphatase is isolated from an organism in which it is naturally expressed.

[0068] Alkaline phosphatase (ALP, ALKP, ALPase, Alk Phos), or basic phosphatase, is a plasma membrane-bound glycoprotein that catalyzes the hydrolysis of phosphate monoesters and is optimally active in alkaline pH environments. Alkaline phosphatase is an 86 kilodalton homodimeric protein enzyme. Each monomer contains five cysteine ​​residues, two zinc atoms, and one magnesium atom that is essential for its catalytic function.

[0069] Non-limiting examples of alkaline phosphatases include bovine (Quick calf intestinal alkaline phosphatase, or CIP, NEB), Pandalus borealis (shrimp alkaline phosphatase, NEB), Antarctic bacteria TAB5 (Antarctic phosphatase, NEB), and E. coli (Takara Bio) phosphatases. Additional non-limiting examples of alkaline phosphatases include placental alkaline phosphatase (PLAP) and human intestinal alkaline phosphatase.

[0070] The non-alkaline phosphatase can be an acid phosphatase. A non-limiting example of a non-alkaline phosphatase is a tartrate-resistant acid phosphatase.

[0071] Exemplary amino acid sequences encoding phosphatases for use in the methods described herein are shown in Table 1, but are not limited to these.

[0072] Table 1. Exemplary alkaline phosphatase sequences TIFF2024546987000002.tif158165TIFF2024546987000003.tif210165TIFF2024546987000004.tif71165

[0073] Polynucleotide Synthesis In some embodiments, a method of synthesizing a polynucleotide includes contacting (e.g., incubating) a polymerase-nucleotide conjugate with a nucleic acid, where the polymerase of the polymerase-nucleotide conjugate extends the nucleic acid using the tethered nucleotide.

[0074] As described above, a method of nucleic acid synthesis is disclosed herein, which comprises contacting (e.g., incubating) a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase. In some embodiments, contacting the conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase is performed before or during a cyclic extension reaction. In some embodiments, the presence of a phosphatase in a stepwise method of nucleic acid synthesis reduces non-termination and processes that lead to the addition of more than one nucleotide per step. In some embodiments, the use of a conjugate reagent that has been treated (e.g., incubated) with a phosphatase reduces non-termination when the conjugate reagent is used in a stepwise method of nucleic acid synthesis, compared to an untreated conjugate reagent.

[0075] Timing of phosphatase addition or removal The polymerase-nucleotide conjugate may be stored with the phosphatase and remain in the system (even during the DNA extension reaction), or the phosphatase may be removed for a specific incubation period before the conjugate is added to the DNA and at the start of the DNA addition reaction.

[0076] In some embodiments, the phosphatase is incubated with a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates. In some embodiments, the incubation of the conjugate reagent with the phosphatase is performed before contacting the sample with the conjugate reagent. In some embodiments, the phosphatase is removed from the conjugate reagent before contacting the sample with the conjugate reagent. In some embodiments, the incubation of the conjugate reagent with the phosphatase is performed after contacting the sample with the conjugate reagent.

[0077] Phosphatase concentration The concentration of phosphatase contacted or incubated with a polymerase-nucleotide conjugate (e.g., a conjugate reagent) can be expressed, for example, in terms of conjugate concentration, units of phosphatase activity, molar concentration, or the stoichiometric ratio of fold increase in phosphatase to conjugate per mg / mL.

[0078] Any suitable stoichiometric ratio of conjugate to phosphatase can be used in the methods described herein. In some embodiments, the stoichiometric ratio of conjugate to phosphatase is about 1:1 to about 1:500. In some embodiments, the stoichiometric ratio of conjugate to phosphatase is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90, about 1:95, about 1:100, about 1:105, about 1:110, about 1:115, about 1:1 20, about 1:125, about 1:130, about 1:135, about 1:140, about 1:145, about 1:150, about 1:155, about 1:160, about 1:165, about 1:170, about 1:175, about 1:180, about 1:185, about 1:190, about 1:195, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, about 1:425, about 1:450, about 1:475, or about 500.

[0079] Any suitable stoichiometric ratio of phosphatase to conjugate can be used in the methods described herein. In some embodiments, the stoichiometric ratio of phosphatase to conjugate is about 1:1 to about 1:500. In some embodiments, the stoichiometric ratio of conjugate to phosphatase is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90, about 1:95, about 1:100, about 1:105, about 1:110, about 1:115, about 1:1 20, about 1:125, about 1:130, about 1:135, about 1:140, about 1:145, about 1:150, about 1:155, about 1:160, about 1:165, about 1:170, about 1:175, about 1:180, about 1:185, about 1:190, about 1:195, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, about 1:425, about 1:450, about 1:475, or about 500.

[0080] Any suitable phosphatase concentration can be used in the methods described herein. In some embodiments, the phosphatase concentration is about 0.01 mg / mL to about 10.5 mg / mL. In some embodiments, the phosphatase concentration is about 0.1 mg / mL, about 0.15 mg / mL, about 0.25 mg / mL, about 0.5 mg / mL, about 0.75 mg / mL, about 1 mg / mL, about 1.25 mg / mL, about 1.5 mg / mL, about 1.75 mg / mL, about 2 mg / mL, about 2.25 mg / mL, about 2.5 mg / mL, about 2.75 mg / mL, about 3 mg / mL, about 3.25 mg / mL, about 3.5 mg / mL, about 3.75 mg / mL, about 4 mg / mL, about 4.25 mg / mL, about 4.5 mg / mL, about 4.75 mg / mL, about 4. ... g / mL, about 5, about 5.25 mg / mL, about 5.5 mg / mL, about 5.75 mg / mL, about 6 mg / mL, about 6.25 mg / mL, about 6.5 mg / mL, about 6.75 mg / mL, about 7, about 7.25 mg / mL, about 7.5 mg / mL, about 7.75 mg / mL, about 8 mg / mL, about 8.25 mg / mL, about 8.5 mg / mL, about 8.75 mg / mL, about 9, about 9.25 mg / mL, or about 9.5 mg / mL, about 9.75 mg / mL, about 10 mg / mL, about 10.25 mg or about 10.5 mg / mL.

[0081] Any suitable fold increase in phosphatase relative to the conjugate may be used in the methods described herein. In some embodiments, the fold increase in phosphatase relative to the conjugate is from about 2-fold to about 500-fold. In some embodiments, the fold increase in phosphatase relative to the conjugate is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, or about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 105-fold, about 110-fold, about 115-fold, about 120-fold, about 130-fold, about 140-fold, about 150-fold, about 160-fold, about 170-fold, about 180-fold, about 190-fold, about 210-fold, about 220-fold, about 230-fold, about 240-fold, about 250-fold, about 260-fold, about 270-fold, about 280-fold, about 290-fold, about 300-fold, about 310-fold, about 320-fold, about 330-fold, about 340-fold, about 350-fold, about 360-fold, about 370-fold, about 380-fold, about 390-fold, about 395-fold, about 395-fold, about 390-fold, about 25-fold, about 130-fold, about 135-fold, about 140-fold, about 145-fold, or about 150-fold, about 155-fold, about 160-fold, about 165-fold, about 170-fold, about 175-fold, about 180-fold, about 185-fold, about 190-fold, about 195-fold, about 200-fold, about 225-fold, about 250-fold, about 275-fold, about 300-fold, about 325-fold, or about 350-fold, about 375-fold, about 400-fold, about 425-fold, about 450-fold, about 475-fold, or about 500-fold.

[0082] Any suitable fold increase of the conjugate to the phosphatase may be used in the methods described herein. In some embodiments, the fold increase of the conjugate to the phosphatase is from about 2-fold to about 500-fold. In some embodiments, the fold increase of the conjugate to the phosphatase is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, or about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 105-fold, about 110-fold, about 115-fold, about 120-fold, about 130-fold, about 140-fold, about 150-fold, about 160-fold, about 170-fold, about 180-fold, about 190-fold, about 200-fold, about 210-fold, about 220-fold, about 230-fold, about 240-fold, about 250-fold, about 260-fold, about 270-fold, about 280-fold, about 290-fold, about 300-fold, about 310-fold, about 320-fold, about 330-fold, about 340-fold, about 350-fold, about 360-fold, about 370-fold, about 380-fold, about 390-fold, about 400-fold, about 400-fold, about 25-fold, about 130-fold, about 135-fold, about 140-fold, about 145-fold, or about 150-fold, about 155-fold, about 160-fold, about 165-fold, about 170-fold, about 175-fold, about 180-fold, about 185-fold, about 190-fold, about 195-fold, about 200-fold, about 225-fold, about 250-fold, about 275-fold, about 300-fold, about 325-fold, or about 350-fold, about 375-fold, about 400-fold, about 425-fold, about 450-fold, about 475-fold, or about 500-fold.

[0083] Any suitable concentration of phosphatase can be used in the methods described herein. In some embodiments, the concentration of the phosphatase is about 0.5 μM to about 500 μM. In some embodiments, the concentration of the phosphatase is about 0.5 μM, about 1 μM, about 2 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, about 100 μM, about 105 μM, about 110 μM, about 115 μM, about 120 μM, about 125 μM. , about 130 μM, about 135 μM, about 140 μM, about 145 μM, about 150 μM, about 155 μM, about 160 μM, about 165 μM, about 170 μM, about 180 μM, about 185 μM, about 190 μM, about 195 μM, about 200 μM, about 225 μM, about 250 μM, about 275 μM, about 300 μM, about 325 μM, about 350 μM, about 375 μM, about 400 μM, about 425 μM, about 450 μM, about 475 μM, or about 500 μM.

[0084] Percentage of non-termination In some embodiments, the presence of a phosphatase in a stepwise method of nucleic acid synthesis reduces non-termination and processes that lead to the addition of more than one nucleotide per step. A polynucleotide or nucleic acid produced by the methods described herein is said to contain an insertion if a non-termination event occurs. In some embodiments, nucleic acid synthesis in the presence of a phosphatase reduces the rate of non-termination by about 50% to about 100% compared to nucleic acid synthesis in the absence of a phosphatase. In some embodiments, the rate of non-termination is reduced by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% compared to nucleic acid synthesis in the absence of a phosphatase. In some embodiments, the total amount of nucleic acid synthesis products with insertions produced in the presence of phosphatase is less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, less than about 0.05%, or less than about 0.01%. In some embodiments, the nucleic acid synthesis products produced in the presence of phosphatase are free of nucleic acid synthesis products with insertions.

[0085] final product In some embodiments, disclosed herein are methods of synthesizing a polynucleotide comprising a predetermined sequence, comprising contacting a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase. In some embodiments, the contacting comprises incubating the conjugate reagent with the phosphatase. In some embodiments, the method produces a heterogeneous population of polynucleotide products comprising the predetermined sequence. The heterogeneous population of polynucleotide products comprising the predetermined sequence may be referred to as a "final product."

[0086] In some embodiments, contacting a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates with a phosphatase prevents nucleotide insertions (i.e., non-terminations) such that these insertions are not present in the predetermined sequence. In some embodiments, the final product comprises nucleic acid, a proportion of which comprises the target sequence and a proportion of which does not comprise the target sequence. In some embodiments, the final product comprises less than about 99%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, less than about 0.05%, or less than about 0.01% compared to polynucleotides comprising sequences that are not the predetermined (i.e., not the "target" sequence). In some embodiments, the final product is substantially free of polynucleotides comprising sequences that are not the predetermined ("target") sequence.

[0087] Final product analysis Any suitable method known in the art can be used to analyze the end product. The end product can be evaluated by analyzing the nucleic acid synthesis products at any time after the initiation of the extension reaction (e.g., over a reaction time course). Analysis can be performed, for example, by capillary electrophoresis (CE), as previously demonstrated (Smith and Nelson. Curr Protoc Nucleic Acid Chem. Chapter 10: Unit 10.9.2003; Durney et al. Anal Bioanal Chem. 407: 6923-6938. 2015). CE can separate and report the abundance of polynucleotide products with single nucleotide resolution. The relative abundance of each nucleic acid product generated by the methods of nucleic acid synthesis provided herein can be analyzed by CE. By comparing the abundance of the starting material (i.e., the initial polynucleotide or oligonucleotide into which the nucleotide has been incorporated) and the extension product, it is possible to determine the extent to which the extension reaction is completed. The change over time of the starting material and extension species indicates the turnover rate, as described herein. This approach to determine conversion rates has been previously demonstrated (Palluk et al. Nat Biotech. 36(7):645-650. 2018). Alternatively, analysis of nucleic acid synthesis products can be performed using reverse-phase high performance liquid chromatography (RP-HPLC) as previously described (Jensen and Davis. Biochemistry. 57(12):1821-1832. 2018).

[0088] CE and RP-HPLC can also be used to determine the purity of each species in the nucleic acid synthesis product by determining the area under the curve of the peaks in the electropherograms and chromatograms of CE and RP-HPLC, respectively. The abundance of each polynucleotide product in the multiple nucleotide products can be determined using any suitable software package suitable for fitting a curve to the electropherograms and chromatograms and calculating the area under the curve (AUC).

[0089] Any suitable polynucleotide sequencing method can be used for analysis (e.g., analysis of sequences of polynucleotide products, e.g., intermediate products, e.g., final products, etc.). For example, the sequencing method can be long-read sequencing, next-generation sequencing, short-read sequencing, shotgun sequencing, Sanger sequencing, high-throughput sequencing, sequencing by synthesis, sequencing by ligation, sequencing by hybridization, and / or sequencing by mass spectrometry. Sequencing can be suitable for identifying a predetermined sequence in the final product. Sequencing can be suitable for determining the proportion of sequences in the final product that are not predefined sequences.

[0090] Any method known in the art for analysis of the size of the final product can be used to determine the purity of the final product, for example, gel electrophoresis and / or mass spectrometry can be used to determine the purity of the final product.

[0091] Conjugates Disclosed herein are compositions comprising a conjugate comprising a nucleotide bound to a polymerase, wherein the purity of the linked polymerase-shielded nucleotide of the composition is greater than about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 99.5%, or about 99.9% shielded nucleotide compared to the total nucleotides in the composition. In some embodiments, the purity of the linked polymerase-shielded nucleotide is substantially free of impurities.

[0092] Provided herein is a method of nucleic acid synthesis using a conjugate, the conjugate comprising a polymerase and a nucleotide, the polymerase and the nucleotide being linked via a linker. In some embodiments, the linker comprises a selectively cleavable bond. In some embodiments, the nucleotide is a modified nucleotide. The linker can be attached to the base, sugar, or phosphate of the nucleotide (e.g., a wild-type nucleotide, e.g., a modified nucleotide comprising one or more modifications relative to the wild-type nucleotide). In some embodiments, the nucleotide is a nucleotide analog. The polymerase portion of the conjugate can extend the nucleic acid by adding its linked nucleotide (i.e., the polymerase can catalyze the attachment of the nucleotide that it is linked to the 3' end of the nucleic acid) and remains attached to the extended nucleic acid via the linker until the linker is cleaved.

[0093] In some embodiments, the polymerase and nucleotide are covalently linked, and the distance between the linking atom of the nucleotide and the polymerase to which it is bound can be, for example, in the range of about 4-100 Å, about 15-40 Å, or about 20-30 Å, or a distance appropriate to the position on the TdT variant to which the nucleotide or nucleotide analog is tethered.

[0094] When a conjugate comprising a polymerase and a nucleotide is incubated with a nucleic acid, the nucleic acid is preferentially extended using the tethered nucleotide (as opposed to using a nucleotide from another conjugate molecule).

[0095] Linker nucleotide bond In some embodiments, the linker of the present disclosure is attached to the "5" position of a pyrimidine or the "7" position of a 7-deazapurine. In other embodiments, the linker may be attached to the exocyclic amine of a nucleobase, for example, by N-alkylating the exocyclic amine of a cytosine with a nitrobenzyl moiety, as discussed below. The linker may be attached to any suitable atom of the nucleotide to form a conjugate, such as the phosphate, sugar, or base of the nucleotide, as would be apparent to one of skill in the art. In some embodiments, the linker is attached to the α-phosphate, sugar, or base of the nucleotide, such that the polymerase remains attached to the nucleotide after addition to the 3' end of the oligonucleotide. In some embodiments, the linker is attached to the β, χ, δ, ε, φ, or γ phosphate of the nucleotide. In some embodiments, the linker is attached to the terminal phosphate of the nucleotide.

[0096] Certain polymerases have high tolerance to modifications of certain portions of nucleotides, e.g., modifications of the 5-position of pyrimidines and the 7-position of purines are well tolerated by some polymerases (see He and Seela, Nucleic Acids Research 30.24(2002):5485-5496.; or Hottin et al., Chemistry. 2017 Feb 10;23(9):2109-2118). In some embodiments, linkers are attached at these positions.

[0097] In some embodiments, a polymerase-nucleotide conjugate is prepared by first synthesizing an intermediate compound (herein referred to as a "linker-nucleotide") that includes a linker and a nucleotide, and then the intermediate compound is attached to a polymerase. As a non-limiting example, in some embodiments, nucleosides having substitutions compared to natural nucleosides, such as pyrimidines having 5-hydroxymethyl or 5-propargylamino substituents, or 7-deazapurines (having 7-hydroxymethyl or 7-propargylamino substituents), may be useful starting materials for preparing linker-nucleotides. An exemplary set of nucleosides having 5- and 7-hydroxymethyl substituents that may be useful for preparing linker nucleotides is shown below. TIFF2024546987000005.tif38165

[0098] An exemplary set of nucleosides having 5- and 7-deaza-7-propargylamino substituents that may be useful in preparing linker nucleotides is shown below. TIFF2024546987000006.tif43165

[0099] These nucleosides are also commercially available as deoxyribonucleoside triphosphates.

[0100] In some embodiments, the method of preparation (e.g., involving intermediate compounds) where the conjugate comprises a linker nucleotide. Any suitable nucleotide may be used. In some embodiments, the linker nucleotide comprises a nucleotide polyphosphate or a modified nucleotide polyphosphate. In some embodiments, the linker nucleotide comprises a nucleotide triphosphate or a modified nucleotide triphosphate. In some embodiments, the linker nucleotide comprises a nucleotide tetraphosphate or a modified nucleotide tetraphosphate. In some embodiments, the linker nucleotide comprises a nucleotide pentaphosphate or a modified nucleotide pentaphosphate. In some embodiments, the linker nucleotide comprises a nucleotide hexaphosphate or a modified nucleotide hexaphosphate. In some embodiments, the linker nucleotide comprises a modified nucleobase. In some embodiments, the linker nucleotide comprises a modified nucleobase. In some embodiments, the modified nucleobase comprises an O- or N-linked modification. In some embodiments, the O- or N-linked modification is removable after incorporation of the nucleotide portion of the linker nucleotide into the polynucleotide. In some embodiments, the O- or N-linked modification is removable by a photolysis process. In some embodiments, the photolysis process comprises exposure to UV light, the UV light comprising wavelengths of 365 nm and / or 405 nm. In some embodiments, the O- or N-linked modifications are removable by a chemical process. In some embodiments, the chemical process is selected from a beta-elimination reaction, a Pd-catalyzed deallylation, and a reduction reaction. In some embodiments, the O- or N-linked modifications are removable by an enzymatic process. In some embodiments, the enzymatic process comprises removal by an alkyltransferase or a methyltransferase.

[0101] In some embodiments, O- or N-linked modification reduces or eliminates Watson-Crick base pairing in the polynucleotide that comprises the modified nucleobase.In some embodiments, O- or N-linked modification reduces or eliminates secondary structure in the polynucleotide that comprises the modified nucleobase.In some embodiments of the method, after removing O- or N-linked modification, the modified nucleobase comprises a natural nucleobase.In some embodiments, the natural nucleobase is guanine, cytosine, adenine, thymine, or uracil.

[0102] Linker polymerase binding The conjugates provided herein include a polymerase tethered to a nucleotide via a linker.

[0103] Any suitable linker for tethering a nucleotide to a polymerase is contemplated for use in the methods described herein. In some embodiments, the linker specifically binds to a cysteine ​​residue of a polymerase using sulfhydryl-specific conjugation chemistry. Exemplary sulfhydryl-specific attachment chemistries include, but are not limited to, ortho-pyridyl disulfide (OPSS), maleimide functional groups, 3-arylpropiolonitrile functional groups, allenamide functional groups, haloacetyl functional groups such as iodoacetyl or bromoacetyl, alkyl halides or perfluoroaryl groups that can react favorably with sulfhydryls surrounded by specific amino acid sequences (Zhang, Chi, et al. Nature chemistry 8, (2015) 120-128.). Other conjugation chemistries for specific labeling of cysteine ​​residues will be apparent to those skilled in the art or are described in the relevant literature and texts (e.g., Kim, Younggyu, et al, Bioconjugate chemistry 19.3 (2008): 786-791.).

[0104] In some embodiments, the linker is attached to the lysine residue via an amine-reactive functional group (e.g., NHS ester, sulfo-NHS ester, tetra- or pentafluorophenyl ester, isothiocyanate, sulfonyl chloride, etc.). In some embodiments, the linker is attached to the polymerase via attachment to a genetically inserted unnatural amino acid, e.g., p-propargyloxyphenylalanine or p-azidophenylalanine, which can undergo azide-alkyne Huisgen cycloaddition, although there are many suitable unnatural amino acids suitable for site-specific labeling and can be found in the literature (e.g., as described in Lang and Chin., Chemical reviews 114.9(2014):4764-4806.).

[0105] In some embodiments, the linker may be specifically attached to the polymerase N-terminus. In some embodiments, the polymerase is mutated to have an N-terminal serine or threonine residue that can be specifically oxidized to generate an N-terminal aldehyde for subsequent coupling to, for example, a hydrazide. In some embodiments, the polymerase is mutated to have an N-terminal cysteine ​​residue that can be specifically labeled with an aldehyde to form a thiazolidine. In some embodiments, the N-terminal cysteine ​​residue can be labeled with a peptide linker via native chemical ligation.

[0106] In some embodiments, peptide tag sequences can be inserted into a polymerase that can be enzymatically specifically labeled with synthetic groups as shown in the literature, for example, using biotin ligase, transglutaminase, lipoic acid ligase, bacterial sortase, and phosphopantetheinyl transferase (e.g., as described in references 74-78 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).

[0107] In some embodiments, the linker is attached to a label domain fused to the polymerase. For example, linkers with corresponding reactive moieties may be used to covalently label SNAP tags, CLIP tags, Halo tags, and acyl carrier protein domains (e.g., as described in references 79-82 of Stephanopoulos & Francis Nat. Chem, Biol. 7, (2011) 876-884).

[0108] In some embodiments, the linker binds to an aldehyde that is specifically generated within the polymerase, as described by Carrico et al. (Nat. Chem. Biol. 3, (2007) 321-322). For example, an amino acid sequence recognized by the enzyme formylglycine generating enzyme (FGE) may be inserted into the polymerase and then exposed to FGE, which specifically converts a cysteine ​​residue in the recognition sequence to formylglycine (i.e., generates an aldehyde). This aldehyde can then be specifically labeled, for example, with a hydrazide or aminooxy moiety of the linker.

[0109] In some embodiments, the linker can be attached to the polymerase via non-covalent binding to a portion of the linker fused to the polymerase. Examples of such attachment strategies include fusing the polymerase to streptavidin, which can bind to the biotin portion of the linker, or fusing the polymerase to anti-digoxigenin, which can bind to the digoxigenin portion of the linker. In some embodiments, site-specific labeling can result in attachment of the linker to the polymerase that can be easily reversed (e.g., an ortho-pyridyl disulfide (OPSS) group that forms a disulfide bond with cysteine ​​that can be cleaved using a reducing agent, e.g., TCEP), while other attachment chemistries generate permanent attachments.

[0110] In some embodiments, the polymerase is mutated to ensure specific attachment of the tethered nucleotide to a specific position of the polymerase, as would be apparent to one skilled in the art. For example, in sulfhydryl-specific conjugation chemistry, such as maleimide or ortho-pyridyl disulfide, accessible cysteine ​​residues in the wild-type polymerase can be mutated to non-cysteine ​​residues to prevent labeling at those positions. In this "reactive cysteine-free" background, cysteine ​​residues can be introduced by mutation at the desired attachment position. These mutations preferentially do not interfere with the activity of the polymerase.

[0111] Other strategies for site-specific attachment of synthetic groups to proteins will be apparent to the skilled artisan and are reviewed in the literature (e.g., Stephanopulos & Francis Nat. Chem. Biol 7 (2011) 876-884).

[0112] shielding As described herein, in some embodiments, a polymerase (e.g., a template-independent polymerase) remains bound to a nucleic acid via tethering to the added nucleotide until it is exposed to some stimulus that causes the cleavage of the bond to the added nucleotide. In this situation, further extension by the polymerase-nucleotide conjugate is prevented (i.e., the nucleotide is "shielded") if: 1) the bound polymerase molecule prevents other conjugates from accessing the 3'OH of the extended DNA molecule, and 2) other nucleotides in the system are prevented from accessing the catalytic site of the polymerase that remains bound to the 3' end of the extended nucleic acid. (The degree of shielding can be described as the degree to which both of these interactions are disrupted.) To allow for subsequent extension, the linker tethering the incorporated nucleotide to the polymerase can be cleaved, releasing the polymerase from the nucleic acid and thus re-exposing its 3'OH group for subsequent extension.

[0113] In some embodiments that employ shielding, the linker can be attached to any atom in the nucleobase, sugar, or α-phosphate, as would be apparent to one of skill in the art.

[0114] Methods for nucleic acid synthesis provided herein that use a shielding effect to achieve termination include an extension step in which the nucleic acid is preferentially exposed to conjugates in the absence of free (i.e., untethered) nucleoside triphosphates, since the termination mechanism of shielding may not prevent their incorporation into the nucleic acid.

[0115] In some embodiments, the termination of further extension may be "complete", meaning that after the nucleic acid molecule is extended by the conjugate, no further extension may occur during the reaction. In other embodiments, the termination of further extension may be "inadequate", meaning that further extension may occur during the reaction, but at a substantially reduced rate compared to the initial extension, for example, 100 times slower, or 1000 times slower, or 10,000 times slower, or more. A conjugate that achieves an incomplete termination can still be used to extend nucleic acids primarily by single nucleotides (e.g., in methods for nucleic acid synthesis and sequencing) when the reaction is stopped after an appropriate amount of time. In some embodiments, the reagent containing the conjugate may further contain a polymerase that does not contain a tethered nucleotide, but these polymerases should not significantly affect the reaction since there are no free dNTPs in the mixture.

[0116] Conjugate-based reagents that use shielding effect to achieve termination preferentially contain only polymerase-nucleotide conjugates in which all polymerases remain folded in active conformation.In some cases, when the polymerase portion of the conjugate is unfolded, its tethered nucleotides may be more accessible to the polymerase portion of other conjugate molecules.In these cases, unshielded nucleotides may be more easily incorporated by other conjugate molecules, avoiding the termination mechanism.

[0117] Polymerase-nucleotide conjugates that use shielding effect to achieve termination are preferably labeled with only a single nucleotide moiety. Polymerase-nucleotide conjugates labeled with multiple nucleotides that can access catalytic sites can, in some cases, incorporate multiple nucleotides into the same nucleic acid. Thus, additional tethered nucleotides can result in additional, undesired nucleotide incorporation into nucleic acid during reaction. Furthermore, since only one tethered nucleotide can occupy the (buried) catalytic site of that polymerase at a time, other tethered nucleotide(s) can increase the accessibility of the polymerase moiety of other conjugate molecules, as discussed below.

[0118] Polymerase-nucleotide conjugates that use a shielding effect to achieve termination preferentially include the shortest possible linker that still allows the nucleotide to frequently access the catalytic site of its tethered polymerase molecule in a productive conformation to allow rapid incorporation of the nucleotide into the nucleic acid. Such conjugates may also preferentially use a linker attachment position to the polymerase that is as close to the catalytic site as possible, allowing the use of shorter linkers. The length of the linker determines the maximum distance from the attachment point that the tethered nucleotide or tethered nucleic acid can reach. Smaller distances may lead to reduced accessibility of the tethering moiety to other polymerase-nucleotide molecules, as described below. In some embodiments, the linker is about 24 and 28 Å long. Short linkers, for example linkers 8-15 Å long, may increase shielding, while longer linkers, for example linkers longer than 50 Å, 70 Å, or 100 Å, may reduce shielding. The shielding effect can be influenced by a combination of factors including, but not limited to, the structure of the polymerase, the length of the linker, the structure of the linker, the attachment position of the linker to the polymerase, the binding affinity of the nucleotide to the catalytic site of the polymerase, the binding affinity of the nucleic acid to the polymerase, the preferred conformation of the polymerase, and the preferred conformation of the linker.

[0119] One contribution to shielding may be steric effects that block the 3'OH of the nucleic acid extended by the conjugate from reaching the catalytic site of the polymerase portion of another conjugate. Steric effects may also prevent the tethered nucleotide from reaching the catalytic site of another polymerase-nucleotide conjugate molecule due to the collision between the conjugates that would occur during such an approach. These steric effects may result in complete termination if they completely block the productive interaction between the tethered nucleotide (or extended nucleic acid) of one conjugate molecule and another conjugate molecule, or they may result in incomplete termination if they only prevent such intermolecular interactions.

[0120] Another contribution to shielding comes from the binding affinity of the tethered nucleotide to the catalytic site of the polymerase. The tethered nucleotide of the conjugate has a high effective concentration relative to the catalytic site of the tethered polymerase, so it can remain bound to the site for a long time. When a nucleotide is bound to the catalytic site of the tethered polymerase molecule, it is not available for incorporation by other polymerase molecules. Thus, tethering reduces the effective concentration of nucleotides available for intermolecular incorporation (i.e., incorporation catalyzed by a polymerase molecule in which the nucleotide is not tethered). This shielding effect can enhance termination by reducing the rate at which the nucleotide portion of one conjugate molecule is used by the polymerase portion of another conjugate molecule to extend the nucleic acid.

[0121] Another contribution to shielding comes from the binding affinity of the 3' region of the nucleic acid molecule to the catalytic site of the polymerase molecule. After extension by the conjugate, the nucleic acid is tethered to the conjugate via its 3' terminal nucleotide, and has a high effective concentration to the catalytic site of the linked polymerase, so the nucleic acid can remain bound to the site for a long time. When the nucleic acid is bound to the catalytic site of its tethered polymerase molecule, it is not available for extension by other conjugate molecules. This effect can enhance termination by reducing the rate at which the nucleic acid extended by the first conjugate is further extended by other conjugate molecules.

[0122] In some embodiments, the polymerase-nucleotide conjugate contains an additional moiety that sterically prevents the tethered nucleotide (or the tethered nucleic acid after extension) from approaching the catalytic site of another conjugate molecule. Such moieties include those and other bulky molecules, such as polypeptide or protein domains that can be inserted into the loop of a polymerase, as well as inserted unnatural amino acids or polymers that can be site-specifically ligated to specific polypeptide tags.

[0123] Polymerase As described above, when a conjugate comprising a polymerase (e.g., a template-independent polymerase) and a nucleotide is incubated with a nucleic acid or polynucleotide, it preferentially extends (i.e., lengthens) the nucleic acid or polynucleotide by incorporating the tethered or modified nucleotide into the nucleic acid or polynucleotide (as opposed to using a nucleotide or modified nucleotide of another conjugate molecule). In some embodiments, the polymerase in the polymerase-nucleotide conjugate is folded in an active conformation. In other embodiments, the polymerase in the polymerase-nucleotide conjugate is not folded.

[0124] Any polymerase that can extend a polynucleotide, incorporate a nucleotide into a polynucleotide, or incorporate a nucleotide analog into a polynucleotide is contemplated for use in the methods described herein. In some embodiments, the polynucleotide is single-stranded. In some embodiments, the polynucleotide is double-stranded. In some embodiments, the polynucleotide is immobilized on a solid support.

[0125] For DNA synthesis applications, specifically, template-independent polymerases may be used, such as terminal deoxynucleotidyl transferase (TdT) or DNA nucleotidylexotransferase, which terms may be used interchangeably to refer to enzymes having activity as described for EC class 2.7.7.31.

[0126] In some embodiments, the methods of the disclosure use a conjugate comprising a template-independent polymerase. In some embodiments, the conjugate comprises a Pol-X family polymerase. In some embodiments, the conjugate comprises a polymerase terminal deoxynucleotidyl transferase (TdT), or a mutant form thereof (e.g., a non-wild type TdT, e.g., a modified TdT). In some embodiments, the template-independent polymerase is TdT or a variant thereof (i.e., a modified TdT). In some embodiments, the TdT or a variant thereof comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:1. In some embodiments of the method, the TdT comprises a sequence identical to SEQ ID NO:1 or a portion thereof. For example, in some embodiments, the TdT comprises a sequence identical to a portion of a particular TdT (e.g., the sequence of SEQ ID NO: 1, etc.). For example, in some embodiments, a given TdT may be truncated relative to the length of a particular TdT, such as that set forth in SEQ ID NO: 1. In some embodiments, the TdT may be a circular permutation of SEQ ID NO: 1). In some embodiments, the TdT variant comprises one or more amino acid substitutions, insertions, deletions, and / or circular permutations thereof relative to a reference TdT (e.g., wild-type TdT, modified TdT, etc.).

[0127] In some embodiments of the method, the polymerase is a fusion protein. In some embodiments of the method, the fusion protein comprises maltose binding protein (MBP).

[0128] In some embodiments of the method, the TdT or variant thereof may be operably linked to a linker moiety comprising a covalent or non-covalent bond; an amino acid tag (e.g., a poly-amino acid tag, a poly-His tag, a 6His tag (SEQ ID NO: 8)); a compound (e.g., polyethylene glycol); a protein-protein binding pair (e.g., biotin-avidin); an affinity coupling; a capture probe; or any combination thereof. The linker moiety may be separate from the TdT variant or may be part of the TdT variant.

[0129] >Terminal deoxynucleotidyl transferase (TdT)

[0130] MGGRDIVDGSEFSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSALAFMRASSVLKSLPFPITSMKDTEGIPSLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATDEEQQLLHKVTDFWKQQGLLLYADILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMSPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA (SEQ ID NO: 1)

[0131] Illustrative examples of polymerases capable of extending single-stranded nucleic acids include, but are not limited to, polymerase Theta (Kent et al., eLife 5(2016):el3740), polymerase mu (Fuarez et al., Nucleic acids rease34.16(2006):4572-4582.; or McElhinny et al., Molecular cell 19.3(2005):357-366.), or polymerases in which template-independent activity is induced, e.g., polymerases induced by insertion of elements of template-independent polymerases (Juarez et al., Nucleic acids research 34.16(2006):4572-4582). In other DNA synthesis applications, the polymerase may be a template-dependent polymerase, i.e., a DNA-directed DNA polymerase (this term is used interchangeably to refer to an enzyme having activity 2.7.7.7 using the IUBMB nomenclature).

[0132] In some embodiments, such as RNA synthesis applications, tethered ribonucleotides may be used. In some such embodiments, an RNA-specific nucleotidyl transferase, such as Escherichia coli poly(A) polymerase (IUBMB EC 2.7.7.19) or poly(U) polymerase, among others, may be used. The RNA nucleotidyl transferase may contain modifications, e.g., single point mutations, that affect substrate specificity for a particular rNTP (Lunde et al., Nucleic acids research 40.19(2012):9815-9824.). In some embodiments, very short tethering between the RNA nucleotidyl transferase and the ribonucleotide may be used to induce a high effective concentration of nucleotides, thereby forcing the incorporation of rNTPs that may not be natural substrates for the nucleotidyl transferase.

[0133] Linker In some embodiments, in conjugates of the present disclosure that include a linker, the linker comprises an atom that connects the nucleotide to the polymerase. The linker can attach the base, sugar, or alpha-phosphate of the nucleotide or modified nucleotide to the polymerase. In some embodiments, the polymerase and the nucleotide are covalently linked, and the distance between the attachment atom of the nucleotide and the polymerase to which it is attached can be, for example, in the range of about 4-100 Å, about 15-40 Å, or about 20-30 Å, or a distance appropriate to the location on the polymerase to which the nucleotide is tethered. Any suitable linker for tethering the nucleotide or modified nucleotide to the polymerase is contemplated in the methods described herein. In some embodiments, the linker comprises a polyether or polyethylene glycol (PEG). In some embodiments, the linker comprises one or more peptide bonds. In some embodiments, the linker comprises one or more sarcosines. In some embodiments, the linker comprises one or more glycines. In some embodiments, the linker comprises one or more prolines. In some embodiments, the linker comprises a carbamate. In some embodiments, the linker is attached to the nucleotide at an atom of the nucleobase that is not involved in base pairing. In such embodiments, the linker is considered to be at least an atom that connects the polymerase to any atom of the monocyclic or polycyclic ring system (e.g., pyrimidine or purine or 7-deazapurine or 8-aza-7-deazapurine) attached to the Γ position of the sugar. In some embodiments, the linker is attached to the nucleotide at an atom of the nucleobase that is involved in base pairing. In some embodiments, the linker is linked to the sugar or α-phosphate of the nucleotide. In some embodiments, the linker is of sufficient length to allow the nucleotide to access the active site of the polymerase to which it is tethered. As described in more detail herein, the polymerase of the conjugate can catalyze the addition of the nucleotide to which it is linked to the 3' end of the nucleic acid.

[0134] Linker cleavage As described herein, linkers can be attached to various positions on nucleotides (e.g., of the conjugates of the present disclosure), and various cleavage strategies can be used.It should be understood that the cleavage strategy is determined by the type of linker that connects nucleotides or modified nucleotides and polymerase.Any suitable method for cleaving linkers is contemplated in the methods described herein.

[0135] In some embodiments, the linker is cleaved and a nucleotide containing a chemical group is formed from the retained portion of the linker (i.e., scar) after cleavage of the linker. Exemplary non-limiting chemical groups (i.e., scars) after cleavage of the linker are shown below. In some embodiments, the chemical group is removed by a chemical, photolytic, or enzymatic process.

[0136] In some embodiments, the linker may be cleaved by exposure to any suitable reducing agent, such as dithiothreitol (DTT), β-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP). For example, a linker containing a 4-(disulfanyl)butanoyloxymethyl group attached to the 5-position of a pyrimidine or the 7-position of a 7-deazapurine may be cleaved by a reducing agent (e.g., DTT) to generate a 4-mercaptobutanoyloxymethyl scar on the nucleobase. This scar can undergo intramolecular thiolactonization to remove the 2-oxothiolane, leaving a smaller hydroxymethyl scar on the nucleobase. An example of such a linker attached to the 5-position of cytosine is shown below, but this strategy is applicable to any suitable nucleobase. TIFF2024546987000007.tif48165

[0137] In other embodiments, the linker can be cleaved by exposure to light. For example, a linker containing a (2-nitrobenzyl)oxymethyl group can be cleaved with 365 nm light, leaving a hydroxymethyl scar, for example, as shown for cytosine below, although the strategy is applicable to any suitable nucleobase: TIFF2024546987000008.tif59165

[0138] In other embodiments, the linker may contain a 3-((2-nitrobenzyl)oxy)carbonyl)aminopropynyl group that can be cleaved with 365 nm light to release a nucleobase bearing a propargylamino scar. This strategy is applicable to any suitable nucleobase. TIFF2024546987000009.tif43165

[0139] In other embodiments, the linker may comprise an acyloxymethyl group that can be cleaved with a suitable esterase to release a nucleobase bearing a hydroxymethyl scar, for example as shown for cytosine below, although the strategy is applicable to any suitable nucleobase. TIFF2024546987000010.tif53165

[0140] In such embodiments, the linker may include additional atoms (included in R' above) adjacent to the ester that increase the activity of the esterase towards the ester bond.

[0141] In other embodiments, the linker may comprise an N-acyl-aminopropynyl group that can be cleaved with a peptidase to release a nucleobase bearing a propargylamino scar, for example as shown for 5-propargylaminocytosine below, although the strategy is applicable to any suitable nucleobase. TIFF2024546987000011.tif48165

[0142] In such embodiments, the linker may contain additional atoms (included in R' above) adjacent to the amide that increase the activity of the peptidase towards the amide bond.

[0143] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments given the teachings provided herein. The scope of the present disclosure is not intended to be limited to the disclosure herein, but rather is as set forth in the appended claims.

[0144] In the claims, articles such as "a," "an," and "the" can mean one or more, unless the contrary is indicated or otherwise clear from the context. A claim or description containing "or" between one or more elements of a group is considered to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless the contrary is indicated or otherwise clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which two or more, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0145] It should also be noted that the term "comprising" is intended to be open, allowing but not requiring the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0146] Where ranges are given, endpoints are included. Moreover, unless otherwise indicated or otherwise clear from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to be capable of assuming, in different embodiments of the present disclosure, any particular value or subrange within the stated range, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0147] The term "about" as used herein refers to a normal error range for each value, which is readily known to those skilled in the art. The term "about" a value or parameter reference herein includes (and describes) the implementation of the value or parameter itself. For example, a description that references "about X" includes a description of "X". In some embodiments, "about" refers to a value up to + / -10% of the recited value, for example, + / -1%, + / -2%, + / -3%, + / -4%, + / -5%, + / -6%, ±8%, ±9%, or ±10%.

[0148] All cited sources, e.g., references, publications, databases, database entries, and techniques cited herein, are incorporated herein by reference, even if not explicitly stated in the citation. In the event of a conflict between the statements in the cited sources and this application, the statements in this application take precedence.

[0149] The section and table headings are not intended to be limiting. EXAMPLES

[0150] Below are examples of specific embodiments for implementing the techniques provided by the present disclosure. These examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0151] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of the art. Such techniques are fully explained in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); ALLehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

[0152] Example 1: Preparation of polymerase-nucleotide conjugates 1. Generation of polymerase (TdT) An inducible plasmid was generated expressing mouse TdT with a single cysteine ​​at position 182 (Palluk et al. for complete protocol, see Nature Biotechnology, 2018). For further details on polymerase-nucleotide conjugate preparation, see also U.S. Patent Publication No. 2019 / 0112627, "Nucleic Acid Synthesis and Sequencing Using Tethered Nucleoside Triphosphates," which is incorporated herein by reference in its entirety.

[0153] 2. Protein Expression and Purification of Polymerase TdT expression was performed using BL21(DE3) Gold cells (Agilent) in TB medium containing antibiotics for the resistance markers of the plasmids. 50 mL of overnight culture was used to inoculate 400 mL expression cultures at 1 / 20 volume. Cells were grown at 37°C and shaken at 200 rpm until they reached an OD of 0.6. IPTG was added to a final concentration of 0.5 mM and expression was performed for 16-20 h at 16°C. Cells were harvested by centrifugation at 8000G for 10 min and resuspended in 20 mL of buffer A (20 mM Tris-HCl, 0.5 M NaCl, pH 8) + 5 mM imidazole. Cell lysis was performed using sonication followed by centrifugation at 30,000G for 20 min. The supernatant was applied to a gravity column containing 1 mL of Ni-NTA agarose (Qiagen). The column was washed with 20 volumes of Buffer A + 40 mM imidazole and bound protein was eluted using 4 mL of Buffer A + 500 mM imidazole. Protein was concentrated to approximately 0.15 mL on a Vivaspin20 column (MWCO 10 kDa, Sartorius) and then dialyzed overnight against 200 mL of TdT stock buffer (100 mM NaCl, 200 mM K2HPO4, pH 6.5) using a Pur-A-Lyzer™ Dialysis Kit Mini 12000 tubing (Sigma).

[0154] The Ni-purified sample was applied to a HiTrap Q HP anion column. Proteins were eluted with a linear gradient from 100% Q buffer A (100 mM NaCl, 20 mM K2HPO4, pH 6.5) to 100% Q buffer B (1 M NaCl, 20 mM K2HPO4, pH 6.5). Fractions that contained TdT were identified using SDS-PAGE analysis, and these samples were pooled and concentrated.

[0155] 3. Attachment of the tethered nucleotide to the polymerase To prepare TdT-nucleotide conjugates, a cleavable linker-nucleotide with a moiety capable of site-specific conjugation to cysteine ​​(i.e., maleimide) was first synthesized. Exemplary linker nucleotides are described in U.S. Patent Publication No. 2019 / 0112627, "Nucleic Acid Synthesis and Sequencing Using Tethered Nucleoside Triphosphates." Equimolar TdT and linker nucleotides were then incubated overnight at 4° C. in 500 mM NaCl, 20 mM K2HPO4, pH 6.5. The TdT conjugates were separated from unreacted linker nucleotides using an S200 size exclusion column (Cytiva) pre-equilibrated in 20 mM Tris acetate, 50 mM potassium acetate (pH 7.9). The resulting conjugates include conjugates that include at least one TdT nucleotide bound (or tethered) to a polymerase.

[0156] Example 2: Phosphatases prevent unwanted insertions during conjugate-based polynucleotide synthesis without affecting synthesis kinetics As described herein, polymerase-nucleotide conjugates can be used to incorporate a single nucleotide of a given conjugate into the free 3' end of an oligonucleotide, while the polymerase can remain attached after incorporation, including preventing subsequent nucleotide addition in a controlled manner. However, insertion of two or more nucleotides can occur when using a conjugate solution for polynucleotide synthesis, which negatively affects the accuracy of conjugate-based polynucleotide synthesis. To test whether phosphatase can improve synthesis accuracy by preventing insertion during conjugate-based polynucleotide synthesis, single nucleotide incorporation reactions were performed into the 3' end of free oligonucleotides using A, T, C, or G nucleotide polymerase conjugates with or without phosphatase, as follows:

[0157] A solution of 1 μM TdT conjugated to the A, T, C, or G linker nucleotide prepared in Example 1 was incubated in Tris or HEPES buffer at pH 8 with a divalent metal (e.g., magnesium or cobalt), 50 mM salt (e.g., potassium acetate or NaCl), 50 nM of one of two starter DNA oligos (5′-6-FAM-T35-3′ (SEQ ID NO: 9) for A, and 5′-6-FAM-T41GCGGCGCGTTTCGCGCCGC-3′ (SEQ ID NO: 10) for T, C, and G were used) in the presence or absence of 2 μM calf alkaline intestinal phosphatase purchased from NEB. The phosphatase was pre-buffer exchanged into a solution containing a pH 8 buffer (e.g., Tris or HEPES buffer) and salt (e.g., 50 mM NaCl or potassium acetate). The reactions were incubated at 24° C. until they were stopped by the addition of 40 mM EDTA at 6.6 s, 11.6 s, 19.2 s, 65 s, 3 min 21 s, 9 min 52 s, and 21 min 41 s.

[0158] After completion of the reaction, the linker connecting the nucleotides and TdT in the conjugate was cleaved using a reagent such as a cleavage enzyme or a reducing agent to remove TdT from the oligonucleotide. The oligonucleotide was then analyzed by capillary electrophoresis to differentiate the reaction products by length. Specifically, the resulting oligonucleotide addition reaction products were sized by detecting the fluorescence of 6-FAM fluorescein attached to the starter oligo. The results are shown in Figure 2A. The results show a first peak (starter oligo), a second peak (starter oligo with a single nucleotide added, i.e., "+1 addition"), and a third peak that appears at a later time point (starter oligo with two nucleotides added, i.e., "+2 addition"). That is, during the initial time point, the proportion of extended +1 oligo (single base incorporation, right peak) increases compared to the starter oligo (left peak) until it reaches full length.

[0159] As shown at later time points, at the final 21 minutes and 41 seconds time point shown again in FIG. 2B, another peak due to the addition of two nucleotides to the starter oligo (+2) begins to appear in the absence of phosphatase. The +2 addition peak is labeled in FIG. 2B and corresponds to an undesired insertion. However, when the reaction is performed in the presence of phosphatase, the appearance of +2 addition is significantly reduced, while the rate of +1 addition is not significantly affected. Thus, synthesis using phosphatase-treated conjugate reagents (e.g., synthesis in the presence of phosphatase) improves the accuracy of single nucleotide addition of A, C, T, and G nucleotides to the substrate by the polymerase-nucleotide conjugate and inhibits undesired insertion.

[0160] Example 3: Several phosphatase variants prevent unwanted insertions during conjugate-based polynucleotide synthesis without affecting synthesis kinetics A 2 μM solution of TdT conjugated to the T linker nucleotide prepared in Example 1 was incubated at 24° C. ( FIG. 3A ) or 37° C. ( FIG. 3B ) with a divalent metal (e.g., magnesium or cobalt), 50 mM salt (e.g., potassium acetate or NaCl), 2 mM phosphatase (or no phosphatase as a control), and 50 nM DNA oligo (5′-6-FAM-T32CCC-3′) (SEQ ID NO: 11) in Tris or HEPES buffer at pH 8. The reaction was quenched at 3.8 s, 11.6 s, 19.2 s, 65 s, 3 min 21 s, 9 min 52 s, 21 min 41 s, and 30 min by adding 40 mM EDTA. The phosphatase was pre-buffer exchanged into a solution containing a buffer of pH 8 (e.g., Tris or HEPES) and salt (e.g., 50 mM NaCl or potassium acetate). The following phosphatase variants were tested: bovine (Quick CIP, NEB), northern shrimp (shrimp alkaline phosphatase, NEB), Antarctic bacteria TAB5 (Antarctic phosphatase, NEB), and E. coli (Takara Bio) phosphatase.

[0161] After completion of the reaction, the linker connecting the nucleotides in the conjugate with the TdT was cleaved using a reagent such as a cleavage enzyme or reducing agent to remove the TdT from the oligonucleotide. The resulting synthetic oligonucleotides in 3A were analyzed by capillary electrophoresis to differentiate the oligo population by length, with "0" assigned to represent the peak of the starter oligo, "+1" representing the peak of the starter oligo with a single incorporated T at its 3' end, and in 3B the results show a visible peak for the oligo with two incorporated nucleotides "+2 addition" in the "no phosphatase" control.

[0162] Specifically, oligonucleotides were analyzed using capillary electrophoresis and fluorescence detection of 6-FAM fluorescein attached to the starting oligo, and reaction products were differentiated by length. Oligonucleotide addition reaction products obtained after variable addition times ranging from 3.8 s to 21 min 41 s at 24 °C are shown in Figure 3A. Oligonucleotide addition reaction products obtained after 30 min addition time at 37 °C are shown in Figure 3B, with the arrow indicating the expected size of the +2 addition.

[0163] As shown, the appearance of the +2 addition is significantly reduced at all time points tested compared to "no phosphatase" (Figure 3B), while the +1 addition is completed at the 21 min 41 s time point in the presence of all phosphatases (Figure 3A). Furthermore, all but the TAB5 phosphatase variants had no visible effect on the kinetics of nucleotide incorporation compared to "no phosphatase". Thus, all phosphatase variants tested are suitable for inhibiting undesired insertions during polynucleotide synthesis, and most variants are also suitable when fast incorporation kinetics are desired.

[0164] Example 4. Phosphatases inhibit unwanted insertions during stepwise controlled synthesis of 50-mer polynucleotides using polymerase-nucleotide conjugates. Circular polynucleotide synthesis using a nucleotide polymerase conjugate is carried out to generate a defined 50-mer sequence: 5'-AACCGACCAAGCTACGGTTCAGAAAATTCGCGATGCAATTCGCGATCAGC-3' (SEQ ID NO: 12) was prepared.

[0165] Synthesis of FAM-labeled DNA oligos (starter oligos) hybridized to surface-bound DNA: 5'-6-FAM-CTGACAGAGATGATGAAGTCACATGAGACATGAACTGAGTCTTTT-3' (SEQ ID NO: 13) The procedure was started from the 3' end of the primer.

[0166] Two 50-mer synthesis reactions were performed, one in the presence of phosphatase and one without. DNA extension was performed on the starting molecule by cyclically adding nucleotides via TdT-nucleotide conjugates. Each DNA extension cycle, which adds one nucleotide to the 3' end of the surface-bound polynucleotide, was performed as follows (at temperatures between 24 and 37 °C):

[0167] 1. Nucleotide addition: A solution of TdT-nucleotide conjugate (corresponding to either A, T, C, or G), divalent metal (e.g., cobalt or magnesium), and 50 mM salt (e.g., potassium acetate or NaCl) in Tris or HEPES buffer, with or without 3 μM E. coli phosphatase, at pH 8, was added to the surface-bound DNA (e.g., starter oligo).

[0168] 2. Removal of oligo-bound TdT: After incubation for a sufficient time to add the TdT-nucleotide conjugate to the surface-bound oligo, the TdT extension reaction was terminated by adding 40 mM EDTA, and the linker connecting the nucleotide and TdT in the conjugate was cleaved using a reagent such as a cleavage enzyme or a reducing agent.

[0169] 3. Regeneration of the surface: A solution of NaOH pH 11 containing 0.5 M NaCl was then used to wash away all unbound reaction components from the surface.

[0170] Steps 1 to 3 were repeated to generate the desired polynucleotide sequence.

[0171] The resulting synthetic polynucleotides were removed from the surface and analyzed by detecting FAM fluorescence on a SeqStudio Genetic Analyzer (ThermoFisher) DNA Analyzer to differentiate the populations of polynucleotides by length. The results are shown in Figure 4.

[0172] As shown in Figure 4, synthesis without phosphatase shows products greater than 50 bases (representing products with one or more non-termination events or insertions) on the order of 40% of all substrates. However, in the presence of phosphatase, synthetic oligonucleotides greater than 50 bases make up only about 10% of the synthetic population, indicating a significant reduction in the rate of insertion at each synthetic step. Furthermore, there is no detectable change in deletions (cycles in which no addition occurred) in the presence or absence of phosphatase.

[0173] The above results show that the presence of phosphatase during conjugate-based polynucleotide synthesis results in a substantial (at least 4-fold) reduction in insertions without affecting the deletion rate in multi-step synthesis reactions compared to reactions without phosphatase, representing a significant improvement in the quality of polynucleotide synthesis by polymerase-nucleotide conjugates.

[0174] Other embodiments The words that have been used are words of description rather than limitation, and it is to be understood that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0175] While the present disclosure has been described at some length and with some specificity with respect to certain illustrated embodiments, it is not intended that it should be limited to any such specificity or embodiment, or to any particular embodiment, but rather should be construed with reference to the appended claims in a manner that provides the broadest possible interpretation of such claims in view of the prior art and thus effectively encompasses the intended scope of the present disclosure.

[0176] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

[Claim 1] The invention described in the specification.

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