Compositions and methods of use for enzymatic polynucleotide synthesis

Abstract

Compositions and methods for enzymatic nucleic acid synthesis are disclosed herein. In particular, provided herein are nucleic acid synthesis reaction buffer compositions and related methods for performing enzymatic nucleic acid synthesis, including the use of components and approaches that improve nucleotide synthesis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 290,310, filed December 16, 2021, the entire disclosure of which is incorporated herein by reference for all purposes.

[0002] Sequence Listing Reference This application contains a Sequence Listing (XML file named ABB-008WO_SL.xml, created on December 13, 2022, size 3,150 bytes) that was submitted electronically and is incorporated herein by reference. [Background technology]

[0003] 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 nucleic acid substrate (e.g., a DNA substrate), incorporation of an added nucleotide (e.g., a protected or blocked nucleotide), followed by a deprotection step to allow for future series of nucleotide incorporation. The rate at which the DNA substrate is extended, as determined by the enzyme kinetics, is a major limitation in the field of DNA synthesis (Figures 1A and 1B). In current methods, each cycle of nucleotide addition requires a tremendous amount of time to ensure the correct incorporation of the nucleotide in the extending polynucleotide. Due to the time required for each nucleotide addition compound, each with an extension reaction, enzymatic nucleotide synthesis of a desired polynucleotide is limiting for many nucleic acid synthesis applications. Minimizing the time of each cycle allows for the production of a desired polynucleotide in a time- and cost-efficient manner.

[0004] There is a need in the art for improved enzymatic polynucleotide synthesis methods having conditions and buffer compositions that minimize the duration of the enzymatic polynucleotide extension reaction. Summary of the Invention

[0005] overview In one aspect, the disclosure provides a method of nucleic acid synthesis, the method comprising: providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, the conjugates comprising a nucleotide or modified nucleotide bound to a polymerase via a linker; and contacting a sample comprising a polynucleotide with the conjugate reagent, the polymerase of the conjugate catalyzing an extension reaction comprising covalently adding a blocked nucleotide of the conjugate to the 3' hydroxyl of the polynucleotide in the presence of at least one divalent cation, the at least one divalent cation concentration being less than about 2500 μM. In some embodiments, the linker is a cleavable linker. In some embodiments, the method described herein further comprises cleaving the linker. In some embodiments, the method described herein further comprises repeating each of the steps described herein to synthesize a polynucleotide. In some embodiments, the polynucleotide comprises a predetermined sequence.

[0006] In some aspects, the present disclosure provides a method for nucleic acid synthesis.In some embodiments, the method for nucleic acid synthesis includes: providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, the conjugate comprises nucleotides linked to polymerase via linker; providing a sample comprising polynucleotide; contacting the sample with the conjugate reagent in a reaction volume, so that the sample and the conjugate reagent are combined and in the presence of each other, the polymerase of the conjugate catalyzes an extension reaction comprising covalently adding the nucleotide of the conjugate to the 3' hydroxyl of the polynucleotide, and at least one divalent cation is present in the reaction volume, and the total concentration of divalent cations present in the reaction volume is about 500 μM or less.

[0007] In some embodiments, the total concentration of divalent cations present in the reaction volume is less than or equal to about 250 μM, 125 μM, or about 50 μM.

[0008] In some embodiments, the extension reaction performed has a faster turnover rate than an extension reaction performed in the presence of a concentration of the same or another divalent cation greater than about 1000 μM.

[0009] In some embodiments, the faster conversion rate is about 1 second faster, about 2 seconds faster, about 3 seconds faster, about 4 seconds faster, about 5 seconds faster, about 6 seconds faster, about 7 seconds faster, about 8 seconds faster, about 9 seconds faster, about 10 seconds faster, about 15 seconds faster, about 20 seconds faster, about 25 seconds faster, about 30 seconds faster, about 35 seconds faster, about 40 seconds faster, about 45 seconds faster, about 50 seconds faster, about 55 seconds faster, or about 60 seconds faster compared to a reference reaction performed in the presence of a divalent cation at a concentration greater than about 1000 μM.

[0010] In some embodiments, the divalent cation present in the reaction volume at the highest concentration relative to the total concentration of divalent cations in the reaction volume is cobalt (Co 2+ ) or zinc (Zn 2+ ).

[0011] In some embodiments, at least one divalent cation is present at a concentration of about 2500 μM or less, and extension reactions performed in the presence of a concentration of about 2500 μM or less have a faster turnover rate than extension reactions performed in the presence of the same or another divalent cation at a concentration greater than about 2500 μM. In some embodiments, the concentration of about 2500 μM or less is about 1 μM, about 2.5 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 100 μM, about 150 μM, about 200 μM, about 250 μM, about 300 μM, about 350 μM, about 400 μM, about 450 μM, about 500 μM, about 750 μM, about 1000 μM, about 1500 μM, about 2000 μM, or up to about 2500 μM.

[0012] In some embodiments, the faster conversion rate is about 60 seconds faster to about 300 seconds faster compared to a reference reaction not performed in the presence of a divalent cation concentration of about 2500 μM or less. In some embodiments, the faster conversion rate is about 1 second faster, about 2 seconds faster, about 3 seconds faster, about 4 seconds faster, about 5 seconds faster, about 6 seconds faster, about 7 seconds faster, about 8 seconds faster, about 9 seconds faster, about 10 seconds faster, about 15 seconds faster, about 20 seconds faster, about 25 seconds faster, about 30 seconds faster, about 35 seconds faster, about 40 seconds faster, about 45 seconds faster, about 50 seconds faster, about 55 seconds faster, or about 60 seconds faster compared to a reference reaction not performed in the presence of a divalent cation concentration of about 2500 μM or less.

[0013] In some embodiments, the at least one divalent cation is Mg 2+ , Ca 2+ , Sr 2+ , B.A. 2+ , Mn 2+ , Co 2+ , Fe 2+ , Ni 2+ , Cu 2+ , and Zn 2+ In some embodiments, the at least one divalent cation is not Co. 2+ In some embodiments, the at least one divalent cation is Zn 2+ In some embodiments, the extension reaction is 2+ It is executed in the absence of

[0014] In one aspect, the disclosure provides a method of synthesizing a polynucleotide comprising repeating one or more of the methods or one or more steps of the methods provided herein one or more times.

[0015] In some embodiments, the polynucleotide comprises a predetermined sequence.

[0016] In some embodiments, the polymerase is a template-independent polymerase. In some embodiments, the polymerase is selected from Pol IV, Pol μ, and terminal deoxyribonucleotidyl transferase (TdT), or a variant thereof. In some embodiments, the polymerase is TdT, or a variant thereof.

[0017] 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.

[0018] In one aspect, the disclosure provides a nucleic acid synthesis reaction buffer comprising at least one divalent metal ion, wherein when used in a synthesis reaction, the concentration of the divalent metal ion present during the synthesis reaction is about 2500 μM or less.

[0019] In some embodiments, at least one divalent cation present in the synthesis reaction is less than about 1 μM, about 2.5 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 100 μM, about 150 μM, about 200 μM, about 250 μM, about 300 μM, about 350 μM, about 400 μM, about 450 μM, about 500 μM, about 750 μM, about 1000 μM, about 1500 μM, about 2000 μM, or up to about 2500 μM.

[0020] In some embodiments, the at least one divalent cation is Mg 2+ , Ca 2+ , Sr 2+ , B.A. 2+ , Mn 2+ , Co 2+ , Fe 2+ , Ni 2+ , Cu 2+ , and Zn 2+ In some embodiments, the at least one divalent cation is selected from Mg 2+In some embodiments, the at least one divalent cation is Ca 2+ In some embodiments, the at least one divalent cation is Sr 2+ In some embodiments, at least one divalent cation is Ba 2+ In some embodiments, the at least one divalent cation is Mn 2+ In some embodiments, the at least one divalent cation is Co 2+ In some embodiments, the at least one divalent cation is Fe 2+ In some embodiments, the at least one divalent cation is Ni 2+ In some embodiments, the at least one divalent cation is Cu 2+ It is.

[0021] In some embodiments, the at least one divalent cation is Zn 2+ In some embodiments, the nucleic acid synthesis reaction buffer further comprises tris(hydroxymethyl)aminomethane. In some embodiments, the nucleic acid synthesis reaction buffer further comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. In some embodiments, the nucleic acid synthesis reaction buffer further comprises a potassium salt. In some embodiments, the nucleic acid synthesis reaction buffer further comprises a non-ionic detergent. In some embodiments, the nucleic acid synthesis reaction buffer further comprises bovine serum albumin. In some embodiments, the nucleic acid synthesis reaction buffer further comprises sodium chloride. In some embodiments, the nucleic acid synthesis reaction buffer further comprises β-mercaptoethanol. In some embodiments, the nucleic acid synthesis reaction buffer further comprises glycerol.

[0022] In one aspect, the disclosure provides a method of nucleic acid synthesis, the method comprising: providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, the conjugates comprising nucleotides or modified nucleotides bound (e.g., covalently linked) to a polymerase via a linker; and contacting a sample comprising a polynucleotide with the conjugate reagent, wherein the polymerase of the conjugate catalyzes an extension reaction comprising covalently adding a blocked nucleotide of the conjugate to a 3' hydroxyl of the polynucleotide in a nucleic acid synthesis reaction buffer according to any one of the preceding claims.

[0023] In one aspect, the disclosure provides a method of nucleic acid synthesis, the method comprising providing 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 being catalyzed by a concentration of cobalt (Co 2+ ), and optionally Mg 2+ catalyzes an extension reaction comprising the covalent addition of a nucleotide of a conjugate to the 3' hydroxyl of the polynucleotide in the absence of Co 2+ The concentration of Co is about 0.250 mM. 2+ , about 0.125 mM Co 2+ , or about 0.050 mM Co 2+ and about 0.250 mM Co 2+ , about 0.125 mM Co 2+ , or about 0.050 mM Co 2+ The extension reaction in the presence of Co at concentrations greater than 0.500 mM 2+ and contacting the reaction, the reaction being faster than that carried out in the presence of

[0024] In one aspect, the disclosure provides a method of nucleic acid synthesis, the method comprising providing 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 being catalyzed by a concentration of zinc (Zn 2+ ), and optionally Mg 2+ catalyzes an extension reaction involving the covalent addition of a nucleotide of a conjugate to the 3' hydroxyl of the polynucleotide in the absence of Zn 2+ The concentration of Zn is about 0.250 mM. 2+ , about 0.125 mM Zn 2+ , or about 0.050 mM Zn 2+ and about 0.250 mM Zn 2+ , about 0.125 mM Zn 2+ , or about 0.050 mM Zn 2+ The extension reaction in the presence of Zn at concentrations above 0.500 mM 2+ and contacting the reaction, the reaction being faster than that carried out in the presence of

[0025] In one aspect, the disclosure provides a method for reducing incorporation of free nucleotides into polynucleotides generated using a nucleic acid synthesis reaction, the method comprising providing 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 being catalyzed by a concentration of cobalt (Co 2+ ), and optionally in the presence of Mg 2+ catalyzing an extension reaction comprising covalently adding a conjugate nucleotide to the 3' hydroxyl of the polynucleotide in the absence of 2+ and about 0.050 mM Co 2+Incorporation of free nucleotides into polynucleotides using nucleotide extension reactions in the presence of Co at concentrations greater than 0.125 mM 2+ and contacting the reaction such that the amount of oxidized carbon dioxide is reduced relative to that in a reaction carried out in the presence of

[0026] In one aspect, the disclosure provides a method of improving a nucleotide synthesis reaction, the improvement comprising performing nucleic acid synthesis in the presence of divalent cations at a divalent cation concentration of about 0.250 mM, about 0.125 mM, or about 0.050 mM, wherein the rate of the extension reaction is faster than the rate of the extension reaction performed in the presence of a divalent cation concentration greater than about 0.500 mM.

[0027] 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. [Brief description of the drawings]

[0028] [Figure 1A] 1 is a scheme illustrating the repetitive cycles of enzymatic polynucleotide synthesis. [Figure 1B] 1 is a scheme illustrating the repetitive cycles of enzymatic polynucleotide synthesis.

[0029] [Figure 2A] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing low cobalt acetate concentrations (0.05 mM CoOAc, FIGS. 2B and 2D) or standard cobalt acetate concentrations (0.5 mM CoOAc, FIGS. 2A and 2C). [Figure 2B]Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing low cobalt acetate concentrations (0.05 mM CoOAc, FIGS. 2B and 2D) or standard cobalt acetate concentrations (0.5 mM CoOAc, FIGS. 2A and 2C). [Figure 2C] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing low cobalt acetate concentrations (0.05 mM CoOAc, FIGS. 2B and 2D) or standard cobalt acetate concentrations (0.5 mM CoOAc, FIGS. 2A and 2C). [Figure 2D] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing low cobalt acetate concentrations (0.05 mM CoOAc, FIGS. 2B and 2D) or standard cobalt acetate concentrations (0.5 mM CoOAc, FIGS. 2A and 2C). [Figure 2E] FIG. 2C is a plot showing quantification and analysis of the products of FIGS. 2A-2D and associated calculated reaction rates (kobs).

[0030] [Figure 3A] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3B]Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3C] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3D] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3E] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3F] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3G] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3H] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3I] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3J] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3K]Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3L] Electropherograms showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various cobalt acetate concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 3M] FIG. 5 is a plot showing quantification and analysis of the products of FIGS. 3A-3L and associated calculated reaction rates (kobs).

[0031] [Figure 4A] FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4B] FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4C]FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4D] FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4E] FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4F] FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4G]FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4H] FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4I] FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4J] FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4K]FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4L] FIG. 1 is an electropherogram showing results from analysis of products at different time points in enzymatic polynucleotide extension reactions performed using polymerase-nucleotide conjugates in reaction buffers containing various zinc acetate (ZnOAc) concentrations (0.05 mM ZnOAc, 0.125 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc). [Figure 4M] FIG. 5 is a plot showing quantification and analysis of the products of FIGS. 4A-4L and associated calculated reaction rates (kobs).

[0032] [Figure 5A] FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5B] FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5C]FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5D] FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5E] FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5F] FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5G]FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5H] FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5I] FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5J] FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5K]FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5L] FIG. 1 is an electropherogram showing results from analysis of products at different time points in an enzymatic polynucleotide extension reaction using free polymerase and free nucleotides performed in reaction buffers containing various cobalt acetate (CoOAc) concentrations (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). [Figure 5M] 6 is a plot showing quantification and analysis of the products of FIG. 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description The present disclosure provides insight based on the surprising discovery that the rate of an extension reaction using a polymerase-nucleotide conjugate in polynucleotide synthesis can be increased by reducing the concentration of divalent cations present in the reaction as compared to the standard divalent cation concentrations used in polynucleotide synthesis reactions using free polymerase and free polynucleotide.

[0034] This disclosure provides compositions and methods related to this discovery. Details of various embodiments of the compositions and methods are set forth in this disclosure. Other features, objects, and advantages of the compositions and methods disclosed herein will be apparent from the description and drawings, and from the claims.

[0035] 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.

[0036] As used herein, the term "nucleoside polyphosphate" refers to a "nucleotide" and may also be referred to as a "nucleotide polyphosphate." For example, a "nucleotide triphosphate" and a "nucleoside triphosphate" both refer to a nucleotide that includes a nucleobase, a sugar, and a polyphosphate consisting of three linked phosphate groups.

[0037] 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.

[0038] 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 component, such as a blocking group) from a phosphatase that can remove the 5' phosphate. In some embodiments, such a nucleotide is likely to inhibit subsequent nucleotide addition after it is added to an oligonucleotide and before removal of the tethered polymerase.

[0039] As used herein, the term "unprotected nucleotide" or "unshielded nucleotide" refers to a nucleotide that is not sterically hindered by a tethered polymerase (or other entity or component, such as a blocking group) from a phosphatase that can remove the 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 the polymerase. The unshielded nucleotide that is not exposed to a phosphatase is more likely to be misadded to a polynucleotide as an insertion after the correct addition of the shielded nucleotide.

[0040] Nucleic acid synthesis A method of nucleic acid synthesis is disclosed herein. Nucleic acid synthesis can refer to the synthesis or production of a product that is a nucleic acid molecule (i.e., a polynucleotide). A method of nucleic acid synthesis can include stepwise synthesis, in which nucleotides are inserted stepwise into a nucleic acid polymer or polynucleotide. A typical process for stepwise synthesis of a polynucleotide includes stepwise addition of nucleotides to an initiating molecule (e.g., an initial oligonucleotide) through cyclic steps of addition of a polymerase and nucleotides (e.g., a polymerase-nucleotide conjugate) to the oligonucleotide, and covalent incorporation of nucleotides into the 3' end of the oligonucleotide catalyzed by the polymerase. The successful incorporation of nucleotides into an oligonucleotide can be referred to as "extension" or "extension reaction".

[0041] In some embodiments, the polymerase and the nucleotide are linked together (i.e., tethered) to form a conjugate (i.e., a polymerase-nucleotide conjugate). In stepwise synthesis using the conjugate, the tethered nucleotide is covalently incorporated (i.e., added) to the 3' end of the oligonucleotide, which is catalyzed by the tethered polymerase. The tethered polymerase may remain tethered to the nucleotide after the covalent incorporation. The covalent incorporation of the nucleotide may be referred to as an extension or addition. The tethered polymerase may be cleaved from the inserted nucleotide to expose the 3' end of the oligonucleotide. These steps may be repeated to synthesize a desired polynucleotide. The desired polynucleotide may have a predetermined (i.e., predefined or targeted) sequence.

[0042] As will be appreciated by those skilled in the art, the methods provided herein, such as nucleic acid synthesis, are carried out in a reaction volume. In a given context, the contents of the reaction volume may change before, after, and during synthesis. For example, in some embodiments, the reaction volume includes one or more of a buffer, a polynucleotide, a polymerase-nucleotide conjugate, a nucleotide initiator molecule, a synthesis product, a phosphatase, and the like. In some such embodiments, the reaction volume may be prepared to include only selected components (e.g., a buffer and a nucleotide initiator molecule), and one or more additional components may be added to the volume at one or more subsequent times. In some embodiments, all components for a given synthesis may be added substantially simultaneously. In some embodiments, one or more components of a synthesis reaction may be pretreated (e.g., pretreatment of a polymerase-nucleotide conjugate with a phosphatase) before being included in the reaction volume for a nucleic acid synthesis reaction.

[0043] In particular, the method of nucleic acid synthesis disclosed herein is carried out in a reaction buffer composition. The reaction buffer composition is an aqueous solution. The reaction buffer composition comprises a set of components suitable for the stability of a polymerase, a nucleotide, a polymerase-nucleotide conjugate, an initiating molecule, a nucleic acid molecule product, and any surface or substrate on which the method disclosed herein is carried out. In some embodiments, the method disclosed herein is carried out on a solid surface, in a vessel such as a test tube, a microplate, or other vessel having one or more vessels suitable for containing a liquid, in a single dot on a solid surface and / or other substrate, etc. Furthermore, the reaction buffer composition comprises a set of components suitable for carrying out the catalytic step (e.g., polynucleotide polymerization carried out by a polymerase) described in the method of nucleic acid synthesis described herein.

[0044] The conditions under which nucleic acid synthesis is carried out can be varied, for example, the time for carrying out each step in the stepwise nucleotide addition cycle can be varied to improve the purity of the multiple products generated by the methods of nucleic acid synthesis described herein.

[0045] In some embodiments, the synthesis reaction occurs in the presence of one or more divalent cations. In some embodiments, the one or more divalent cations are magnesium (Mg 2+ ), Calcium (Ca 2+ ), Strontium (Sr 2+ ), Barium (Ba 2+ ), Manganese (Mn 2+ ), Cobalt (Co 2+ ), iron (Fe 2+ ), Nickel (Ni 2+ ), Copper (Cu 2+ ), and / or zinc (Zn 2+ In some embodiments, the one or more divalent cations are present at a concentration of 2.5 mM or less.

[0046] In some embodiments, the concentration of any given divalent cation present in nucleic acid synthesis reaction volume is less than about 1 μM, about 2.5 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 100 μM, about 150 μM, about 200 μM, about 250 μM, about 300 μM, about 350 μM, about 400 μM, about 450 μM, about 500 μM, about 1000 μM, about 1500 μM, about 2000 μM, or about 2500 μM.In some embodiments, the concentration of divalent cation in synthesis reaction is 0 μM or substantially 0 μM.In some such embodiments, such divalent cation is considered to be absent in such reaction.

[0047] In some embodiments, the concentration of divalent cations present in a synthesis reaction and / or reaction volume comprises a total concentration of divalent cations, which may be comprised of one or more divalent cations. In some embodiments, the one or more divalent cations are magnesium (Mg 2+ ), Calcium (Ca 2+ ), Strontium (Sr 2+ ), Barium (Ba 2+ ), Manganese (Mn 2+ ), Cobalt (Co 2+ ), iron (Fe 2+ ), Nickel (Ni 2+ ), Copper (Cu 2+ ), and zinc (Zn 2+ In some embodiments, the concentration of divalent cations (e.g., of a synthesis reaction, e.g., in a reaction volume) includes one or more divalent cations. In some embodiments, one divalent cation (e.g., Co 2+ , e.g. Zn 2+ ) is present in a higher concentration (e.g., by a certain percentage) than any other divalent cation. In some embodiments, one divalent cation (e.g., Co 2+ , e.g. Zn 2+ ) is present at the highest concentration relative to the concentration of one or more other divalent cations (e.g., Mg2+). In some embodiments, one divalent cation (e.g., Co 2+ , e.g. Zn2+ ) is the only divalent cation present (i.e., e.g. Mg 2+ For example, by way of non-limiting example, in some embodiments, the reaction volume may comprise a total volume, or the synthesis reaction may occur in a total volume, in which the total concentration of divalent cations in the volume is, for example, Co 2+ and Mg 2+ In some such embodiments, one of the two divalent cations is present at a higher concentration (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more) than the other divalent cation. In some embodiments, the reaction volume contains a single divalent cation (e.g., Co 2+ , Zn 2+ ) includes the total concentration of divalent cations.

[0048] The methods of nucleic acid synthesis disclosed herein can be used to generate a nucleic acid molecule product (i.e., a polynucleotide product). In some embodiments, the nucleic acid molecule product (i.e., a polynucleotide product) has a target (i.e., a predetermined) sequence. A "target" or "predetermined" sequence refers to a desired polynucleotide sequence that is intentionally produced by the methods of nucleic acid synthesis. The predetermined sequence can include any number of nucleotides that include nucleobases (e.g., adenine, thymine, guanine, cytosine, and / or uracil). In some embodiments, the nucleotides are modified nucleotides (i.e., nucleotide analogs). In some embodiments, the nucleobases are modified nucleobases. In some embodiments, the predetermined sequence includes one or more designated positions that may be random nucleobases. The inclusion of positions with random nucleobases can be useful, for example, to introduce randomized mutations into the polynucleotide product.

[0049] The nucleic acid molecule product or polynucleotide product generated by the methods described herein may include a plurality of products. In some embodiments, the plurality of products includes nucleic acid molecules that include a target (i.e., predetermined) sequence. In some embodiments, the plurality of products includes nucleic acid molecules that include a sequence that is not a target sequence. In some embodiments, the plurality of products includes nucleic acid molecule products that include a target sequence and nucleic acid molecule products that are not a target sequence. The "purity" of a plurality of products may refer to, for example, the ratio of the abundance of nucleic acid molecule products with a target sequence to the abundance of nucleic acid molecule products without a target sequence, taking into account all products and what proportion of the products do or do not include a target sequence. Such purity measurements may be expressed to show the ratio of one to the other, such as the ratio of one to the other, such as those with or without a target sequence, to all products.

[0050] Nucleic acid synthesis reaction buffer composition The present disclosure provides reaction buffer compositions (i.e., nucleic acid synthesis reaction buffers) for carrying out polynucleotide extension reactions. The polymerase enzymes used in the disclosed methods require divalent cation cofactors to carry out the covalent addition of nucleotides during enzymatic nucleic acid synthesis.

[0051] In some embodiments, nucleic acid synthesis reaction buffer comprises at least one divalent metal ion. In some embodiments, the concentration of at least one divalent metal ion is less than about 2500 μM. In some embodiments, the concentration of at least one divalent cation is less than about 1 μM, about 2.5 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 100 μM, about 150 μM, about 200 μM, about 250 μM, about 300 μM, about 350 μM, about 400 μM, about 450 μM, about 500 μM, about 1000 μM, about 1500 μM, about 2000 μM, or about 2500 μM. In some embodiments, the concentration of a given divalent cation in a nucleic acid synthesis reaction is 0 μM or substantially 0 μM. In some such embodiments, such divalent cation is considered to be absent from such reaction.

[0052] In some embodiments, the at least one divalent cation is magnesium (Mg 2+ ), Calcium (Ca 2+ ), Strontium (Sr 2+ ), Barium (Ba 2+ ), Manganese (Mn 2+ ), Cobalt (Co 2+ ), iron (Fe 2+ ), Nickel (Ni 2+ ), Copper (Cu 2+ ), and zinc (Zn 2+ ), or a combination thereof. In some embodiments, the at least one divalent cation is selected from Mg 2+ In some embodiments, the at least one divalent cation is Ca 2+ In some embodiments, the at least one divalent cation is Sr 2+ In some embodiments, at least one divalent cation is Ba 2+ In some embodiments, the at least one divalent cation is Mn 2+ In some embodiments, the at least one divalent cation is Co 2+In some embodiments, the at least one divalent cation is Fe 2+ In some embodiments, the at least one divalent cation is Ni 2+ In some embodiments, the at least one divalent cation is Cu 2+ In some embodiments, the at least one divalent cation is Zn 2+ It is.

[0053] In some embodiments, the at least one divalent cation is Mg 2+ In some embodiments, the at least one divalent cation is not Ca 2+ In some embodiments, the at least one divalent cation is not Sr 2+ In some embodiments, the at least one divalent cation is not Ba 2+ In some embodiments, the at least one divalent cation is not Mn 2+ In some embodiments, the at least one divalent cation is not Co. 2+ In some embodiments, the at least one divalent cation is not Fe 2+ In some embodiments, the at least one divalent cation is not Ni 2+ In some embodiments, the at least one divalent cation is not Cu. 2+ In some embodiments, the at least one divalent cation is not Zn 2+ isn't it.

[0054] In some embodiments, Mg 2+ Nucleic acid synthesis reactions that contain at least one divalent cation that is not Mg 2+ (either alone or in the presence of one or more other divalent cations) proceeds at a faster rate than the identical reaction involving

[0055] In some embodiments, the nucleic acid synthesis reaction occurs in the absence of magnesium or in the presence of a concentration that is lower than the concentration of at least one other divalent cation.

[0056] In some embodiments, the nucleic acid synthesis reaction buffer comprises a pH buffering component. The buffering component is used in a concentration of 1 mM to 1 M in the nucleic acid synthesis reaction buffer. In some embodiments, the buffering component is at a concentration of about 10 mM to about 100 mM. In some embodiments, the buffering component is at a concentration of about 100 to about 200 mM. In some embodiments, the buffering component is at a concentration of about 50 mM to about 100 mM. In some embodiments, the buffering component is at a concentration of about 10 mM to about 50 mM. In some embodiments, the buffering component is at a concentration of about 20 mM. Exemplary buffering components include, but are not limited to, Tris (tris(hydroxymethyl)aminomethane), Tricine, Bicine, Bis-Tris, CAPS, EPPS, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES, MOPS, PIPES, TAPS, and TES. In some embodiments, the nucleic acid synthesis reaction buffer comprises Tris. In some embodiments, the nucleic acid synthesis reaction buffer comprises HEPES.

[0057] In some embodiments, the nucleic acid synthesis reaction buffer has a pH of about pH 6.0 to about pH 8.5. In some embodiments, the pH is about pH 6.0 to about pH 8.5. In some embodiments, the pH is about pH 6.5 to about pH 8.0. In some embodiments, the pH is about pH 7 to about pH 7.5. In some embodiments, the pH is about pH 7.5 to about pH 8.0. In some embodiments, the pH is about pH 8.

[0058] In some embodiments, the nucleic acid synthesis reaction buffer comprises a monovalent cation. The monovalent cation may be a salt. In some embodiments, the monovalent cation is selected from sodium, potassium, lithium, rubidium, cesium, ammonium, or any combination thereof. In some embodiments, the monovalent cation is at a concentration of about 100 to about 200 mM.

[0059] In some embodiments, the nucleic acid synthesis reaction buffer comprises a detergent, surfactant, or non-ionic surfactant, hi some embodiments, the detergent, surfactant, or non-ionic surfactant is selected from TRITON X-100®, Nonidet P-40 (NP-40), Tween 20, P20, and Brij 35, or any combination thereof.

[0060] In some embodiments, the nucleic acid synthesis reaction buffer comprises one or more stabilizing agents. In some embodiments, the one or more stabilizing agents are bovine serum albumin and / or glycerol.

[0061] In some embodiments, the nucleic acid synthesis reaction buffer comprises one or more reducing agents, hi some embodiments, the reducing agent is selected from dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), and β-mercaptoethanol.

[0062] Formulation of nucleic acid synthesis reaction buffer compositions To prepare the nucleic acid synthesis reaction buffer compositions described herein, the reagent components are mixed at working concentrations to form a solution suitable for immediate use with or without dilution or addition of further reagents. The water used in the formulations of the present disclosure can be distilled, deionized, and sterile filtered (through a 0.1-0.2 micrometer filter) and is free of contamination by DNase and RNase enzymes. Such water is commercially available, for example, from Sigma Chemical Company (Saint Louis, Mo.), or may be made as needed according to methods well known to those of skill in the art.

[0063] Methods for Nucleic Acid Synthesis In particular, the present disclosure provides a method for nucleic acid synthesis.In some embodiments, the method for nucleic acid synthesis comprises: providing a conjugate reagent comprising a plurality of polymerase-nucleotide conjugates, the conjugates comprising nucleotides or modified 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 catalyzes an extension reaction comprising covalently adding the blocked nucleotide of the conjugate to the 3' hydroxyl of the polynucleotide in the nucleic acid synthesis reaction buffer disclosed herein.

[0064] In some embodiments, a method of nucleic acid synthesis includes contacting a polynucleotide with a polymerase and a nucleotide, wherein the effective concentration of the nucleotide relative to the polymerase is artificially increased. In some embodiments, the method is carried out in a nucleic acid synthesis reaction buffer as disclosed herein. As a non-limiting example, the effective concentration of the nucleotide relative to the polymerase can be artificially increased, for example, by engineering the polymerase to have a higher affinity for the nucleotide to be incorporated by the polymerase in an extension reaction, or by tethering the nucleotide to the polymerase.

[0065] In some embodiments, the extension reaction has a faster turnover rate than an extension reaction performed in the presence of at least one divalent cation at a concentration greater than about 1 μM, about 2.5 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 100 μM, about 150 μM, about 200 μM, about 250 μM, about 300 μM, about 350 μM, about 400 μM, about 450 μM, about 500 μM, about 1000 μM, about 1500 μM, about 2000 μM, or about 2500 μM.

[0066] In some embodiments, the faster conversion speed is between about 1 second faster and about 60 seconds faster, in some embodiments, the faster conversion speed is about 1 second faster, about 2 seconds faster, about 3 seconds faster, about 4 seconds faster, about 5 seconds faster, about 6 seconds faster, about 7 seconds faster, about 8 seconds faster, about 9 seconds faster, about 10 seconds faster, about 15 seconds faster, about 20 seconds faster, about 25 seconds faster, about 30 seconds faster, about 35 seconds faster, about 40 seconds faster, about 45 seconds faster, about 50 seconds faster, about 55 seconds faster, or about 60 seconds faster.

[0067] In some embodiments, the faster conversion rate is about 60 seconds faster to about 300 seconds faster. In some embodiments, the faster conversion rate is about 70 seconds faster. In some embodiments, the faster conversion rate is about 80 seconds faster. In some embodiments, the faster conversion rate is about 90 seconds faster. In some embodiments, the faster conversion rate is about 100 seconds faster. In some embodiments, the faster conversion rate is about 110 seconds faster. In some embodiments, the faster conversion rate is about 120 seconds faster. In some embodiments, the faster conversion rate is about 130 seconds faster. In some embodiments, the faster conversion rate is about 140 seconds faster. In some embodiments, the faster conversion rate is about 150 seconds faster. In some embodiments, the faster conversion rate is about 160 seconds faster. In some embodiments, the faster conversion rate is about 170 seconds faster. In some embodiments, the faster conversion rate is about 180 seconds faster. In some embodiments, the faster conversion rate is about 190 seconds faster. In some embodiments, the faster conversion rate is about 200 seconds faster. In some embodiments, the faster conversion speed is about 210 seconds faster. In some embodiments, the faster conversion speed is about 220 seconds faster. In some embodiments, the faster conversion speed is about 230 seconds faster. In some embodiments, the faster conversion speed is about 240 seconds faster. In some embodiments, the faster conversion speed is about 250 seconds faster. In some embodiments, the faster conversion speed is about 260 seconds faster. In some embodiments, the faster conversion speed is about 270 seconds faster. In some embodiments, the faster conversion speed is about 280 seconds faster. In some embodiments, the faster conversion speed is about 290 seconds faster. In some embodiments, the faster conversion speed is about 300 seconds faster.

[0068] Determining the conversion speed The methods of nucleic acid synthesis provided herein include faster conversion rates for extension reactions that include the nucleic acid synthesis reaction buffers described herein. The conversion rate refers to the time required to extend an oligonucleotide by at least one nucleotide.

[0069] Any suitable method known in the art can be used to determine the conversion rate. The conversion rate can be assessed by analyzing the nucleic acid synthesis products over time after initiating the extension reaction (i.e., the time course of the reaction). The 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 starting material (i.e., the initial polynucleotide or oligonucleotide into which a nucleotide is being incorporated) with the abundance of the expected polynucleotide product, it is possible to determine the extent to which the extension reaction is complete. The change over time in the starting material and extension species indicates the conversion 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).

[0070] 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).

[0071] Conjugates Described herein are methods of nucleic acid synthesis using a conjugate comprising a polymerase and a nucleotide, where the polymerase and the nucleotide are linked via a linker that comprises a cleavable linkage. The polymerase portion of the conjugate can extend a nucleic acid using the linked nucleotide (i.e., the polymerase can catalyze the joining of the nucleotide to which it is attached to the nucleic acid) and remains attached to the extended nucleic acid via the linker until the linker is cleaved.

[0072] Overview When a conjugate comprising a polymerase and a nucleoside polyphosphate is incubated with a nucleic acid, it causes the nucleic acid to be preferentially extended using the tethered nucleotide (as opposed to using the nucleotide of another conjugate molecule). As described above, the polymerase remains attached to the nucleic acid via its tethering to the added nucleotide until it is subsequently exposed to some stimulus that causes the linkage to the added nucleotide to be cleaved. In this situation, further extension by the polymerase-nucleotide conjugate is prevented (i.e., the nucleotide is "shielded") when: 1) the bound polymerase molecule prevents other conjugates from accessing the 3'OH of the extended DNA molecule, and 2) other nucleoside polyphosphates in the system are prevented from accessing the catalytic site of the polymerase that remains attached to the 3' end of the extended nucleic acid. (The degree of shielding can be described as the extent to which both of these interactions are disrupted.) To allow subsequent extension, the linker tethering the incorporated nucleotide to the polymerase is cleaved, releasing the polymerase from the nucleic acid and thus re-exposing its 3'OH group for subsequent extension.

[0073] The 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.

[0074] 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 "abort", 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 abort termination may still be used to extend nucleic acids primarily by a single nucleotide (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 that includes the conjugate may further include a polymerase that does not have a tethered nucleoside polyphosphate.

[0075] 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 nucleoside polyphosphate 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.

[0076] Polymerase-nucleotide conjugates that use shielding effect to achieve termination are preferably labeled with only a single nucleoside polyphosphate moiety. Polymerase-nucleotide conjugates labeled with multiple nucleoside polyphosphates that can access the catalytic site can, in some cases, incorporate multiple nucleoside polyphosphates into the same nucleic acid. Thus, additional tethered nucleotides can result in additional, undesired nucleotide incorporation into the nucleic acid during the reaction. Furthermore, since only one tethered nucleoside polyphosphate can occupy the (buried) catalytic site of the polymerase at a time, other tethered nucleoside polyphosphate(s) can increase the accessibility of the polymerase moiety of other conjugate molecules, as discussed below.

[0077] Polymerase-nucleotide conjugates that use shielding effect to achieve termination preferentially include the shortest possible linker that still allows the nucleoside polyphosphate to frequently access the catalytic site of its tethered polymerase molecule in productive conformation to allow rapid incorporation of nucleotides into nucleic acid. Such conjugates may also preferentially use the linker attachment position to the polymerase as close as possible to the catalytic site, allowing the use of shorter linkers. The length of the linker will determine the maximum distance from the attachment point that the tethered nucleoside polyphosphate 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 approximately 24 and 28 Å long. Shorter linkers, e.g., linkers between 8-15 Å in length, may increase shielding, while longer linkers, e.g., linkers greater than 50 Å, 70 Å, or 100 Å, may decrease shielding. The shielding effect may 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 nucleoside polyphosphate to the catalytic site of the polymerase, the binding affinity of the nucleic acid to the polymerase, the preferred conformation of the polymerase, and / or the preferred conformation of the linker.

[0078] 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 part of the other conjugate.Steric effects may also prevent the tethered nucleoside polyphosphate from reaching the catalytic site of the other 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 nucleoside polyphosphate (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.

[0079] Another contribution to shielding comes from the binding affinity of the tethered nucleoside polyphosphate to the catalytic site of the polymerase. The tethered nucleoside polyphosphate of the conjugate will have a high effective concentration relative to the catalytic site of the tethered polymerase, and can remain bound to the site for a long time. When a nucleoside polyphosphate is bound to the catalytic site of the tethered polymerase molecule, it is not available for extension by other polymerase molecules. Thus, tethering reduces the effective concentration of nucleoside polyphosphate 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 using the nucleoside polyphosphate portion of one conjugate molecule to reduce the rate at which the nucleic acid is extended by the polymerase portion of another conjugate molecule.

[0080] 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 tethered polymerase, so the nucleic acid can remain bound to the site for a long time. When a nucleic acid is bound to the catalytic site of the 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.

[0081] In some embodiments, the polymerase-nucleotide conjugate contains an additional moiety that sterically prevents the tethered nucleoside polyphosphate (or the tethered nucleic acid after extension) from approaching the catalytic site of another conjugate molecule. Such moieties include polypeptide or protein domains that can be inserted into the loop of the polymerase, as well as molecules such as polymers and other bulky molecules that can be site-specifically ligated to inserted unnatural amino acids or specific polypeptide tags.

[0082] In some embodiments, the linker 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 herein. In other embodiments, the linker may be attached to any suitable atom of a nucleotide to form a conjugate, such as the phosphate, sugar, or base of the nucleotide, as would be apparent to one skilled 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.

[0083] Certain polymerases have high tolerance to modifications of certain portions of nucleotides, for example, modifications of the 5-position of pyrimidines and the 7-position of purines are well tolerated by some polymerases (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.

[0084] 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 that have substitutions compared to natural nucleosides, such as pyrimidines with 5-hydroxymethyl or 5-propargylamino substituents, or 7-deazapurines (with 7-hydroxymethyl or 7-propargylamino substituents), can be useful starting materials for preparing linker-nucleotides. An exemplary set of nucleosides with 5- and 7-hydroxymethyl substituents that can be useful for preparing linker nucleotides is shown below. TIFF2024545958000002.tif36165

[0085] An exemplary set of nucleosides having 5- and 7-deaza-7-propargylamino substituents that may be useful in preparing linker nucleotides is shown below. TIFF2024545958000003.tif43165

[0086] These nucleosides are also commercially available as deoxyribonucleoside polyphosphates.

[0087] In some embodiments, the method of preparation (eg, involving intermediate compounds) wherein the conjugate comprises a linker nucleotide.

[0088] In some embodiments, the linker-nucleotide comprises a nucleotide. 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. Any suitable nucleotide may be used. A nucleotide is understood to comprise a nucleobase (e.g., adenine, guanine, cytosine, thymine, or uracil), a sugar (e.g., ribose or deoxyribose), and a polyphosphate. A nucleoside is understood to comprise a nucleobase (e.g., adenine, guanine, cytosine, thymine, or uracil) and a sugar (e.g., ribose or deoxyribose).

[0089] In some embodiments, the linker-nucleotide comprises a nucleotide polyphosphate. In some embodiments, the linker-nucleotide comprises a modified nucleotide polyphosphate. It is understood that the polyphosphate portion of the nucleotide can be a monophosphate, diphosphate, triphosphate, tetraphosphate, heptaphosphate, or pentaphosphate. In some embodiments, the nucleotide polyphosphate comprises a nucleoside triphosphate or a modified nucleoside 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.

[0090] 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 a polynucleotide. In some embodiments, the O- or N-linked modification is removable by a photolytic process. In some embodiments, the photolytic 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 modification is 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 modification is removable by an enzymatic process. In some embodiments, the enzymatic process comprises removal by an alkyltransferase or a methyltransferase.

[0091] In some embodiments, O- or N-linked modification reduces or eliminates Watson-Crick base pairing in the polynucleotide that comprises modified nucleobase.In some embodiments, O- or N-linked modification reduces or eliminates secondary structure in the polynucleotide that comprises modified nucleobase.In some embodiments of the method, after removing O- or N-linked modification, modified nucleobase comprises natural nucleobase.In some embodiments, natural nucleobase is guanine, cytosine, adenine, thymine or uracil.

[0092] Linker polymerase binding The conjugates provided herein comprise a polymerase tethered to a nucleotide via a linker.

[0093] Any suitable linker for tethering nucleoside polyphosphate to polymerase is contemplated for use in the methods described herein. In some embodiments, the linker specifically binds to the cysteine ​​residue of the polymerase using sulfhydryl-specific binding chemistry. Exemplary sulfhydryl-specific binding chemistry includes, but is not limited to, ortho-pyridyl disulfide (OPSS), maleimide functional group, 3-arylpropiolonitrile functional group, allenamide functional group, haloacetyl functional group such as iodoacetyl or bromoacetyl, alkyl halide or perfluoroaryl group 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.).

[0094] 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 a bond to a genetically inserted unnatural amino acid, e.g., p-propargyloxyphenylalanine or p-azidophenylalanine, which can undergo an azide-alkyne Huisgen cycloaddition, although there are many suitable unnatural amino acids suitable for site-specific labeling, which can be found in the literature (e.g., Lang and Chin., Chemical reviews 114.9(2014):4764-4806.).

[0095] 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.

[0096] In some embodiments, peptide tag sequences may be enzymatically inserted into polymerases that can be 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).

[0097] In some embodiments, the linker is attached to a label domain fused to the polymerase. For example, SNAP tags, CLIP tags, Halo tags, and acyl carrier protein domains may be covalently labeled using linkers with corresponding reactive moieties (e.g., references 79-82 of Stephanopoulos & Francis Nat. Chem. Biol. 7 (2011) 876-884).

[0098] 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 cysteine ​​residues in the recognition sequence to formylglycine (i.e., producing an aldehyde). This aldehyde may then be specifically labeled, for example, with a hydrazide or aminooxy moiety of the linker.

[0099] In some embodiments, the linker may be attached to the polymerase via non-covalent attachment of a portion of the linker to a portion 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 may 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., using TCEP), while other attachment chemistries will result in permanent attachment.

[0100] In some embodiments, the polymerase is mutated to ensure specific binding of the tethered nucleotide to a specific position of the polymerase, as will be apparent to those skilled in the art. For example, in sulfhydryl-specific binding chemistries such as maleimide or ortho-pyridyl disulfide, accessible cysteine ​​residues in wild-type polymerases may be mutated to non-cysteine ​​residues to prevent labeling at those positions. In this "reactive cysteine-free" context, cysteine ​​residues may be introduced by mutation at the desired binding position. These mutations preferentially do not interfere with the activity of the polymerase.

[0101] 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., Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).

[0102] 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, the nucleic acid or polynucleotide is preferentially elongated (i.e., extended) 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.

[0103] 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.

[0104] 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.

[0105] In some embodiments, the disclosed method uses 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 variant thereof (e.g., a non-wild type TdT, e.g., a modified TdT). In some embodiments of the method, the template-independent polymerase is TdT or a variant thereof (i.e., a modified TdT). In some embodiments of the method, the TdT or a variant thereof comprises a sequence that shares 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 with 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, e.g., the sequence of SEQ ID NO:1). For example, in some embodiments, a given TdT may be truncated relative to the length of a particular TdT, such as that shown in SEQ ID NO: 1. In some embodiments, the TdT may be a circular permutation of SEQ ID NO: 1. In some embodiments, a TdT variant includes one or more amino acid substitutions, insertions, or deletions relative to and / or is a circular permutation of a reference TdT (e.g., wild-type TdT, modified TdT, etc.).

[0106] 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).

[0107] 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:2)); 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.

[0108] >Terminal deoxynucleotidyl transferase (TdT)

[0109] MGGRDIVDGSEFSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSALAFMRASSVLKSLPFPITSMKDTEGIPSLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATDEEQQLLHKVTDFWKQQGLLLYADILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMSPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA (SEQ ID NO: 1)

[0110] 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 (Juarez et al., Nucleic Acids Research 34.16(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).

[0111] In some embodiments, such as RNA synthesis applications, tethered ribonucleotides (e.g., ribonucleoside polyphosphates) may be used. In some such embodiments, an RNA-specific nucleotidyl transferase, such as E. coli poly(A) polymerase (IUBMB EC 2.7.7.19) or poly(U) polymerase, among others, may be used. The RNA nucleotidyl transferase may include 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 ribonucleotides (e.g., ribonucleoside triphosphates) may be used to induce a high effective concentration of ribonucleotides (e.g., ribonucleoside polyphosphates), thereby forcing the incorporation of rNTPs that may not be natural substrates for the nucleotidyl transferase.

[0112] Linker In some embodiments, the conjugates of the present disclosure include a linker. In some such embodiments, the linker includes at least an atom that connects the nucleotide to the polymerase. The linker can be attached to the base, sugar, or alpha phosphate of the nucleotide or modified nucleotide to the polymerase. In some embodiments, the polymerase and the nucleotide are linked with a linker. In some such embodiments, the polymerase and the nucleotide are covalently linked (via a linker), 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 (e.g., nucleoside polyphosphate) 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 includes a polyether or polyethylene glycol (PEG). In some embodiments, the linker includes one or more peptide bonds. In some embodiments, the linker includes 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 attached to the sugar or α-phosphate of the nucleotide. In some embodiments, the linker is long enough to allow the nucleotide (e.g., nucleoside polyphosphate) to access the active site of the polymerase to which it is tethered.As detailed herein, the polymerase of the conjugate is capable of catalyzing the addition of a nucleotide to which it is linked to the 3' end of a nucleic acid.

[0113] Linker cleavage As described herein, linker may be attached to various positions on nucleotide (e.g., of the conjugate of the present disclosure), and various cleavage strategies may be used. It should be understood that the cleavage strategy is determined by the type of linker that connects nucleotide or modified nucleotide and polymerase. Any suitable method for cleaving linker is contemplated in the methods described herein.

[0114] In some embodiments, the linker is cleaved to form a nucleotide that includes a chemical group from the retained portion of the linker (i.e., scar) after cleavage of the linker. For example, exemplary non-limiting chemical groups (i.e., scars) after cleavage of the linker are shown below. In some embodiments, for example, the chemical group is removed by a chemical, photolytic, or enzymatic process.

[0115] 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 may 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. TIFF2024545958000004.tif47165

[0116] In other embodiments, the linker may be cleaved by exposure to light. For example, a linker containing a (2-nitrobenzyl)oxymethyl group may be cleaved with 365 nm light to leave a hydroxymethyl scar, for example, as shown below for cytosine, although this strategy is applicable to any suitable nucleobase: TIFF2024545958000005.tif54165

[0117] In other embodiments, the linker may contain a 3-(((2-nitrobenzyl)oxy)carbonyl)aminopropynyl group that may be cleaved with 365 nm light to release a nucleobase bearing a propargylamino scar. This strategy is applicable to any suitable nucleobase. TIFF2024545958000006.tif48165

[0118] In other embodiments, the linker may comprise an acyloxymethyl group that may be cleaved with a suitable esterase to release a nucleobase bearing a hydroxymethyl scar, for example as shown below for cytosine, although this strategy is applicable to any suitable nucleobase. TIFF2024545958000007.tif45165

[0119] In such embodiments, the linker may contain additional atoms (included in R' above) adjacent to the ester that increase the activity of the esterase towards the ester bond.

[0120] In other embodiments, the linker may comprise an N-acyl-aminopropynyl group that may be cleaved with a peptidase to release a nucleobase bearing a propargylamino scar, for example as shown below for 5-propargylaminocytosine, although this strategy is applicable to any suitable nucleobase. TIFF2024545958000008.tif39165

[0121] 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.

[0122] 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 in accordance with the compositions and methods described 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.

[0123] In the claims, articles such as "a", "an" and "the" may 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 members of a group is considered satisfied if one, more than one, or all of the members of the group are present, used, or otherwise relevant in 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, used, or otherwise relevant in a given product or process. The present disclosure includes embodiments in which two or more, or all of the members of a group are present, used, or otherwise relevant in a given product or process.

[0124] 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.

[0125] Where ranges are given, the endpoints are included. Additionally, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges are understood to contemplate any particular value or subrange within the range recited in different embodiments of the present disclosure to one tenth of the unit of the lower limit of the range, unless otherwise clearly indicated by the context.

[0126] 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. Reference to a value or parameter herein with the term "about" includes (and describes) the incorporation of the value or parameter itself. For example, a description of "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%.

[0127] All sources, e.g., references, publications, databases, database entries, and techniques cited herein, are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a conflict between the statements in the source and this application, the statements in this application take precedence.

[0128] The section and table headings are not intended to be limiting. EXAMPLES

[0129] Below are examples of specific embodiments for carrying out 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 numbers used (e.g., amounts, temperatures, etc.), but it is understood that some experimental error and deviation are allowed for.

[0130] The practice of the embodiments of the present disclosure may employ conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology within the skill of the art, unless otherwise indicated. Such techniques are fully explained in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A Lehninger, 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).

[0131] Example 1: Effect of cobalt concentration on the rate of nucleotide incorporation by DNA polymerase-nucleotide conjugates Reducing the concentration of divalent metals increases the rate of nucleotide incorporation In this example, nucleotide incorporation rates were analyzed for TdT enzyme conjugated to G nucleotides using standard magnesium acetate and cobalt acetate concentrations and compared to low cobalt acetate concentrations (see Figures 2A-2E).

[0132] Enzymatic polynucleotide synthesis was carried out in a buffer containing 20 mM Tris acetate, 50 mM potassium acetate, 50 μM (Figures 2B and 2D) or 0.5 mM cobalt acetate (Figures 2A and 2C), 0 mM (Figures 2B and 2D) or 10 mM (Figures 2A and 2C) magnesium acetate, 50 nM DNA oligo substrate, 1 μM TdT enzyme-nucleotide conjugate, pH 7.9. Polymerase-nucleotide conjugate kinetic activity was measured after quenching the reaction with EDTA and DNA fragment analysis by capillary electrophoresis.

[0133] Figures 2A-2D show the abundance of DNA oligo substrates (Substrate) and products (Product) containing incorporated nucleotides at various times in polynucleotide extension reactions containing TdT enzyme conjugated to G nucleotides in reaction buffers containing standard concentrations of: (i) magnesium acetate (10 mM MgOAc) and cobalt acetate (0.5 mM CoOAc) (see Figures 2A and 2C), or (ii) low cobalt acetate (0.05 mM CoOAc) in the absence of magnesium acetate (see Figures 2B and 2D). Surprisingly, in extension reactions without magnesium acetate and in low cobalt acetate conditions, the nucleotide was fully incorporated into the polynucleotide after 30 seconds. In contrast, extension reactions containing standard magnesium acetate and cobalt acetate concentrations were still incomplete after 67 seconds.

[0134] The results of the synthesis / extension reactions shown in Figures 2A and 2B were quantified (across all time points) and reaction rates were determined for low and high cobalt buffer conditions (Figure 2E). obs The calculated reaction rate for (k = 0.4625) obs ) was obtained using high cobalt acetate conditions (10 mM MgCl2, 0.5 mM CoOAc, k obs = 0.04664), which was several orders of magnitude better than the calculated reaction rate.

[0135] These results indicate that, compared to standard concentrations of cobalt acetate and magnesium acetate, low concentrations of cobalt acetate in the absence of magnesium promote faster nucleotide incorporation rates for polynucleotide extension reactions using DNA polymerase-nucleotide conjugates.

[0136] Reducing divalent metal concentrations increases the rate of nucleotide incorporation In this example, nucleotide incorporation rates for TdT enzyme conjugated to G nucleotides were analyzed using various cobalt acetate or zinc acetate concentrations in the absence of magnesium acetate.

[0137] Enzymatic polynucleotide synthesis was carried out in a buffer containing 20 mM Tris acetate, 50 mM potassium acetate, 50 μM-2.5 mM cobalt acetate or 50 μM-2.5 mM zinc acetate, 50 nM DNA oligo substrate, 1 μM TdT enzyme-nucleotide conjugate, pH 7.9. Polymerase-nucleotide conjugate kinetic activity was measured after quenching the reaction with EDTA and DNA fragment analysis by capillary electrophoresis.

[0138] Analysis by capillary electrophoresis was performed using cobalt acetate or zinc acetate at concentrations ranging from 50 μM to 2.5 mM cobalt acetate (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc; see Figures 3A-3L and Table 1) or 50 μM to 2.5 mM zinc acetate (0.05 mM ZnOAc, 0.125 mM Figure 4 shows the abundance of DNA oligo substrates (Substrate) and products containing incorporated nucleotides (Product) at various time points in polynucleotide extension reactions containing TdT enzyme conjugated to G nucleotides in reaction buffers containing various concentrations of ZnOAc (0.01 mM ZnOAc, 0.25 mM ZnOAc, 0.75 mM ZnOAc, 1.25 mM ZnOAc, and 2.5 mM ZnOAc; see Figures 4A-4L). The results show that the rate of nucleotide incorporation using TdT-nucleotide conjugates increases as a function of decreasing divalent metal ion concentration. This was observed for both cobalt acetate and zinc acetate, as shown by the increased abundance of product observed at faster time points at lower concentrations of divalent metal ions. In extension reactions containing standard cobalt acetate concentration (2.5 mM), the DNA oligo substrate is not fully converted after 139 seconds. Under conditions using lower cobalt acetate conditions, the extension reaction is completed in less than 139 seconds (see Table 1).

[0139] The results of the synthesis reactions shown in Figures 3A-3L were quantified and the reaction rates were determined for each of the buffer conditions (Figure 3M). The calculated reaction rates (k obs ) increased with decreasing CoOAc concentration.

[0140] The results in Figures 4A-4L were quantified to determine the reaction rate for each of the buffer conditions (Figure 4M). Conversion under these conditions occurred at an initial fast rate and a slower rate (k slow ) is biphasic. The amplitude of the initial fast velocity is shown in the table in FIG. 4M. As the concentration of ZnOAc decreased, both the initial fast and slow velocities increased.

[0141] Table 1. Nucleotide incorporation rates at various CoOAc concentrations. TIFF2024545958000009.tif53162

[0142] Decreasing the concentration of divalent metals reduces the rate of free nucleotide incorporation In this example, the nucleotide incorporation rates for TdT enzyme-catalyzed incorporation of free G nucleotides into DNA oligos were analyzed using various cobalt acetate concentrations in the absence of magnesium acetate (FIGS. 5A-5L).

[0143] Enzymatic polynucleotide synthesis was carried out in a buffer containing 20 mM Tris acetate, 50 mM potassium acetate, 50 μM-2.5 mM cobalt acetate (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc; see Figures 5A-5L), 50 nM DNA oligo substrates, 1 μM TdT enzyme-nucleotide conjugate, free nucleotide substrate (ddGTP, 0.5 mM), pH 7.9. Polymerase-nucleotide conjugate kinetic activity was measured after quenching the reaction with EDTA and DNA fragmentation analysis by capillary electrophoresis.

[0144] Figures 5A-5L show the abundance of DNA oligo substrates (Substrate) and products containing incorporated nucleotides (Product) at various times in polynucleotide extension reactions involving TdT enzyme and free ddGTP in reaction buffers containing 50 μM to 2.5 mM cobalt acetate (0.05 mM CoOAc, 0.125 mM CoOAc, 0.25 mM CoOAc, 0.75 mM CoOAc, 1.25 mM CoOAc, and 2.5 mM CoOAc). When the results of Figures 5A-5L are quantified and plotted, the results show a slower reaction rate for the lowest concentration of CoOAc (Figure 5M).

[0145] Unlike nucleotide incorporation mediated by TdT-nucleotide conjugates, which artificially increased the effective nucleotide concentration for the polymerase, reducing the cobalt acetate concentration in the presence of free TdT and free nucleotide dramatically reduced the rate of nucleotide incorporation (Table 2).

[0146] Table 2. Nucleotide incorporation rates at various CoOAc concentrations. TIFF2024545958000010.tif53164

[0147] Summary of results Taken together, the results surprisingly show that low divalent metal ion concentrations improve the rate of nucleotide incorporation in the extension of polynucleotides by TdT-nucleotide conjugates, including by increasing the rate of nucleotide incorporation into polynucleotides, compared to standard divalent metal ion concentrations. That is, in contrast to previously recorded results showing that the rate of nucleotide incorporation increases at standard concentrations of divalent metal ions (about 2.5 mM for most divalent metal ions), the present results provide an unexpected finding that low divalent metal ion concentrations increase the rate of nucleotide incorporation in extension reactions. The standard concentrations are supported by findings in the literature (Kato et al. J Biol. Chem. 242 (11). 1967) and are also suggested by protocols included with commercially available polymerases and corresponding reaction buffers (see, for example, protocols and buffers at ThermoFisher https: / / www.thermofisher.com / order / catalog / product / 16314015). In contrast, the results herein show that lower than standard concentrations of divalent metal ions increase nucleotide incorporation using polymerase-nucleotide conjugates.

[0148] The examples shown here were performed with dGTP. Similarly, low concentrations of divalent metal ions improved the extension reaction for other nucleobases (data not shown). Furthermore, the effect of low cobalt and zinc concentrations with TdT-nucleotide conjugates is consistent with other polymerase-nucleotide conjugates and other suitable divalent cations used in nucleotide incorporation reactions (e.g., Mg 2+ , Ca 2+ , Sr 2+ , B.A. 2+ , Mn 2+ , Co 2+ , Fe 2+ , Ni 2+ , Cu 2+ , Zn 2+ )

[0149] Other embodiments The language that has been used is one of description rather than limitation, and it is 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.

[0150] While the present disclosure has been described at some length and with some specificity with respect to certain embodiments described, 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 order to provide the broadest possible interpretation of such claims in view of the prior art and thus effectively encompass the intended scope of the present disclosure.

[0151] 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, 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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