A novel nucleotide complex that can improve DNA yield

Abstract

The present invention relates to novel nucleotide complexes that enhance the enzymatic production of DNA. The nucleotide complexes contain divalent cations in a ratio of 0.2-1.5 divalent cations per nucleotide, with minimal monovalent ions (other than hydrogen or hydronium ions from any solvent). The nucleotide complexes have desirable properties for the synthesis of deoxyribonucleic acid (DNA), preferably on a large or industrial scale, and in particular for the cell-free enzymatic synthesis of DNA. Additionally, the present invention includes improved processes for preparing said nucleotide complexes. Further complexes are disclosed that have unique properties in accelerating DNA synthesis at higher concentrations, and these complexes also contain zwitterionic molecules.

Description

[Technical field]

[0001] The present invention relates to novel nucleotide complexes that enhance the enzymatic production of DNA. The nucleotide complexes contain divalent cations present in a ratio of 0.2-1.5 divalent cations per nucleotide, with minimal monovalent ions (other than hydrogen or hydronium ions from any solvent). The nucleotide complexes have desirable properties for the synthesis of deoxyribonucleic acid (DNA), particularly cell-free enzymatic synthesis of DNA, preferably on a large or industrial scale, with improved yield and / or improved efficiency. Furthermore, the present invention includes an improved process for preparing said nucleotide complexes. The process results in a unique nucleotide complex, where the complexes counter-ionize only divalent cations in a ratio of 0.2-1.5 divalent cations per nucleotide. Substantially all of the moieties that provide the monovalent cations from the starting material are removed. Further complexes are disclosed that have unique properties in accelerating DNA synthesis at higher concentrations, and these complexes also contain zwitterionic molecules. [Background technology]

[0002] Amplification of deoxyribonucleic acid (DNA) can be carried out by using cell-based methods, for example by growing bacterial cultures that grow DNA in fermenters. Cell-free enzymatic methods for amplifying DNA from a starting template have also been described, including the polymerase chain reaction and strand displacement reaction.

[0003] In the past, DNA amplification on a test scale was performed using equipment based on microtiter plates and robotically controlled pipettes that add reaction components as needed. Such equipment and methods are suitable for producing small quantities of DNA for test purposes, but do not yield sufficient quantities for other purposes. Large-scale amplification and production of specific nucleic acids and proteins is mostly performed by cell-based methods. Such methods are generally effective at producing very large amounts of product, but are costly to construct. Furthermore, for clinical and therapeutic purposes, it is preferable to synthesize DNA in a cell-free environment. For amplification of plasmids using methods conventional in the art, such as fermentation, a commercial scale operation may be capable of producing 2.6 g / l. This is considered "industrial scale" by those skilled in the art.

[0004] To date, biocatalysts such as polymerases have not been routinely utilized for industrial-scale production of DNA products in vitro, and reactions are largely limited to microliter volumes. Scaling up processes that use enzymatic synthesis of DNA has proven problematic, particularly as yields of DNA products have been disappointing.

[0005] The applicants have been working on whether it is possible to scale up using commercially available nucleotides. As described in WO2016 / 034849, which is incorporated herein by reference, a new method has been developed that involves adding fresh nucleotides to the reaction mixture when nucleotides are exhausted or the product concentration reaches a threshold value. However, it has been demonstrated that even higher yields can be achieved, and the inventors have developed a nucleotide complex that is associated with a mixture of divalent and monovalent cations (PCT / GB2021 / 050366). Included in this application is a further improvement of this nucleotide complex.

[0006] Enzymatic DNA synthesis generally requires the use of a polymerase or polymerase-like enzyme to catalyze the addition of nucleotides to a nascent nucleic acid chain. Generally, a template DNA is required that is amplified in the reaction. However, it is also possible to perform template-free DNA synthesis, where incorporation occurs de novo.

[0007] It is important to note that due to the highly charged nature of nucleic acids, they are always surrounded by counterions that neutralize most of the charges, which relieves electrostatic repulsion between sections of the sequence, so that nucleic acids can be condensed into neat, compact structures in cells. Nucleotides, the building blocks of nucleic acids, are also ionic species and require the presence of positive counterions to maintain electrical neutrality. Thus, most, but not all, nucleotides are provided as salts with positive counterions. In the absence of positive counterions from salts, nucleotides appear as free acid forms, where electrical neutrality is maintained by hydrogen ions. Since nucleotides have four negative charges, salts are typically prepared with two divalent cations or four monovalent cations. It will be clear to those skilled in the art that nucleotides (salts or acids) can dissociate into anionic and cationic components in solution as soon as they are dispersed in water or other solvents.

[0008] Generally, nucleotides are provided as either lithium or sodium salts for DNA synthesis, amplification, or sequencing. Lithium is generally preferred because lithium salts provide greater solubility than sodium salts, stability against repeated freezing and thawing cycles, remain sterile due to lithium's bacteriostatic activity against various microorganisms, and provide higher reliability and longer shelf life. The use of these salts is so routine that it is believed that the counter ions present with the nucleotides are not an issue for those skilled in the art. In fact, all nucleotides used in the examples of WO2016 / 034849 are lithium salts of nucleotides, because lithium salts of nucleotides are sold as a good choice for those skilled in the art. Nucleotides provided as salts of only divalent cations, such as magnesium ions, are highly desirable because divalent cations are required as cofactors during enzymatic DNA synthesis. Unfortunately, nucleotides provided as magnesium salts are highly insoluble, which limits the use of such nucleotides. Instead, magnesium is typically provided to the reaction separately as a chloride salt, in conjunction with nucleotides that are counterion-bound to monovalent cationic species.

[0009] The inventors have previously found that the species of cationic species present as counterions in nucleotide salts is important to the yield, efficiency, and accuracy of high-yield enzymatic DNA synthesis reactions, as detailed in WO2020 / 035698 and WO2021 / 161051 (PCT / GB2021 / 050366), which are incorporated herein by reference. Summary of the Invention

[0010] The inventors have developed a new family of novel nucleotide complexes that can be powdered or present in solution. These nucleotide complexes share an important feature, at least one divalent cation is present in a ratio of 0.2-1.5 divalent cations per nucleotide. Very simply, the novel nucleotide complexes contain only divalent cations present in a ratio of 0.2-1.5 divalent cations per nucleotide, and no other cations are present in counterion association with the complex. Those skilled in the art will understand that this definition of cation does not apply to ions potentially present in any solvent (e.g., hydrogen and hydronium ions present in water). To this simplest nucleotide complex, further entities such as zwitterionic molecules may be added. Nucleotide complexes containing a mixture of divalent cations and zwitterions have been demonstrated by the inventors to have particular utility in producing large yields of DNA very quickly.

[0011] The inventors have now developed a means to increase even further the maximum tolerable starting nucleotide concentration for a DNA synthesis reaction by using a process that removes polyatomic moieties from monovalent cations from nucleotide complexes that are effectively counterionized with a mixture of divalent and monovalent cations, or by starting the process by removing monovalent entities from nucleotide complexes that are associated with only monovalent cations, and then combining nucleotide complexes that are associated with only divalent cations. The starting material for the process can be a mixed counterion nucleotide complex, as described in WO2021 / 161051 (PCT / GB2021 / 050366), in which the monovalent cation is effectively polyatomic and the polyatomic moiety is volatile under suitable conditions, such as vacuum.

[0012] The starting material may also be a single counterion nucleotide complex, such as that described in WO2020 / 035698, which contains, for example, four ammonium ions. When using this starting material, the divalent counterion bound nucleotide complex may be added after the monovalent entity is removed. The monovalent cation is polyatomic, and the polyatomic moiety may be volatile under suitable conditions, such as vacuum.

[0013] Either method may further allow for the addition of a zwitterionic molecule before, during, or after removal of the multiatomic moiety from the monovalent cation. Using the process of the present invention, the inventors have found that it is possible to further remove "monovalent cations" from the nucleotide complex by means of resuspending the dried nucleotide complex in a solvent without the use of a buffer. Most notably, this was completely unexpected, since it is already known that nucleotide salts with divalent cations such as magnesium are poorly soluble. The net effect is to reduce the amount of monovalent cations present even further than described in WO2021 / 161051 (PCT / GB2021 / 050366). This is important, since it is hypothesized by the inventors that monovalent cations are inhibitory to DNA synthesis at large concentrations. This inhibition by monovalent cations can occur regardless of the source of the cation (nucleotide, buffer component, etc.). Furthermore, by also providing the nucleotide with a divalent cation as a counterion, particularly in the case of magnesium, manganese, or calcium, this can also effectively provide a cofactor required by the synthetic enzyme. Thus, there is no need to provide further or additional divalent cations. Because magnesium or manganese are typically added to reactions as salts (containing two negative charges on one or more anions), including them instead as counterions to nucleotides essentially reduces the amount of anion present. Such a reduction can be beneficial to downstream DNA processing enzymes, as it may allow for fewer steps at the end of the synthesis reaction to prepare the DNA for further processing.

[0014] Furthermore, the inventors have surprisingly shown in the examples that the addition of additional molecules, such as zwitterions, to the novel nucleotide complexes of the present invention leads to additional new nucleotide complexes with altered properties with respect to DNA synthesis. These complexes are believed to be particularly beneficial for accelerating the rate of DNA synthesis, especially at starting concentrations above 100 mM.

[0015] The data presented in the examples demonstrate that the novel nucleotide complexes are superior in terms of yield and efficiency to previously prepared nucleotide complexes, especially at much higher concentrations of nucleotide entities (especially above 60 mM, and even above 100 mM). Of note, DNA is used as a template to produce large amounts of RNA, especially mRNA, and thus producing DNA on a commercial scale is important for mass production of, for example, RNA vaccines. Thus, the need for clean and efficient DNA production on an industrial scale is currently growing exponentially. The DNA produced using the present invention can be used as a template to produce SARS-CoV-2 mRNA vaccines, etc. The DNA can be used in the preparation of viral particles, such as lentiviruses and adeno-associated viruses.

[0016] 〔overview〕 The present invention relates to novel nucleotide complexes, all of which require the presence of divalent cations, present in a ratio of 0.2 to 1.5 divalent cations per nucleotide, and no additional cations are actually present or provided for "counterion bonding" with the nucleotide.

[0017] These divalent cations are preferably divalent metal cations, optionally one or more of magnesium, manganese, or calcium. The divalent cations may be a single ion (e.g., magnesium) or a mixture of ions (e.g., magnesium and calcium).

[0018] In the simplest nucleotide complexes, the novel nucleotide complexes contain divalent cations present in a ratio of 0.2 to 1.5 divalent cations per nucleotide, with substantially no other cations present for counterion binding to the complex.

[0019] One of ordinary skill in the art will appreciate that this definition of cation does not apply to ions that occur naturally in any solvent (eg, hydrogen and hydronium ions present in water).

[0020] To this simplest nucleotide complex, further entities may be added, such as zwitterionic molecules. Nucleotide complexes containing a mixture of divalent cations and zwitterions have been demonstrated by the present inventors to have particular utility for making large amounts of DNA very rapidly.

[0021] The present invention relates to nucleotide complexes comprising nucleotides associated with divalent cations present in a ratio of 0.2 to 1.5 divalent cations per nucleotide and zwitterionic molecules present in a ratio of 0 to 4 zwitterionic molecules per nucleotide. Thus, the presence of zwitterionic molecules is optional.

[0022] The present invention relates to a nucleotide complex comprising a nucleotide associated with a divalent cation present in a ratio of 0.2 to 1.5 divalent cations per nucleotide, and optionally further associated with a zwitterionic molecule present in an amount of 4 or less zwitterionic molecules per nucleotide.

[0023] The present invention relates to at least one process for the preparation of novel nucleotide complexes, which is a process in which the previously described nucleotide complexes (e.g., those in WO2021 / 161051) are dried to effectively further reduce the volatile base of the monovalent cation contained in the starting material. Such a process is carried out when the monovalent cation selected for the starting material is polyatomic and becomes volatile under suitable conditions, such as vacuum. The nucleotide complexes after the drying or evaporation step may be partially or completely dried, for example having most or all of the solvent removed. It will therefore be understood that the starting nucleotide complexes may be in solution. The starting material for this process may simply be a mixture of nucleotide complexes associated with divalent cations and nucleotide complexes associated with polyatomic monovalent cations.

[0024] The invention allows for mixing the nucleotide associated with the divalent cation and the nucleotide associated with the multi-atomic monovalent cation at any suitable time, which may be before, during or after the multi-atomic moiety is removed. Removal of the multi-atomic moiety is carried out by making it volatile under suitable conditions.

[0025] The process of the present invention can be considered "drying" or "evaporating", especially when the starting nucleotide complex or the second nucleotide complex are in solution. The drying process and subsequent resuspension can produce enhanced DNA compared to current methods, i.e., can carry out enzymatic DNA synthesis in an environment with increased or higher yield, more efficient process, and fewer additional components than is considered possible under current methods.This significantly increases productivity while simultaneously reducing the cost of synthesizing DNA, especially on a large scale.In particular, it is shown that such nucleotide complexes are suitable for use at particularly high starting concentrations of nucleotide, for example 60 mM or even 100 mM or more.

[0026] To achieve high yields on an industrial scale, it is necessary to utilize high concentrations of the "building blocks" of DNA, i.e., nucleotides (especially dNTPs). In general, the inventors have found that merely changing the parameters of the reaction conditions does not allow one to significantly increase the yield from an enzymatic reaction to make it suitable for industrial purposes.

[0027] Considering that nucleotides are provided to the enzymatic reaction as salts, increasing the amount of nucleotides results in a significant increase in the ionic strength of the reaction mixture. Ionic strength is a function of the concentration of all ions present. An important consideration is that the enzymes that catalyze DNA synthesis reactions are proteins, and an increase in ionic strength can result in protein unfolding and thus inactivation of enzyme activity.

[0028] Furthermore, it can be considered that the presence of salts can also affect the pH of the reaction mixture. Depending on the acid-base properties of the component ions, salts can dissolve in water to result in a neutral solution (strong acid / strong base), a basic solution (weak acid / strong base) or an acidic solution (strong acid / weak base). Thus, by increasing the concentration of nucleotide salts or any other salts (magnesium chloride as an example) to the reaction mixture, this can also affect the pH and further limit the pH stabilization performance of any buffers present. Thus, the addition of higher concentrations of nucleotide salts can, for example, result in suboptimal pH control, which affects enzymatic DNA synthesis, in particular in terms of reducing DNA yield or adversely affecting the accuracy of DNA synthesis. Thus, there are many considerations regarding the "scaling up" of enzymatic DNA production, and the inventors have devised a means to increase the yield without adversely affecting the DNA synthesis reaction.

[0029] Without wishing to be bound by theory, the inventors hypothesize that the phosphate group of the nucleotide complex may provide some buffering capacity in solution.Furthermore, as the DNA synthesis reaction proceeds, the reaction may self-buffer due to the release of phosphate from the nucleotide complex.Measurements performed show that the pH decreases after the nucleotide complex is prepared, but not as much as expected, considering the possible protonation of nucleotides when volatile parts are removed.

[0030] Therefore, the present invention relates to a method for further improving the nucleotide complexes described previously by subjecting the nucleotide complexes described previously to a drying process, optionally to a point where no solvent remains.The starting material for the preparation of the nucleotide complexes of the present invention may be a mixed counterion nucleotide complex, such as those described in WO2021 / 161051 (PCT / GB2021 / 050366). Such may include the presence of divalent cations and polyatomic monovalent cations that become volatile under suitable conditions, such as vacuum.The starting material may be a single counterion nucleotide complex or a salt described in WO2020 / 035698.

[0031] The present invention relates to a process for the preparation of a nucleotide complex, comprising the step of applying heat and / or vacuum to a starting nucleotide complex, said starting nucleotide complex comprising a nucleotide associated with a multi-atom monovalent cation, wherein the multi-atom moiety is volatile under heat and / or vacuum.

[0032] The present invention relates to a process for the preparation of a nucleotide complex, comprising the step of evaporating a multiatomic moiety present in a starting nucleotide complex, said starting nucleotide complex comprising a nucleotide in association with a multiatomic monovalent cation.

[0033] The starting nucleotide complex according to any aspect of the invention may also include a divalent cation. Alternatively, a nucleotide complex comprising a nucleotide associated with a divalent cation may be added to the starting material before or after the process is performed.

[0034] Alternatively, the polyatomic moiety may be described as "volatisable." Alternatively stated, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: A process for the preparation of a nucleotide conjugate comprising the steps of: (i) providing a starting nucleotide complex comprising a nucleotide associated with a multi-atom monovalent cation, the multi-atom portion of the cation being volatilizable; (ii) applying any one or more of heat, vacuum, and / or a change in pH to the starting nucleotide complex to allow a proportion of the multi-atom moiety to evaporate; (iii) before, during or after step (ii), mixing the starting nucleotide complex with a second nucleotide complex, the second nucleotide complex being in association with a divalent cation; The present invention provides a method for producing the same.

[0035] Polyatomic moieties are volatilizable. This means that they can be volatile under suitable conditions, but generally are not volatile under normal / standard conditions. Suitable conditions can be the application of heat, vacuum, and / or changing the pH of the nucleotide complex. The pH can be changed to increase or decrease the pH, preferably to increase the pH.

[0036] The method comprises: (iv) mixing the starting nucleotide mixture with a zwitterionic molecule before, during or after step (ii); It may further include.

[0037] The starting nucleotide complex can be a nucleotide associated with a polyatomic monovalent cation present in a ratio of between 0.2 and 4 polyatomic monovalent cations per nucleotide, optionally between 0.5 and 3, between 1 and 2, or any ratio in between. The cations can be a single species of polyatomic monovalent cation or can be a mixture of different species.

[0038] The second nucleotide complex can be a nucleotide associated with divalent cations present in a ratio of between 0.2 and 4 divalent cations per nucleotide, optionally between 0.5 and 3, between 1 and 2, or any ratio in between. The cations can be a single species of divalent cation or can be a mixture of different species.

[0039] The present invention relates to a process for the preparation of a nucleotide complex by evaporating a polyatomic moiety present in a starting nucleotide complex, said starting nucleotide complex being associated with between 0.2 and 2 divalent cations and between 0.2 and 2.5 polyatomic monovalent ions per nucleotide.

[0040] The novel nucleotide complexes may be used as prepared according to any method of the present invention. Alternatively, the novel nucleotide complexes may be provided with at least one zwitterionic molecule before or after the process of evaporating the polyatomic moiety. Furthermore, the present invention relates to enzymatic DNA synthesis using a nucleotidyl transferase, such as a polymerase enzyme or other DNA synthesizing enzyme, either of which can be optionally engineered to impart specific properties, and any of the nucleotide complexes prepared according to the present invention.

[0041] Depending on the nucleotidyl transferase used, the present invention can relate to DNA synthesis from a nucleic acid template or to template-free de novo DNA synthesis. The invention may relate to isothermal methods of synthesizing DNA that do not require temperature cycling by heating and cooling during amplification, although the use of heat may initially denature the template, if present.The invention preferably relates to the use of polymerase enzymes that are capable of replicating a nucleic acid template via strand displacement replication, independent of or with the aid of other enzymes.

[0042] The process of the present invention involves the use of starting material (starting nucleotide complex) and optionally a second nucleotide complex, which are nucleotides in the form of complex with associated ions, sometimes referred to herein as counterions. Nucleotide complexes are generally present in solution, and thus associated counterions may or may not be dispersed in solution. Depending on the nature of their preparation, counterions may be effectively "shared" between nucleotides, resulting in a ratio of counterions to nucleotides that is not an integer. In a solution containing nucleotides, the ionic species are divalent counterions and polyatomic monovalent counterions, resulting in partial or complete charge balance of each nucleotide being provided by a mixture of monovalent and divalent cations. It may be a condition that the complexes can be electrically neutral or have a net negative charge. The starting nucleotide complexes can be provided in solution or dispersed in solution by adding solid nucleotide complexes to the solution.

[0043] Alternatively, the starting complex may be a nucleotide associated with only monovalent cations that is mixed with a nucleotide associated with only divalent cations during the process. Due to the nature of their preparation, the counterions may be effectively "shared" between the nucleotides, resulting in a non-integer ratio of counterions to nucleotides.

[0044] The starting nucleotide complex comprises a nucleotide (also described herein as a nucleotide ion or ionic species) present in solution and associated with at least two different positive counterions (cations). One of these counterions is preferably a polyatomic monovalent cation, i.e., has a single positive charge due to the loss of one electron. The polyatomic portion of the monovalent cation should be capable of becoming volatile under suitable conditions, such as under vacuum. It is volatizable or volatilizable. It will be understood that at normal temperature and pressure (NTP is 20°C and 1 atm), the polyatomic portion of the polyatomic monovalent cation is preferably substantially non-volatile, and suitable conditions are applied to make said portion volatile. One of these counterions in the starting nucleotide complex is preferably a divalent cation, i.e., has a double positive charge due to the loss of two electrons. For the avoidance of doubt, these divalent counterions are not lost, removed, or evaporated during the process of the present invention. Alternatively, the divalent counterion may be provided during the process by addition in the form of a nucleotide complex in association with a divalent cation.

[0045] The starting nucleotides can be provided in complexes with provided mixed counterions that are polyatomic monovalent and divalent cations.The starting nucleotides can be provided in complexes with provided counterions that are polyatomic monovalent cations.

[0046] It will be understood that polyatomic monovalent cations may be capable of becoming volatile under suitable conditions, such as vacuum, for processing using the present invention. Thus, simple metal monovalent cations such as lithium, sodium, and potassium are not suitable for use as counterions in the starting material, since they are neither polyatomic nor capable of becoming volatile under suitable conditions, such as vacuum. Due to contaminants in the original manufacturing process, a very small proportion of monovalent counterions present in the nucleotide complex may be such metal ions.

[0047] Thus, the starting nucleotide complex may essentially consist of nucleotides, associated monovalent polyatomic cations, and optionally divalent cations. Small amounts of monovalent metal ion contaminants may be present, but at concentrations that are not critical in the present invention. The insignificant amounts may be less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the concentration of the nucleotide complex. In some embodiments, the starting nucleotide complex is in solution, and additional entities may be added to aid in evaporating or volatising the polyatomic moiety, or to provide additional stability to the new nucleotide complex formed. Such entities generally do not contain metal ions, but are instead polyatomic, preferably zwitterionic molecules. Such entities are preferably not volatilizable under the same conditions as the polyatomic moiety.

[0048] It will be understood that since nucleotides have four negative charges, it is customary to provide four positive charges, generally by four monovalent cations, to maintain electrical neutrality, and most commercial nucleotide salts are obtained on this basis. When the nucleotide complex is present in solution as a starting material, less than four positive charges may be provided by polyatomic monovalent and / or divalent cations used as counterions. Without wishing to be bound by theory, the inventors assume that the remaining charges, if required, may be provided by entities such as hydronium ions to reach electrical neutrality. When additional entities such as zwitterions are present, they are believed by the inventors to help stabilize the complex. When the nucleotide complex is subjected to the process of the present invention, a certain percentage or substantially all of the polyatomic moieties are removed or evaporated. A certain percentage may be 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% removed or evaporated (when compared to the starting nucleotide complex). Preferably 100% of the moiety is removed or evaporated. Preferably less than 0.2, optionally less than 0.1 polyatomic monovalent cations per nucleotide remain, preferably no polyatomic monovalent cations remain.

[0049] The polyatomic moieties of polyatomic monovalent cations are volatilizable and can become volatile under any suitable conditions. The polyatomic moieties are preferably not volatile under normal temperature and pressure. The polyatomic moieties are described herein as becoming volatile. Alternative means of describing such moieties are evaporable, vaporizable, vaporizable, or volatilizable. Volatility is indicated by the vapor pressure of the substance. A substance with a higher vapor pressure can vaporize more easily under suitable conditions than a substance with a lower vapor pressure. At a given temperature and pressure, a substance with a high vapor pressure is more likely to exist as a vapor, and a substance with a low vapor pressure is more likely to be a liquid or solid. Suitable conditions are selected to vaporize the substance.

[0050] Suitable conditions for rendering a moiety volatile are temperature (e.g., application of heat) or pressure (e.g., application of a vacuum). Some polyatomic entities can be rendered volatile by a change in pH, e.g., by increasing the pH (e.g., by including a basic amino acid). The most suitable conditions for rendering a polyatomic moiety volatile are heat and / or vacuum.

[0051] The polyatomic monovalent cations can be volatile under vacuum. The temperature at which the cations become volatile under vacuum can be any suitable temperature, and thus the complex in solution can be at room temperature (approximately 20°C), cooled or heated. As used in the examples, heat can be applied to help volatilize the polyatomic monovalent cations. Heat can be applied at a range of 20-80°C, 25-75°C, 30-70°C, 35-65°C, or any suitable temperature therebetween, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80°C.

[0052] A vacuum can create a region with a gas pressure below atmospheric pressure. Therefore, the gas pressure can be reduced by 10 5 Pascal(Pa)~10 -10The vacuum can be any suitable pressure in the range of less than 10 Pa. Ideally, the vacuum is 5 Pascals (Pa) can be created at pressures between 100 Pa. Standard atmospheric pressure is 101,325 Pa. The vacuum can be created using any suitable technique, including the use of a vacuum pump, or by any other method of reducing pressure, such as the use of a liquid flowing at a high velocity.

[0053] The polyatomic monovalent cations can become volatile upon exposure to heat, which can be applied at temperatures ranging from 40 to 100°C, 45 to 95°C, 50 to 90°C, 55 to 85°C, or any suitable temperature therebetween, for example, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C.

[0054] A monovalent ion as described herein is an ion that is polyatomic and therefore consists of two or more atoms. One of the atoms may be nitrogen, e.g., a protonated nitrogen atom. An exemplary ion is the ammonium ion (NH + ).

[0055] A polyatomic monovalent cation can be described as the conjugate acid of a base, where the base is the volatile moiety. For example, for ammonium under vacuum:

[0056]

number

[0057] In this example, the ammonium ion is the conjugate acid of ammonia (both indicated with an *). Thus, the polyatomic monovalent cation can be described as the conjugate acid of the base, where the base is the volatile part. In general, the base is the species that is formed after the conjugate acid donates a proton. Alternatively, the monovalent cation can be described as a Bronsted-Lowry acid, where the conjugate base is volatile. It is assumed that the proton is donated to the nucleotide entity in the process of the present invention. Thus, the part that is evaporated or removed in the process is the base, referred to herein as the "polyatomic part".

[0058] Exemplary conjugate bases and acids include ethylamine base and ethylammonium conjugate acid, and methylamine base and methylammonium conjugate acid, both of which are derivatives of ammonium and lose a proton to form a volatile conjugate base (a multiatomic moiety).

[0059] Thus, the polyatomic monovalent cation may include a nitrogen atom, preferably a protonated nitrogen atom. The polyatomic monovalent cation may be ammonium or a derivative thereof. Derivatives of ammonium are also included, a non-limiting and exemplary list of which includes monoalkylammonium or dialkylammonium. Those skilled in the art will recognize the derivatives of ammonium with a single positive charge suitable for use in the present invention.

[0060] Alternatively, polyatomic monovalent cations can be described as organic cations. Although ammonium is commonly described as an inorganic ion, many of its derivatives are organic molecules, and the ammonium ion can be included in the same group as its derivatives that are organic molecules. Organic nitrogen occurs in many forms, including organic solvents, peptides, and proteins.

[0061] Thus, there is provided a process for preparing a novel nucleotide complex, comprising a step of drying the nucleotide complex, said complex comprising nucleotides and divalent cations present in a ratio of between 0.2 and 2 divalent cations and polyatomic monovalent cations present in a ratio of between 0.2 and 2.5 polyatomic monovalent cations per nucleotide. The drying step is preferably carried out under heat and / or vacuum, the polyatomic moieties of the monovalent cations being volatile under these conditions. Preferably, there are between 0.2 and 1 divalent cation per nucleotide.

[0062] Thus, there is also provided a method for preparing a novel nucleotide complex, comprising removing a polyatomic moiety from a starting nucleotide complex, said starting nucleotide complex comprising nucleotides and polyatomic monovalent cations present in a ratio of between 0.2 and 4 polyatomic monovalent cations per nucleotide. The starting nucleotide complex may be mixed with a second nucleotide complex, said second nucleotide complex comprising nucleotides and divalent cations present in a ratio of between 0.4 and 2 divalent cations per nucleotide. This mixing step may be before or after removal of the polyatomic moiety.

[0063] Thus, there is also provided a process for preparing novel nucleotide complexes comprising the step of removing a polyatomic moiety from a starting nucleotide complex, said starting complex comprising nucleotides and divalent cations present in a ratio of between 0.2 and 2 divalent cations and polyatomic monovalent cations present in a ratio of between 0.2 and 2.5 polyatomic monovalent cations per nucleotide.

[0064] The removal is preferably carried out under altered pH, heat, and / or vacuum, where the polyatomic moieties of the monovalent cations become volatile under these conditions. According to any process of the present invention, drying, evaporation or removal of volatile moieties is preferably carried out under vacuum.

[0065] Those skilled in the art will understand that the nucleotide complex used as the starting material will generally be in a solution that allows it to be dried or that allows the volatile moieties to be evaporated or removed.

[0066] The term nucleotide, as used herein, may be interpreted as a nucleotide ion or nucleotide ion species, which will be understood to be a nucleotide entity without any counterion.

[0067] moreover, Provided is a cell-free process for the enzymatic synthesis of DNA, comprising obtaining a nucleotide complex in solution, said complex being prepared according to any process or method described herein, and adding a nucleotidyl transferase.

[0068] After preparation, the nucleotide complex of the present invention is considered to be substantially free of the monovalent cations originally associated therewith.Thus, the level is expected to be reduced to between 0 and 1 monovalent cation present per nucleotide, preferably between 0 and 0.5 monovalent cations per nucleotide, optionally less than 0.2 monovalent cations per nucleotide, such as 0.2, 0.1 or 0 monovalent cations per nucleotide.Thus, protons or hydronium ions are not considered to be monovalent cations for the purposes of the present invention.

[0069] Thus, the present invention provides a novel nucleotide complex. The novel nucleotide complex is a nucleotide associated with a divalent cation present at a ratio of between 0.5 and 1.5 divalent cations per nucleotide and a multiatomic monovalent cation present at a ratio of less than 0.5 monovalent cations per nucleotide. Such a novel nucleotide complex may be present in a solvent such as water. The nucleotide complex in solution may be substantially free of additional cations other than residual metal monovalent cations that may be manufacturing contaminants from the nucleotide itself. These suitable tolerable levels are disclosed herein.

[0070] Thus, the present invention provides a novel nucleotide complex solution consisting essentially of nucleotides, divalent cations present in a ratio of between 0.5 and 1.5 divalent cations per nucleotide, and monovalent cations present in a ratio of less than 0.5 monovalent cations per nucleotide.

[0071] Preferred are novel nucleotide complex solutions in which the divalent cations are present in a ratio between 0.5-1.5, 0.6-1.4, 0.7-1.3, 0.8-1.2, 0.9-1.1 per nucleotide, or simply one divalent cation per nucleotide.

[0072] Preferred are novel nucleotide complexes in which monovalent cations are present in a ratio of less than 0.5, 0.4, 0.3, 0.2, 0.1 per nucleotide, or in which there are virtually no monovalent cations present.

[0073] Any combination of the above is possible using the process of the present invention. The divalent cation is preferably calcium, magnesium, or manganese. The inventors have demonstrated that if the "charge" of the associated divalent cation does not balance the charge of the nucleotide, then a hydronium ion (HO +) provide an additional positive charge. Thus, for all nucleotide complexes described herein, hydronium ions may be present. These ions may be present in a ratio of 0.5-3.5, optionally 1-3, optionally 1.5-2.5, optionally approximately 2 ions per nucleotide. They are not actively added, but are formed when the nucleotide complexes are prepared according to the methods of the present invention.

[0074] The process of the invention may further comprise the step of adding one or more zwitterionic molecules to the starting nucleotide complex or to the new nucleotide complex, such zwitterionic molecules preferably not being volatile under the conditions used in the process of the invention.

[0075] Zwitterions contain both positively and negatively charged groups and have a net charge of 0. Some zwitterions cannot isomerize to no longer contain positively and negatively charged groups, but most can lose all charge and become completely neutral, often at neutral pH (pH 7), but not always. At various pHs, zwitterionic molecules can gain or lose hydronium ions in solution, which makes them charged and converts the molecule to a form that is not zwitterionic. Zwitterionic molecules can be amino acids, where the negative charge comes from a carboxylate ion and the positive charge comes from an ammonium ion. Zwitterionic molecules can be ylides. An ylide is a neutral dipolar molecule that contains a formally negatively charged atom (usually a carbanion) directly bonded to a heteroatom (usually nitrogen, phosphorus, or sulfur) that has a formal positive charge. Ylides are a subclass of zwitterions that are polar, where the positively and negatively charged atoms are taken together, i.e., X + -Y - Both amino acids and ylides are attached to the nucleotide complexes exemplified herein.

[0076] In particular, for amino acids, any suitable enantiomer may be selected. The L-enantiomer of histidine is exemplified herein. The L-enantiomer is commonly found in biology and may therefore be preferred. Preferred amino acids are Lewis acids:bases. Zwitterions that are Lewis acids:bases may be suitable for the present invention.

[0077] The maximum number of zwitterions that can be associated with a nucleotide complex, assuming one single positively charged group, is four, although additional zwitterions may be present in solution. Thus, the present invention provides a novel nucleotide complex. The novel nucleotide complex is a nucleotide associated with a zwitterionic molecule present in an amount of 4 or less zwitterionic molecules per nucleotide, a divalent cation present in a ratio of between 0.5 and 1.5 divalent cations per nucleotide, and optionally a monovalent ion present in less than 0.5 monovalent cations per nucleotide. Such a novel nucleotide complex may be present in a solvent such as water. The nucleotide complex in solution may be substantially free of further or additional cations or ions other than residual metal monovalent cations that may be manufacturing contaminants from the nucleotide itself.

[0078] Thus, the present invention provides novel nucleotide complex solutions consisting essentially of nucleotides, zwitterionic molecules present in an amount of 4 or less zwitterionic molecules per nucleotide, divalent cations present in an amount between 0.5 and 1.5 divalent cations per nucleotide, and optionally monovalent cations present in an amount of less than 0.5 monovalent cations per nucleotide.

[0079] Preferred are novel nucleotide complex solutions in which the divalent cations are present in a ratio between 0.5-1.5, 0.6-1.4, 0.7-1.3, 0.8-1.2, 0.9-1.1 per nucleotide, or simply one divalent cation per nucleotide.

[0080] Preferred are novel nucleotide complexes in which monovalent cations are present in a ratio of less than 0.5, 0.4, 0.3, 0.2, 0.1 per nucleotide, or in which there are virtually no monovalent cations present.

[0081] Preferred are novel nucleotide complexes in which the zwitterionic molecules are present in a ratio of 0-4, 0-3, 0.5-2.5, 1-2, or 1.5 zwitterionic molecules present per nucleotide. As used herein, zwitterionic and zwitterionic are used interchangeably.

[0082] Any combination of the above is possible using the process of the present invention. The divalent cation is preferably calcium, magnesium, or manganese, or a mixture thereof.

[0083] The zwitterionic molecule is preferably an amino acid or an ylide, with preferred amino acids being histidine, lysine, and arginine. Novel nucleotide complexes are provided that include nucleotides associated with divalent cations present in a ratio of between 0.5 and 1.5 divalent metal cations per nucleotide and zwitterionic molecules present in a ratio of between 0.5 and 4 zwitterionic molecules per nucleotide.

[0084] Preferred are novel nucleotide complexes that include nucleotides associated with between 0.5 and 1.5 calcium, magnesium, and / or manganese per nucleotide, and between 0.5 and 4 molecules selected from any one or more of histidine, lysine, arginine, or dimethylsulfoxonium-(isobutanoyl)methylide.

[0085] It will be understood that any of the above ratios are applicable to these novel nucleotide complexes.Novel nucleotide complexes can also essentially consist of the listed entities, in addition to solvent if necessary.Other ions are preferably absent or present at very low levels as discussed above (less than 5%, 4%, 3%, 2%, or 1% of the nucleotide concentration are contaminating ions).

[0086] The present invention further extends to a cell-free process for the enzymatic synthesis of DNA, comprising the use of any of the novel nucleotide complexes described herein. A cell-free process for enzymatic synthesis of DNA, comprising obtaining a nucleotide complex in solution, said complex being a nucleotide in association with a divalent cation present in a ratio of between 0.2 and 1.5 divalent cations and a monovalent cation present in a ratio of between 0 and 1 monovalent cation per nucleotide, in combination with a nucleotidyl transferase. Optionally, the nucleotide complex may also be in association with a zwitterionic molecule present in an amount of up to 4 zwitterionic molecules per nucleotide.

[0087] A cell-free process for enzymatic synthesis of DNA using a nucleotidyl transferase, comprising combining the enzyme with a nucleotide complex, the complex being a nucleotide associated with a divalent cation present at a ratio of between 0.2 and 1.5 divalent cations per nucleotide and a monovalent cation present at a ratio of between 0 and 1 monovalent cations per nucleotide. Optionally, the nucleotide complex may also be associated with a zwitterionic molecule present at a ratio of between 0 and 4 zwitterionic molecules per nucleotide.

[0088] Also provided is a novel nucleotide complex in solution comprising a nucleotide and a divalent cation present in a ratio of between 0.2 and 1.5 divalent cations and a monovalent cation present at between 0 and 1 monovalent cations per nucleotide. Optionally, the nucleotide complex may also be associated with a zwitterionic molecule present at between 0 and 4 zwitterionic molecules per nucleotide.

[0089] Also provided is a novel nucleotide complex in solution consisting essentially of a nucleotide and between 0.2 and 1.5 divalent cations and between 0 and 1 monovalent cation per nucleotide. Optionally, the nucleotide complex may also be associated with between 0 and 4 zwitterionic molecules per nucleotide.

[0090] The term nucleotide, as used herein, may be interpreted as a nucleotide ion or nucleotide ion species, which will be understood to be a nucleotide entity without any counterion.

[0091] It will be understood that in solution, the ions forming the complex may or may not be dissociated. With respect to nucleotide complexes, references to monovalent cations as used herein generally do not include protonation by or association with hydronium ions, either of which may be present in nucleotide complexes in solution to balance the charge present.

[0092] Enzymatic DNA synthesis is preferred for the production of DNA on a larger scale, i.e. not for laboratory scale amplification (ng-mg scale per liter), but for therapeutic or prophylactic use (described as several grams per liter of reaction mixture). In scaling up this laboratory scale amplification, the inventors have found that it is not as simple as providing more substrate and other components and finding that the yield follows. Thus, the process generally involves the use of nucleotide complexes at concentrations of 30 mM or more, said concentration being determined when the nucleotide complex is combined with the nucleotidyl transferase. Mixing the nucleotide complexes with the enzyme results in the formation of a reaction mixture. The nucleotide complex is dried (or the volatile polyatomic moieties are evaporated), so that it may simply be added to a solution containing the nucleotidyl transferase. The concentration is determined in the reaction mixture in which the process is carried out. Thus, the concentration of the nucleotide complex is determined in the reaction mixture when the nucleotide complex is added. Thus, the concentration is the initial concentration or the concentration at the beginning of the process.

[0093] Thus, there is provided a cell-free process for enzymatic DNA synthesis, comprising the use of a nucleotide complex at a concentration of at least 30 mM in solution, said complex being prepared according to any of the methods described herein.

[0094] Optionally, the complex may be present at a concentration of at least 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, or 120 mM. Optionally, the complex is present at a concentration of 40 mM to 160 mM, 50 mM to 150 mM, 60 mM to 140 mM, 70 mM to 130 mM, or 80 mM to 120 mM, or any range between these values.

[0095] Without wishing to be bound by theory, the nucleotide complexes prepared according to the methods described herein contain nucleotides associated with between 0.2 and 1.5 divalent cations and between 0 and 1 monovalent cation, said monovalent cations being polyatomic. Optionally, the nucleotide complexes may also be associated with between 0 and 4 zwitterionic molecules per nucleotide. All of these may be described as being present in the nucleotide complex.

[0096] According to any embodiment of the present invention, the nucleotide complex is substantially free of any monovalent metal cations, such as lithium (at a concentration of less than 1% of the nucleotide concentration), sodium (less than 5%), and potassium (less than 5%). If a denaturing agent, such as sodium hydroxide, is required during DNA synthesis, it can be added to the reaction mixture once the nucleotide complex is prepared.

[0097] Thus, there is provided a cell-free process for enzymatic synthesis of DNA, comprising obtaining a nucleotide complex in solution at a concentration of at least 30 mM, said complex comprising nucleotides associated with divalent cations present at a ratio of between 0.2 and 1.5 divalent cations and polyatomic monovalent cations present at a ratio of between 0 and 1 polyatomic monovalent cation per nucleotide, and adding a nucleotidyl transferase. Optionally, the nucleotide complex may also be associated with zwitterionic molecules present at a ratio of between 0 and 4 zwitterionic molecules per nucleotide. Optionally, the complex may be present at a concentration of at least 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, or 120 mM. Optionally, the complex is present at a concentration of 40 mM to 160 mM, 50 mM to 150 mM, 60 mM to 140 mM, 70 mM to 130 mM, or 80 mM to 120 mM, or any range between these values.

[0098] Enzymatic DNA synthesis can involve any enzyme capable of synthesizing DNA, especially nucleotidyl transferase, and in this specification, the definition of any enzyme capable of synthesizing DNA includes all enzymes that can transfer nucleotides to the end of a nascent polynucleotide chain based on a template or de novo.Nucleotidyl transferase can include polymerase or modified polymerase, such as DNA polymerase or RNA polymerase.Polymerase can be from any of the known families of DNA polymerase, including family A, B, C, D, X, Y, and RT.One example of a DNA polymerase from family X is terminal deoxynucleotidyl transferase.

[0099] The nucleotidyl transferase may be present in solution, or the nucleotide complex may be added to the enzyme in solution as a solid preparation, for example as a lyophilized powder. Enzymatic DNA synthesis may be carried out de novo, without the use of a template.

[0100] Enzymatic DNA synthesis may involve a template, for example a nucleic acid template, including a DNA template. Enzymatic DNA synthesis may be carried out in a reaction mixture containing the components described herein.

[0101] In other words, there is provided a cell-free process for synthesizing DNA in solution, comprising contacting a template with at least one nucleotidyl transferase in the presence of one or more nucleotide complexes, said nucleotide complexes comprising nucleotides associated with divalent cations present at a ratio of between 0.2 and 1.5 divalent cations and polyatomic monovalent cations present at a ratio of between 0 and 1 polyatomic monovalent cations per nucleotide. Optionally, the nucleotide complexes may also be associated with zwitterionic molecules present at a ratio of between 0 and 4 zwitterionic molecules per nucleotide. Optionally, the concentration of said nucleotide complexes is at least 30 mM, preferably 40 mM. Optionally, the complexes may be present at a concentration of at least 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, or 120 mM. Optionally, the complex is present at a concentration of 40 mM to 160 mM, 50 mM to 150 mM, 60 mM to 140 mM, 70 mM to 130 mM, or 80 mM to 120 mM, or any range between these values.

[0102] In other words, the nucleotide complex comprises a mixture of divalent cations and polyatomic monovalent cations along with the nucleotide itself. Optionally, the nucleotide complex may also be associated with zwitterionic molecules present in a ratio of between 0 and 4 zwitterionic molecules per nucleotide. Thus, there is provided a cell-free process for synthesizing DNA, comprising contacting a template with at least one nucleotidyl transferase in the presence of one or more nucleotide complexes to form a reaction mixture, the nucleotide complex being present at a concentration of at least 30 mM and comprising nucleotides associated with divalent cations present in a ratio of between 0.2 and 1.5 divalent cations and monovalent cations present in a ratio of between 0 and 1 monovalent cation. Optionally, the nucleotide complex may also be associated with zwitterionic molecules present in a ratio of between 0 and 4 zwitterionic molecules per nucleotide.

[0103] When referring to the concentration of nucleotide or nucleotide complex, it is preferably the concentration of nucleotide (or its complex) when the DNA synthesis process starts, i.e., the starting or initial concentration of nucleotide (or nucleotide complex). It is therefore the concentration after addition to the reaction mixture. It will be understood that addition of other components can be made during the process, and such addition may dilute the concentration of nucleotide / nucleotide complex unless additional nucleotide / nucleotide complex is provided to replenish the concentration. Furthermore, the concentration of nucleotide / nucleotide complex will decrease as the process proceeds, as nucleotide / nucleotide complex will be used or consumed by the process, i.e., DNA synthesis reaction. In certain embodiments, additional nucleotide / nucleotide complex may be added as the process proceeds to replenish substrate for enzymatic reaction. Ideally, the concentration of nucleotides in a DNA synthesis reaction can be kept in the range of 40 mM to 160 mM, 50 mM to 150 mM, 60 mM to 140 mM, 70 mM to 130 mM, or 80 mM to 120 mM, or any range or value between these values. Constant maintenance of nucleotide concentration can be beneficial to a DNA synthesis reaction.

[0104] The inventors have surprisingly found that if nucleotide complexes containing a mixture of polyatomic monovalent and divalent cations are dried under heat and / or vacuum and then resuspended, the resulting nucleotide complexes can be used in DNA synthesis reactions with polymerases, and the efficiency of conversion of nucleotides to DNA remains constant at various concentrations. The examples acknowledge that the reaction efficiency using these novel nucleotides remains around 55-65% regardless of the tested concentrations between 40 mM and 120 mM. Such an effect has not been observed before by the inventors, who have previously acknowledged that other complexes have a specific "peak" that they prefer to function. This further improvement is an improvement compared to conventional nucleotide salts or mixed nucleotide complexes with four monovalent cations described so far. Obtaining the same conversion efficiency of the nucleotide complexes at 120 mM is particularly exciting, as it results in the ability to scale up without loss of efficiency.

[0105] The inventors further demonstrated in the examples that the novel nucleotide complexes of the present invention, especially those containing one magnesium ion (with or without zwitterionic molecules), outperform nucleotide ammonium salts (four ammonium ions), especially at concentrations above 60 mM, resulting in higher yields of DNA.

[0106] It was observed that the addition of zwitterionic molecules to the nucleotide complexes of the invention accelerates the production of DNA, resulting in higher yields being achieved more rapidly, especially at concentrations of the nucleotide complexes above 100 mM. This was observed for both zwitterionic molecules tested. Indeed, according to Example 7, 16 g / l of DNA was synthesized in just 48 hours.

[0107] Accordingly, the present invention provides a cell-free process for enzymatically producing at least 15 g / L of DNA in 40-55 hours, comprising the use of a nucleotidyl transferase and a nucleotide complex comprising a nucleotide associated with about one magnesium or manganese ion and at least one zwitterionic molecule, optionally histidine, lysine, arginine, or dimethylsulfoxonium-(isobutanoyl)methylide.

[0108] All components of this nucleotide complex are as previously described. The inventors have also found that the association of magnesium or manganese cations with the nucleotide complex means that no additional magnesium or manganese is required in the reaction mixture to synthesize DNA. This is beneficial in terms of reducing components and therefore costs. Furthermore, this means that the ionic strength of the reaction mixture can be further reduced compared to the prior art, which the inventors have found to be favorable for DNA synthesis reactions. Such conditions may be ideally suited for various synthesis reactions, such as the production of single-stranded DNA. The inventors have found that in the studies carried out to date, the nucleotide complexes described herein for the first time appear to produce more single-stranded DNA than the parent nucleotide complexes from which they are produced.

[0109] Convention dictates, for example, that magnesium (a divalent cation) is present in DNA synthesis reactions in a minimum ratio of at least 1:1 with the nucleotide. This is because magnesium may be required for the active site of some nucleotidyl transferase enzymes, and magnesium may form a complex with the nucleotide prior to incorporation, and may also form its own salt with the phosphate species released during DNA synthesis. The benefit of including magnesium or manganese in the nucleotide complex is that it reduces or eliminates the need for additional magnesium or manganese. This helps maintain an approximately 1:1 ratio of magnesium or manganese to nucleotide (or a ratio less than 1:1), which the inventors have previously identified as desirable for large-scale DNA synthesis reactions. Reducing the components involved in DNA synthesis, among other things, reduces costs, but this is also important because higher concentrations of magnesium (greater than 1:1) are associated with reduced fidelity in DNA synthesis. Some DNA synthesis enzymes utilize calcium instead of magnesium, and these include DP04. For these enzymes, including calcium as a divalent ion is attractive.

[0110] The divalent cation associated with the nucleotide in the complex is magnesium (Mg 2+ ), Beryllium (Be 2+ ), Calcium (Ca 2+ ), Strontium (Sr 2+ ), Manganese (Mn 2+ ), or zinc (Zn 2+ ), preferably Mg 2+ or Mn 2+The divalent metal cation may include one or more metals selected from the list consisting of: The ratio between the divalent metal cation and the nucleotide (nucleotide ion or nucleotide ion species) may be about 1:1 in the solution, but is preferably between 0.2:1 and 2:1, and optionally 0.5:1 and 1.5:1. Ratios higher than 1:1 may cause some imprecision in DNA synthesis, so ratios lower than 1:1 are desirable and preferred in DNA synthesis. Thus, the provision of divalent cations in relation to the nucleotide complex may reduce or eliminate the need to add additional divalent cations to the reaction mixture. However, if still required, these divalent cations may be provided to the enzymatic DNA synthesis in the form of any suitable salt.

[0111] Furthermore, the method developed by the inventors herein can be carried out in a wide range of conditions with respect to other components present. These conditions range from conventional buffer levels to no additional buffering provided, in effect carrying out the reaction in water with the required components. Increasing the concentration of buffering agent can directly improve pH control by increasing buffering capacity, but chemical buffering substances can also chelate various metal ions, including magnesium ions, which can adversely interfere with the balance of monovalent and divalent cations required for optimal DNA yield. Therefore, it may be desirable to use the concentration of buffering agent at the lowest possible level while maintaining the pH within an acceptable range for optimal DNA production, in balance with other essential reaction components. Those skilled in the art will understand that some of the counterions proposed herein may have buffering capacity themselves, or entities released or generated during DNA synthesis (e.g. pyrophosphate and phosphate) may also help buffer excessive pH changes.

[0112] The required components may include, with or without the provision of a buffer, an enzyme that synthesizes DNA, such as a polymerase (nucleotidyl transferase), a nucleotide complex, and any additional components required depending on the conditions of the reaction, selected from a divalent metal cation provided as a salt, a template, a denaturant, a pyrophosphatase, or one or more primers / primases. These components may form a reaction mixture. Thus, in its most basic form, the reaction mixture is simply the prepared nucleotide complex plus a nucleotidyl transferase. It will be understood that it is desirable for the reaction mixture to be free of extra ionic species, since such entities may have undesirable effects on DNA synthesis. Other than the ions present in the nucleotide complex, other ions may be present in minimal amounts, for example in a denaturant (e.g., sodium hydroxide or potassium hydroxide) or in a buffer. Depending on the nucleotide complex selected and the enzymes involved in the reaction, it may be necessary to further supplement with magnesium or manganese salts. Preferably, the concentration of "additional ions" in the reaction mixture, e.g., at the start of the reaction, may be kept at a minimum level, e.g., less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, or less than 10 mM. Such additional ions are ions other than those provided with the nucleotide complex or derived therefrom during the course of the reaction.

[0113] Thus, the process, i.e., the provision of nucleotides as complexes with reduced mixed counterion provision to the reaction mixture, is advantageous because it surprisingly allows for improved DNA yields and / or improved efficiency of conversion of nucleotides to DNA. These improvements may be improvements compared to similar reaction mixtures in which all nucleotides are provided as conventional salts with the required monovalent cation alone. The provision of novel nucleotide complexes to replace conventionally used nucleotide salts has several additional surprising advantages, such as the ability to reduce the concentration of buffers in the reaction mixture, in some cases to zero, and / or the ability to reduce, reduce, or completely eliminate the need for additional provision of divalent cation cofactors, most notably magnesium, to the reaction mixture, which are typically added as salts. Considering that this salt may no longer be required, the present invention has the effect of reducing the ionic strength of the reaction mixture, since when no divalent cation salt is added, there is no associated anion provision to the reaction mixture (e.g. MgCl2, thus avoiding the addition of two chloride ions). The ionic strength of a solution is a measure of the concentration of ions in that solution. Ionic compounds dissociate into ions when dissolved in water. As used herein, the unit of measurement is molar concentration (mol / L). The inventors hypothesize that a reduction in the ionic strength of the reaction mixture may be beneficial to the process. Monovalent cations provided with nucleotide entities may be inhibitory to the process at high concentrations, and therefore further improvement is possible by further removing them according to the process of the present invention. Alternatively or additionally, in standard processes, magnesium or manganese is provided as a salt, and high concentrations of associated anions from this salt, such as chloride ions, may also be inhibitory to the process. Furthermore, when it is desired to manipulate the DNA generated using the present invention, the inventors have confirmed that the enzymes introduced into the reaction mixture, such as enzymes that cleave and ligate target sequences (DNA processing enzymes), prefer conditions of lower ionic strength, since these enzymes are generally added at the end of the DNA synthesis reaction, when conventional nucleotides and divalent salts may significantly increase the ionic strength.Thus, the products from such DNA synthesis reactions may be suitable for further enzymatic processing (eg, for use as templates to produce RNA using an RNA polymerase).

[0114] In one embodiment, the template directs the enzymatic DNA synthesis in the process. The template can be any nucleic acid template, such as a DNA or RNA template. The template can be a natural nucleic acid, an artificial nucleic acid, or a combination of the two. The amplification of the template is preferably carried out via strand displacement. The amplification of the template is preferably isothermal, i.e., there is no need to cycle between low and high temperatures to proceed with the amplification. In this scenario, heat can be used at the beginning to denature the template, if required, or the template can be denatured by chemical means. However, once the template is denatured, the temperature can be maintained in a temperature range that does not affect the denaturation of the template and the product, if appropriate, to allow any primers or indeed primase enzymes to penetrate between the double-stranded template. Isothermal temperature conditions require that the reaction is not heated to the point where the template and the product denature (compared to PCR, which requires thermal cycling to denature the template and the product). Generally, such reactions are carried out at a constant temperature, depending on the preferences of the enzyme itself. The temperature can be any temperature suitable for the enzyme.

[0115] The cell-free method preferably involves the amplification of the template via strand-displacement replication. This synthesis releases single-stranded DNA, which can then be copied into double-stranded DNA using a polymerase. The term strand-displacement refers to the ability to displace downstream DNA encountered during synthesis, where the polymerase cleaves double-stranded DNA to extend the nascent single strand. DNA polymerases with varying degrees of strand-displacement activity are commercially available. Alternatively, strand-displacement can be achieved by providing a DNA polymerase and a separate helicase. The replicative helicase can cleave double-stranded DNA and facilitate the progression of the leading strand polymerase.

[0116] Independently, optional features of any of the aspects of the invention may be as follows: The template may be circular. DNA may be synthesized by amplification of the template, optionally by strand displacement replication. Strand displacement amplification of said DNA template may be performed by rolling circle amplification (RCA). The polymerase may be Phi29 or a variant thereof. Amplification of DNA may be performed isothermally, i.e. at a constant temperature. Primers or primase may be used to initiate amplification. Nickase may be used to generate primers "in situ" in the double-stranded circular template. One or more primers may be random primers. A pair or set of primers may be used. The synthesized DNA may comprise concatemers comprising tandem units of the DNA sequence amplified from the DNA template. The DNA template may be a closed linear DNA, preferably the DNA template is incubated under denaturing conditions to form a closed circular single-stranded DNA.

[0117] The amount of DNA which can be synthesized is at least 3 g per liter of reaction mixture, in particular at least 16 g / l, preferably up to at least 25 g / l. The novel nucleotide complexes prepared according to the methods of the present disclosure may enable DNA synthesis at high starting concentrations, for example, 90 mM, 100 mM, or even 120 mM or more.

[0118] The amount of DNA that can be synthesized can be more than 60% of the maximum yield calculated for the reaction mixture.Preferably, the amount of DNA that can be synthesized can be more than 80% of the maximum yield calculated.The maximum yield calculated is based on the theoretical yield when all nucleotides are incorporated into the product, which can be calculated by those skilled in the art.

[0119] The efficiency of DNA synthesis from a nucleotide (or nucleotide complex) can be described as the percentage of the nucleotide or complex provided to the reaction mixture that is successfully incorporated into the product over the course of the reaction.

[0120] The efficiency of DNA synthesis may also be maintained over a wider range of nucleotide concentrations by the present invention, resulting in an improved concentration range in which DNA synthesis can be performed. The process for preparing the novel nucleotide complex requires at least one starting nucleotide complex. Any suitable number of phosphate groups may be present as required. However, the nucleotide / nucleotide complex is preferably a deoxyribonucleoside triphosphate (dNTP) or a derivative or modified version thereof. The nucleotide is one or more of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and their derivatives. Each individual starting nucleotide complex may be, but need not be, charge-balanced by various cations providing four positive charges to maintain electrical neutrality. The starting nucleotide complex used in the drying process may include one or more polyatomic monovalent cations, i.e., one or more species of polyatomic monovalent cations, and one or more divalent cations, i.e., one or more species of divalent cations. It will be understood that the number of cations associated with each nucleotide complex need not be an integer, since these may dissociate in solution, and thus ions may dissociate in solution. The starting nucleotide complex may be considered to be a salt if the charges are perfectly balanced.

[0121] The starting nucleotide complex may be as described in WO2021 / 161051 (PCT / GB2021 / 050366). The starting nucleotide complex in solution is used to prepare a nucleotide complex with a reduced supply of monovalent cations. This is accomplished by applying heat and / or vacuum so that the polyatomic moieties from the monovalent cations become volatile and are removed from the solution. They are effectively evaporated. Solvent, e.g., water, may also be removed from the solution. This further reduction in the supply of monovalent cations results in a new nucleotide complex that does not require a buffer for resolubilization.

[0122] In general, the inventors mixed two different nucleotide complexes (one with only divalent cations and the other with only monovalent cations) together to prepare the starting nucleotide complex used in the method of the present invention. Alternatively, these two nucleotide complexes may be mixed during or after the process of removing monovalent entities. The nucleotide complexes each independently include complexes in which not all negative charges are balanced. This has several advantages. Nucleotides complexed with divalent cations have low solubility and are therefore not routinely used in any application. Nucleotides complexed with magnesium ions present a particular problem, as they are not in solution and cannot be used in their original form. However, when mixed with a nucleotide complex associated with one or more monovalent cations, the mixture is soluble and forms a solution. This therefore provides a simple method of utilizing nucleotide complexes that have been desired but not previously processable.

[0123] The polyatomic monovalent ion may be a single type of polyatomic ion or a mixture of different types of polyatomic ions. The divalent ion may be a single type of ion or a mixture of different types of ions. The divalent ion may be magnesium.

[0124] The polyatomic monovalent ions become volatile under suitable conditions, such as under heat and / or vacuum or with a change in pH. Thus, the polyatomic monovalent cation may be the conjugate acid of a base, where the base is the volatile moiety. Thus, the deprotonated form of the polyatomic monovalent cation may be described as the volatile moiety. This may be referred to as the "polyatomic moiety."

[0125] Volatility refers to the tendency of a moiety to evaporate under given conditions and is also described as the degree to which a moiety vaporizes easily. This is at a given temperature and pressure. Volatility is generally described in terms of the moiety's boiling point (especially for liquids). The boiling point is the temperature at which the vapor pressure of a liquid equals the surrounding pressure, causing the liquid to rapidly evaporate or boil. It is pressure dependent. Generally, volatile moieties may have boiling points below 100°C.

[0126] The drying process, removal or evaporation, may be carried out under a vacuum. A vacuum can create a region with a gas pressure below atmospheric pressure. Thus, the gas pressure can be as low as 10 5 The pressure may be any suitable pressure ranging from 10 Pascal (Pa) to 100 Pa. 5 Pa, 10 4 Pa, 10 3 Pa, 10 2 Pa or 10 1 A low vacuum can be 10 Pa, 100 Pa, or any pressure in between. 5 ~3×10 3 The medium vacuum can be achieved in 3×10 Pa. 3 ~10 -1 A high vacuum can occur at 10 Pa. -1 ~10 -10 The vacuum can be created using any suitable technique, including the use of a vacuum pump, or by any other method of reducing pressure, such as the use of a liquid flowing at high speed. The vacuum can be created using any suitable laboratory equipment, aspirators, for example, traditional rotary vane pumps, combination pumps, piston pumps, diaphragm pumps and scroll pumps, and oil-free vacuum pumps. The vacuum is designed to reduce the pressure in the system, thereby lowering the boiling point of the moieties. By lowering the boiling point, the moieties will evaporate at a much lower temperature.

[0127] The drying process, removal or evaporation can be carried out at any suitable temperature. Those skilled in the art will recognize that the boiling point of the volatile moiety can determine the set of conditions, such as pressure and temperature, suitable for drying. The process or method can be carried out at room temperature. Alternatively, heat can be applied to the starting nucleotide complex. The temperature can be increased to 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90°C, or any temperature range therebetween. The temperature required to make the moiety volatile will depend on the pressure at which drying is carried out.

[0128] It may be preferred that the suitable conditions are the application of heat and vacuum. Equipment is available for combinations of treatments as illustrated in the examples. Alternatively or additionally, the moiety may be made volatile by altering the pH, such as by raising the pH by adding a basic component.

[0129] Drying or treatment of the starting nucleotide conjugate under vacuum can complete the equilibration between the conjugate acid and the volatile base, resulting in the evaporation of substantially all of the volatile moieties. Drying or processing of the starting nucleotide complex can result in the removal of substantially all of the polyatomic monovalent moieties from the complex, leaving less than 0.2 polyatomic moieties per nucleotide.Ideally, substantially all of the polyatomic moieties are removed.Without being bound by theory, the inventors believe that the nucleotide entity is protonated, which allows the volatile base to evaporate freely.Nucleotides can be protonated at the nitrogen or phosphate group.Alternatively, water molecules are protonated to produce hydronium ions.

[0130] Drying, removal or evaporation can result in the formation of a dry nucleotide complex, also described as a powder. As used herein, a powder is a dry solid composed of many very fine particles that can flow freely when shaken or rocked. The powder can be crystalline. Drying can result in the removal of substantially all of the solvent present in the starting nucleotide complex solution. Generally, the solvent can be water. Water or other solvents can evaporate during the drying process. Ideally, all of the solvent can be removed so that the resulting nucleotide complex is in a dry state (0% starting volume). Alternatively, a residual amount of solvent may remain, resulting in a level of solvent in the product of approximately less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% starting volume. Such can be visually observed as a "sticky mass". In the examples, the nucleotide complex was dried under vacuum from a starting volume of more than 1 ml to a final volume of approximately 20 μl.

[0131] Generally, the polyatomic monovalent cations are present in a ratio of 0.2 to 4, or 0.2 to 2.5, of polyatomic monovalent cations relative to the starting nucleotide complex according to the invention. This range includes 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, and 2.5, or 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4 polyatomic monovalent ions per nucleotide complex. The ions can be shared between the nucleotide ions. These polyatomic moieties are effectively or substantially removed or evaporated using the drying process described herein. During the process of the present invention, a certain percentage of polyatomic moieties is removed. This percentage can be 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Preferably, 100% is removed. After the drying process, the level of polyatomic moieties (volatile bases of conjugate acids) is greatly reduced, preferably to a level of less than 0.2, less than 0.1, or even 0 polyatomic moieties per nucleotide.

[0132] The process referred to as the drying process described herein results in a nucleotide complex that, when subsequently resuspended in water, has a reduced pH.This reduction in pH is believed to be due to the protonation of the nucleotides in the complex during the drying process.In the examples, the inventors found that the resuspended nucleotide complex is not as acidic as expected, which may be due to the ability of the nucleotide complex to act as a buffering agent.The inventors did not require the use of a buffering agent to resolubilize or dissolve the nucleotide complex before use.

[0133] The polyatomic monovalent cation preferably includes a protonated nitrogen atom. Suitable polyatomic monovalent cations include ammonium and its derivatives. It is this proton that is transferred to the nucleotide entity during vaporization of the polyatomic moiety.

[0134] Once substantially or completely dry, the nucleotide complex can be stored under appropriate conditions, such as in a freezer, until required for use. Upon use, the prepared nucleotide complex may require resolubilization, for example, in water or another solvent. Such may be provided alone or in combination with a nucleotidyl transferase to form a reaction mixture. Other components may be added to the reaction mixture.

[0135] The solvent for resolubilization can be water. The solvent can be any suitable solvent. The inventors have surprisingly found that resolubilization does not require a buffering agent, despite the loss of volatile parts. Therefore, it is hypothesized that the nucleotide complex can independently buffer pH, potentially through the presence of one or more phosphate groups.

[0136] Resolubilization may allow for the addition of other entities, such as the zwitterionic molecules mentioned above. These may be present in the solvent or added separately. Upon drying process or evaporation, the starting nucleotide complex can be at any suitable concentration.

[0137] The advantage of using a drying process or evaporation is that it leads to novel nucleotide complexes that are particularly advantageous for use in the enzymatic synthesis of DNA. It is preferred that the concentration of nucleotides or their complexes in the enzymatic synthesis of DNA, i.e. in the reaction mixture, can be more than 30 mM and up to at least 160 mM. Such concentrations are important in the production of higher yields of DNA, which can be as high as 9.75 g / l to 52 g / l for the two concentrations shown. It is preferred that the concentrations of nucleotides or their complexes described are those at the start of the synthesis reaction, i.e. the starting or initial concentration of nucleotides or their complexes in the reaction mixture that also contains the enzymes necessary for DNA synthesis. Subsequent addition of further components may reduce this concentration, and their use by DNA synthesis enzymes may also reduce the concentration from the starting concentration. The skilled person will know how to calculate the concentration of nucleotides / nucleotide complexes when the recipe is prepared based on the other components used and the volume of the stock nucleotide complex solution / powder. As described above, it may be beneficial to keep the concentration of nucleotide complexes in DNA synthesis reactions constant, for example at 100 mM, 110 mM, 120 mM, 130 mM, or 140 mM throughout the reaction, by continuously monitoring and adding additional nucleotides if the concentration decreases, or by constant addition of nucleotide complexes.

[0138] It should be noted that in the art of DNA synthesis or amplification, the term "nucleotide" is used when authors mean "nucleotide salt," since it is currently not possible to provide and use nucleotides without any form of counterion in DNA synthesis.

[0139] The process can be a batch process or a continuous flow process.The batch can be a closed batch (i.e., all reaction components are provided at the beginning of DNA synthesis), or, as described in WO2016 / 034849, which is incorporated herein by reference, additional components can be added to the reaction as needed during the process.If additional addition is required, this can dilute the concentration of nucleotide or nucleotide complex, unless additional nucleotide complex is added to replenish or increase the concentration.

[0140] Enzymatic cell-free synthesis of DNA with such ions can be performed in a minimal buffer, where no additional salts or detergents are added that have been shown to enhance DNA synthesis or aid primer binding. This minimal buffer may include an agent (buffer) that stabilizes the pH. The minimal buffer may contain a small amount of cations provided by the presence of chemicals used to denature the template, such as sodium hydroxide, potassium hydroxide, or ammonium hydroxide. In the examples, 5 mM sodium hydroxide or potassium hydroxide was used as the denaturing agent, but this concentration may be modified to suit the conditions of the reaction, within the skill of those used to perform DNA denaturation, and the amount of sodium hydroxide, potassium hydroxide, or ammonium hydroxide can be provided from 2.5 mM, up to 5 mM, up to 10 mM, and 15 mM, 20 mM, or 25 mM or more may be used depending on the nature of the template. Thus, the reaction mixture may contain small or minimal amounts of cations and anions that were not originally associated with the nucleotide complex.

[0141] Further advantages are described below. The invention is further described below with reference to exemplary embodiments and the accompanying drawings. [Brief description of the drawings]

[0142] [Figure 1]1 is a plasmid map of the DNA template used in Examples 1 to 3. Various sequence components of the DNA template are indicated. [Diagram 2] 1 is a plasmid map of the DNA template used in Examples 4 to 6. Various sequence components of the DNA template are indicated. [Diagram 3] 1 is a graph showing the yields from DNA synthesis experiments performed using the DNA templates in Examples 2 and 3. It is a plot of the nucleotide complexes and concentration (mM) versus yield (g / L). Results from reactions in water or with added buffer are compared. [Figure 4] 1 is a graph showing the peak yields obtained from DNA synthesis experiments performed using the DNA template in Example 4. This is a plot of nucleotide complex description and concentration (mM) versus peak yield (g / L). These are the highest yields obtained over various incubation times. [Diagram 5] 1 is a graph showing the yields obtained from DNA synthesis experiments carried out using the DNA template in Example 5. It is a plot of the yield (g / L) versus the length of incubation (days) versus the description of nucleotide complexes. These are the yields obtained over various incubation periods at a dNTP concentration of 100 mM. [Figure 6] 1 is a graph showing the yields obtained from DNA synthesis experiments carried out using the DNA template in Example 5. It is a plot of the yield (g / L) versus the length of incubation (days) versus the description of nucleotide complexes. These are the yields obtained over various incubation periods at a dNTP concentration of 120 mM. [Figure 7] 1 is a graph showing the yields obtained from DNA synthesis experiments carried out using the DNA template in Example 6. It is a plot of the nucleotide complex description and concentration (mM) versus yield (g / L). These are the yields obtained after 5 days of incubation with various nucleotide complexes. [Figure 8]1 is a graph showing the yields obtained from DNA synthesis experiments carried out using the DNA template in Example 6. It is a plot of the nucleotide complex description and concentration (mM) versus yield (g / L). These are the yields obtained after 10 days of incubation with various nucleotide complexes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0143] The present invention relates to a cell-free process for the large-scale synthesis of DNA. The process of the present invention may allow for high-throughput synthesis of DNA. The deoxyribonucleic acid (DNA) synthesized according to the present invention may be any DNA molecule. The DNA may be single-stranded or double-stranded. The DNA may be linear. The DNA may be processed to form a circle, in particular a minicircle, a single-stranded closed circle, a double-stranded closed circle, a double-stranded open circle, or a closed linear double-stranded DNA. The DNA may be capable of forming or may be processed to form a specific secondary structure, such as, but not limited to, a hairpin loop (stem loop), an incomplete hairpin loop, a pseudoknot, or any one of various types of double helices (A-DNA, B-DNA, or Z-DNA). The DNA may also form hairpin and aptamer structures.

[0144] The synthesized DNA may be of any suitable length. Using the method of the present invention, lengths up to 77 kilobases or more may be possible. More specifically, the length of DNA that can be synthesized according to the method of the present invention may be up to 60 kilobases, or up to 50 kilobases, or up to 40 kilobases, or up to 30 kilobases or so. Preferably, the synthesized DNA may be 100 bases to more than 77 kilobases, 500 bases to 60 kilobases, 200 bases to 20 kilobases, more preferably 200 bases to 15 kilobases, and most preferably 2 kilobases to 15 kilobases.

[0145] The amount of DNA synthesized according to the process of the present invention can be more than 9.75 g / l. The amount of DNA synthesized is preferably more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g / l or more. The preferred amount of DNA synthesized is 5 g / l. The amount of DNA produced can be described as industrial or commercial quantities in large-scale or mass production. The DNA produced by the process of the present invention can be uniform in quality, i.e., DNA length and sequence. Thus, the process can be suitable for large-scale synthesis of DNA. The process can be uniform in accuracy of synthesis.

[0146] Alternatively, the amount of DNA produced in the synthesis reaction may be compared with the theoretical maximum yield that can be achieved if 100% of nucleotides are incorporated into the synthesized DNA. The method of the present invention improves not only the total yield obtained but also the efficiency of the process, which means that more of the supplied nucleotides are incorporated into the synthesized DNA product than in previous methods. The yield obtainable by the method of the present invention is more than 50% of the theoretical maximum, and up to and beyond 90% of the theoretical maximum. Thus, the percentage of the theoretical maximum yield achieved by the method of the present invention includes 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or more. Traditionally, when using commercially available nucleotide salts, the yield achieved may be disappointing due to the effect of ions that may be inhibitory to the process.

[0147] DNA is synthesized in an enzymatic reaction. This enzymatic synthesis can involve the use of any enzyme that synthesizes DNA, nucleotidyl transferases that can add nucleotides to a nascent polynucleotide chain, most notably polymerase enzymes or modified polymerase enzymes. These are discussed further below. DNA synthesis can be de novo and does not require a template. Enzymatic synthesis can also require the use of a template for DNA synthesis. The template can be any suitable nucleic acid depending on the polymerase, but is preferably a DNA template.

[0148] The template can be any suitable template that simply provides instructions for DNA synthesis by containing a specific sequence. The template can be single-stranded (ss) or double-stranded (ds). The template can be linear or circular. The template can contain natural, artificial, or modified bases or mixtures thereof.

[0149] The template can include any sequence, naturally occurring or artificial. The template can be of any suitable length. In particular, the template can be up to 60 kilobases, or up to 50 kilobases, or up to 40 kilobases, or up to 30 kilobases. Preferably, the DNA template can be 10 bases to 100 bases, 100 bases to 60 kilobases, 200 bases to 20 kilobases, more preferably 200 bases to 15 kilobases, and most preferably 2 kilobases to 15 kilobases.

[0150] The template can be provided in sufficient quantity for use in the method by any method known in the art, for example, the template can be generated by PCR. In the process, the entire template or a selected portion of it can be amplified.

[0151] The template may comprise sequences for expression. The DNA may be for expression in cells (i.e. transfected cells in vitro or in vivo) or in cell-free systems (i.e. protein synthesis). The sequences for expression may be for therapeutic purposes, i.e. gene therapy or DNA vaccines. The sequences for expression may be genes, said genes may code for DNA vaccines, therapeutic proteins, etc. The sequences may comprise sequences that are transcribed into active RNA forms, i.e. small interfering RNA molecules (siRNA). The sequences may comprise sequences that are transcribed into mRNA, most particularly mRNA for producing vaccines. The sequences may comprise sequences for the preparation of viral particles, e.g. lentiviruses or adeno-associated viruses.

[0152] If required, the template may be contacted with at least one polymerase, as described below. Enzymatic DNA synthesis reactions may require at least one DNA polymerase (nucleotidyl transferase). Preferably, the DNA polymerase is a polymerase. The polymerase links nucleotides together to form a DNA polymer. One, two, three, four, or five different enzymes and / or polymerases may be used. The polymerase may be any suitable polymerase from any family of polymerases that synthesizes a polymer of DNA. The polymerase may be a DNA polymerase. Any DNA polymerase may be used, including any commercially available DNA polymerase. Two, three, four, five or more different DNA polymerases may be used, for example, one DNA polymerase that provides a proofreading function and one or more other DNA polymerases that do not. DNA polymerases with different mechanisms may be used, for example, strand-displacing polymerases and DNA polymerases that replicate DNA by other methods. A suitable example of a DNA polymerase that does not have strand-displacing activity is T4 DNA polymerase. A template-independent polymerase, such as a terminal transferase, may also be used.

[0153] Modified polymerases may also be used: these may have been engineered to modify their characteristics, for example to remove their template dependence, to change their temperature dependence, or to stabilize the enzyme for in vitro use.

[0154] The polymerase may be highly stable such that its activity is not substantially decreased by long-term incubation under process conditions. Thus, the enzyme preferably has a long half-life under various process conditions, including but not limited to temperature and pH. It is also preferred that the polymerase has one or more characteristics suitable for the manufacturing process. The polymerase preferably has high accuracy, for example by having proofreading activity. In addition, the polymerase may have high processivity, high strand displacement activity, and low K for dNTPs and DNA. m Preferably, the polymerase exhibits one or more of the following: The polymerase may be capable of using circular and / or linear DNA as a template. The polymerase may be capable of using dsDNA or ssDNA as a template. The polymerase preferably does not exhibit DNA exonuclease activity unrelated to its proofreading activity. Furthermore, the polymerase may be capable of using alternative nucleic acids as templates.

[0155] Those skilled in the art will appreciate that commercially available polymerases, such as Phi29 (New England Biolabs, Inc., Ipswich, Mass., US), Deep Vent® (New England Biolabs, Inc.), Bacillus stearothermophilus (Bst) DNA polymerase I (New England Biolabs, Inc.), Klenow fragment of DNA polymerase I (New England Biolabs, Inc.), M-MuLV reverse transcriptase (New England Biolabs, Inc.), Vent® (exo-minus) DNA polymerase (New England Biolabs, Inc.), Vent® DNA polymerase (New England Biolabs, Inc.), Deep Vent® (exo-) DNA polymerase (New England Biolabs, Inc.), and Bst DNA polymerase large fragment (New England Biolabs, Inc.), are known to those skilled in the art. Whether a given polymerase exhibits the above-defined characteristics can be determined by comparison with the characteristics exhibited by the polymerase enzyme (Stanford Biolabs, Inc.) When referring to high processivity, this typically refers to the average number of nucleotides added by the polymerase enzyme per association / dissociation with the template, i.e., the length of the nascent extension obtained from a single association event.

[0156] Strand displacement polymerases are preferred. Preferred strand displacement polymerases are Phi29, Deep Vent, and Bst DNA polymerase I, or any variants thereof. "Strand displacement" describes the ability of a polymerase to displace a complementary strand when it encounters a region of double-stranded DNA during synthesis. Thus, the template is amplified by displacing the complementary strand and synthesizing a new complementary strand. Thus, during strand displacement replication, the newly replicated strand is displaced to provide a means for the polymerase to replicate additional complementary strands. The amplification reaction begins when the 3' free end of a primer or single-stranded template anneals to a complementary sequence of the template (both are priming events). As DNA synthesis proceeds, and if the polymerase encounters additional primers or other strands annealed to the template, the polymerase displaces it and continues its strand extension. Strand displacement can release single-stranded DNA that can act as a template for more priming events. Priming of the newly released DNA can result in hyperbranching, and high-yield products. It should be understood that strand displacement amplification differs from PCR-based methods in that cycles of denaturation are not essential for efficient DNA amplification, since double-stranded DNA does not impede the continued synthesis of new DNA strands. Strand displacement amplification may only require one initial round of heating to denature the initial template, if it is double-stranded, and to allow the primer to anneal to the primer binding site, if a primer is used. After this, no further heating or cooling is required, so the amplification can be described as isothermal amplification. In contrast, PCR requires cycles of denaturation (i.e., raising the temperature to 94 degrees Celsius or higher) during the amplification process to melt the double-stranded DNA and provide new single-stranded templates. During strand displacement, the polymerase displaces a strand of DNA that has already been synthesized. Furthermore, the polymerase uses the newly synthesized DNA as a template, ensuring rapid DNA amplification.

[0157] The strand displacement polymerase used in the method of the invention preferably has a processivity of at least 20 kb, more preferably at least 30 kb, at least 50 kb, or at least 70 kb or more. In one embodiment, the strand displacement DNA polymerase has a processivity comparable to or higher than that of phi29 DNA polymerase.

[0158] Therefore, strand displacement replication is preferred. During strand displacement replication, the template is amplified by displacing the already replicated strand, which is synthesized by the action of polymerase, and the polymerase displaces another strand, which may be the original complementary strand of the double-stranded template or a newly synthesized complementary strand, the latter being synthesized by the action of polymerase on the initial primer annealed to the template. Thus, the amplification of the template may be performed by displacement of the replicated strand via strand displacement replication of the other strand. This process may be described as strand displacement amplification or strand displacement replication.

[0159] A preferred strand displacement replication method is loop-mediated isothermal amplification, or LAMP. LAMP generally uses 4-6 primers that recognize 6-8 separate regions of the template DNA. Briefly, a strand-displacing DNA polymerase initiates synthesis, and two primers form a loop structure to facilitate subsequent rounds of amplification. An internal primer containing the sequences of the sense and antisense strands of the target DNA initiates LAMP. Subsequent strand-displacement DNA synthesis primed by the external primer releases a single-stranded DNA. This serves as a template for DNA synthesis primed by a second internal and external primer that hybridizes to the other end of the target, generating a stem-loop DNA structure. In subsequent LAMP cycles, one internal primer hybridizes to the loop of the product and initiates displacement DNA synthesis, resulting in the original stem-loop DNA and a new stem-loop DNA whose stem is twice as long. Modified LAMP procedures, in which fewer internal primers are required, can also be employed.

[0160] A preferred strand displacement replication method is rolling circle amplification (RCA). The term RCA describes the ability of RCA-type polymerases to proceed continuously around a circular DNA template strand while extending a hybridized primer. A "primer" may be added, either created by primase or generated by nicking one of the strands of the double-stranded template. This amplification results in the formation of linear single-stranded products with multiple repeated sequences of the amplified DNA. The sequence (single unit) of the circular template is repeated multiple times within the linear product. With respect to the circular template, the initial products of strand displacement amplification are single-stranded concatemers that are either sense or antisense depending on the polarity of the template. These linear single-stranded products serve as the backbone for multiple events of hybridization, primer extension, and strand displacement, resulting in the formation of concatemeric double-stranded DNA products, which also contain multiple repeated sequences of the amplified DNA. Thus, in the concatemeric double-stranded DNA product, there are multiple copies of each amplified "single unit" DNA. RCA polymerases are particularly preferred for use in the methods of the invention. The products of RCA-type strand displacement replication methods may require processing to release single unit DNA. This is desirable when single units of DNA are required. Typical strand displacement conditions using Phi29 DNA polymerase include high levels of magnesium ions, e.g., 10 mM magnesium (usually as the chloride salt) combined with 0.2-4 mM nucleotides (as typically shown as lithium or sodium salts).

[0161] To allow amplification, according to some embodiments, one or more primers may be required by enzymatic DNA synthesis. If no template is used, the primers are designed to provide a starting point for DNA synthesis and initiate the synthesis reaction. If a template is used, the primers may be non-specific (i.e., random in sequence) or specific to one or more sequences contained within the template. Alternatively, a primase or modified polymerase enzyme may be provided to generate the primers de novo. If the primers are random sequences, they allow non-specific initiation at any site of the template. This allows for highly efficient amplification through multiple initiation reactions from each template strand. Examples of random primers are hexamers, heptamers, octamers, nonamers, decamers, or longer lengths, e.g., 12, 15, 18, 20, or 30 nucleotides in length. Random primers can be 6-30, 8-30, or 12-30 nucleotides in length. Random primers are typically provided as a mix of oligonucleotides representing all possible combinations of, for example, hexamers, heptamers, octamers, or nonamers in the template.

[0162] In one embodiment, all primers or one or more primers are specific.This means that the primer has a sequence that is complementary to the sequence in the template from which it is desired to start amplification.In this embodiment, a pair of primers can be used to specifically amplify the part of the DNA template that is inside the two primer binding sites.Alternatively, a single specific primer can be used.A set of primers can be used.

[0163] A primer may be of any nucleic acid composition. A primer may be unlabeled or may contain one or more labels, such as a radionuclide or a fluorescent dye. A primer may also contain chemically modified nucleotides. For example, a primer may be capped, i.e., by chemical or physical means, to prevent the initiation of DNA synthesis until the cap is removed. Primer length / sequence may typically be selected based on temperature considerations, i.e., as one that can bind to the template at the temperature used in the amplification step. A primer may be an RNA primer, e.g., one synthesized by primase.

[0164] In certain embodiments, the template may be contacted with the synthesis enzyme and one or more primers under conditions that promote annealing of the primer to the template. The conditions include the presence of single-stranded nucleic acid that allows hybridization of the primer. The conditions also include temperature and buffer substances that allow annealing of the primer to the template, as is conventional. Appropriate annealing / hybridization conditions may be selected depending on the nature of the primer. One example of conventional annealing conditions that may be used in the present invention includes a buffer substance that includes 30 mM Tris-HCl pH 7.5, 20 mM KCl, 8 mM MgCl2. In the examples, the reaction with the nucleotide complex of the present invention is carried out in 30 mM Tris pH 8.0 as the sole buffer. However, the inventors have described herein conditions that use reduced buffer substances and divalent metal ion components that still allow primer binding, which are discussed further below. Annealing may be carried out using heat after denaturation, followed by stepwise cooling to the desired reaction temperature.

[0165] However, amplification using strand displacement replication can also be performed without primers, and therefore does not require hybridization and primer extension to occur. Instead, the single-stranded template self-primes by forming a hairpin with a free 3' end available for extension. The remaining steps of amplification remain the same. Alternatively, the double-stranded template can be nick to allow strand displacement replication to use one strand of the template itself as a primer. Those skilled in the art are aware of all the methods to provide the initiation of amplification from the template.

[0166] The template and / or polymerase also contact with nucleotides as nucleotide complexes as defined herein. The combination of the template, nucleotidyl transferase, and nucleotide complexes may be described as forming a reaction mixture. The reaction mixture may also include one or more primers or primases. The reaction mixture may also independently include one or more divalent metal cations if not fully provided with the nucleotide complexes. The reaction mixture may further include a chemical denaturing agent. Such a denaturing agent may be potassium hydroxide, ammonium hydroxide, or sodium hydroxide. The reaction mixture may further include an additional enzyme, such as a helicase or pyrophosphatase. The reaction mixture may contain a pH buffer, and in some embodiments, does not contain an additionally added pH buffer.

[0167] A nucleotide is a monomer, or single unit, of a nucleic acid; it is composed of a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. Any suitable nucleotide may be used.

[0168] Nucleotides exist as complexes and are therefore associated with divalent cations and minimal, or deficient, amounts of monovalent cations. Divalent cations are ionic species that have a double positive charge and can be metal ions or polyatomic ions.

[0169] A counterion is an ion that accompanies or associates with an ionic species (a nucleotide in the present invention) to partially or completely balance the charge of that ionic species.

[0170] A complex is generally understood to be a molecular entity formed by loose association of two or more component molecular entities (ionic or uncharged) or corresponding chemical species. Complexes are formed by the association of simpler substances (as compounds or ions) and can be either ions or electrically neutral molecules held together by chemical forces rather than physical forces (i.e., depending on the specific properties of the particular atomic structure). The bonds between the components are usually weaker than covalent bonds.

[0171] The nucleotide complexes prepared as described herein may include a divalent cation. The divalent cation associated with the nucleotide in the complex may be Mg 2+ , B.E. 2+ , Ca 2+ , Sr 2+ , Mn 2+ , or Zn 2+ , preferably Mg 2+ or Mn 2+ The divalent metal cation may include one or more metals selected from the list consisting of: The ratio between the divalent metal cation and the nucleotide (nucleotide ion or nucleotide ion species) may be about 1:1 in the solution, but is preferably between 0.2:1 and 2:1, and optionally 0.5:1 and 1.5:1. A ratio higher than 1:1 may cause some imprecision in DNA synthesis, so a ratio lower than 1:1 is desirable and preferred in DNA synthesis. Thus, the provision of divalent cations in relation to the nucleotide complex may reduce or eliminate the need to add additional divalent cations to the reaction mixture. However, if still required, these divalent cations may be provided to the enzymatic DNA synthesis in the form of any suitable salt.

[0172] Divalent cations may be present in the nucleotide complex in a ratio between 0.2 and 2 divalent cations per nucleotide. This range includes 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2 divalent cations per nucleotide complex. Those skilled in the art will understand that non-integer numbers represent the sharing of divalent ions between nucleotide free acids.

[0173] Optionally, the nucleotide complex may include a nucleotide associated with a zwitterionic molecule present in a ratio of between 0 and 4 zwitterionic molecules per nucleotide. This range includes 0.0, 1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4 zwitterionic molecules per nucleotide. One of skill in the art will understand that non-integer numbers represent the sharing of molecules between the nucleotide free acids.

[0174] The nitrogenous bases can be adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The nitrogenous bases can also be modified bases, such as 5-methylcytosine (m5C), pseudouridine (Ψ), dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G). The nitrogenous bases can also be artificial bases. The concentration of the nucleotide complex can include any combination of various nitrogenous bases.

[0175] It is preferred that the pentose is deoxyribose and, consequently, the nucleotide is a deoxynucleotide. Nucleotides may be in the form of deoxynucleoside triphosphates, designated dNTPs. This is a preferred embodiment of the present invention. Suitable dNTPs may include dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate), dUTP (deoxyuridine triphosphate), dCTP (deoxycytidine triphosphate), dITP (deoxyinosine triphosphate), dXTP (deoxyxanthosine triphosphate), and derivatives and modified versions thereof. Preferably, dNTPs include one or more of dATP, dGTP, dTTP, or dCTP, or modified versions or derivatives thereof. Preferably, a mixture of dATP, dGTP, dTTP, and dCTP, or modified versions thereof, is used. Any suitable ratio of these dNTPs may be used according to the needs of the reaction.

[0176] The nucleotide complex may be already in solution before mixing with the nucleotidyl transferase, or may be provided as a solid, e.g., powder, and need to be dispersed in the solution. The nucleotide complex may include modified nucleotides. The nucleotide complex may be provided in a mixture of one or more suitable bases, preferably one or more of adenine (A), guanine (G), thymine (T), cytosine (C). Two, three, or preferably all four nucleotides (A, G, T, and C) are used in the process of synthesizing DNA. All of these nucleotide complexes may be present in substantially equal amounts, or one or two may be provided in greater amounts depending on the nature of the DNA to be synthesized.

[0177] All nucleotides may be natural nucleotides (i.e. unmodified), modified nucleotides that act like natural nucleotides and are biologically active (i.e. LNA nucleotide-locked nucleic acids), modified and biologically inactive, mixtures of unmodified and modified nucleotides, and / or mixtures of biologically active and biologically inactive nucleotides. Each type of nucleotide (i.e. base) may be provided in one or more forms, i.e. unmodified and modified, or biologically active and biologically inactive forms. All of these nucleotides are capable of forming suitable complexes.

[0178] In one embodiment of the present invention, the nucleotide or nucleotide complex is present at a concentration of at least 30 mM. According to this embodiment, the nucleotide or nucleotide complex can be present in the reaction mixture at a concentration of more than 30 mM, more than 35 mM, more than 40 mM, more than 45 mM, more than 50 mM, more than 55 mM, more than 60 mM, more than 65 mM, more than 70 mM, more than 75 mM, more than 80 mM, more than 85 mM, more than 90 mM, more than 95 mM, more than 100 mM, more than 110 mM, and more than 120 mM. Such concentrations are given as the concentration of the nucleotide complex when the process begins or at the beginning of the process. The concentrations are given after the addition of the nucleotide / nucleotide complex, where the addition can be to the reaction mixture. The nucleotide complex can be any suitable mixture of nucleotide complexes with various nitrogenous bases. The concentrations apply to the total nucleotide complexes present at the beginning of the process, regardless of the composition of the nucleotide complex. Thus, for example, the nucleotide salts at a concentration of 30 mM can be any mixture of dCTP, dATP, dGTP, and dTTP counterionized with appropriate monovalent and divalent cations.

[0179] It will be understood that the nucleotide provided as a complex may dissociate in water and other solvents to form an anionic nucleotide entity (nucleotide ion, nucleotide ion species) and any associated cation, and optionally a zwitterionic (zwitterion) molecule, if present. Because the nucleotide complex may be protonated due to the drying process, it may remain or form a hydronium ion with any water molecule. According to the definition used in the present invention, neither the proton nor the hydronium ion is considered to be a polyatomic monovalent cation as described herein.

[0180] It is a preferred part of any embodiment of the present invention that the nucleotide complex is formed with a mixture of counterions and zwitterions. Enzymatic DNA synthesis may be maintained under conditions that promote the synthesis of DNA, which will depend on the particular method selected.

[0181] Amplification of the template via strand displacement is preferred. Preferably, the conditions promote amplification of the template by displacement of the replicated strand via strand displacement replication of another strand. Conditions include the use of any temperature that allows amplification of DNA, typically in the range of 20 to 90 degrees Celsius. Preferred temperature ranges can be about 20 to about 40 or about 25 to about 35 degrees Celsius. A preferred temperature for LAMP amplification is about 50 to about 70 degrees Celsius.

[0182] Typically, the appropriate temperature for enzymatic DNA synthesis is selected based on the temperature at which a specific polymerase has optimal activity. This information is generally available and forms part of the general knowledge of the skilled artisan. For example, if phi29 DNA polymerase is used, the suitable temperature range may be about 25 to about 35 degrees Celsius, preferably about 30 degrees Celsius. However, thermostable phi29 can function at higher constant temperatures. The skilled artisan will be able to routinely identify the temperature suitable for efficient amplification according to the process of the invention. For example, to identify the optimal temperature range for a given polymerase, the process may be carried out at various temperatures and the yield of amplified DNA monitored. The amplification may be carried out at a constant temperature, and it is preferred that the process is isothermal. Since strand displacement amplification is preferred, there is no need to change the temperature to separate the DNA strands. Thus, the process may be an isothermal process.

[0183] Other conditions that promote DNA synthesis are conventionally believed to include the presence of a suitable buffer / pH and other factors required for enzyme performance or stability. Suitable conventional conditions include any conditions used to provide activity of polymerase enzymes known in the art.

[0184] For example, the pH of the reaction mixture may be in the range of 3 to 10, preferably 5 to 8, or about 7, for example about 7.5. Although not necessary for the use of the novel nucleotide complex, the pH may be maintained in this range by the use of one or more buffers (also called pH buffers). The function of the buffer is to prevent changes in pH. Such buffer substances (buffers) include, but are not limited to, MES, Bis-Tris, ADA, ACES, PIPES, MOBS, MOPS, MOPSO, Bis-Trispropane, BES, TES, HEPES, DIPSO, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, phosphate, citrate-sodium hydrogen phosphate, citrate-sodium citrate, sodium acetate-acetic acid, imidazole, and sodium carbonate-sodium bicarbonate. As discussed above, buffers that do not contribute additional cations to the reaction mixture or to complexes with metal cations present in the reaction mixture are preferred.

[0185] Buffer substances are generally defined by a mixture of reaction components. Usually, they include a buffering agent to maintain a stable pH to ensure optimal activity or stability of enzymes; one or more additional salts composed of cationic and anionic species, i.e., sodium chloride, potassium chloride; and / or surfactants, such as Triton-X-100. Minimal buffer substances are composed only of buffering reagents that do not provide additional salts or surfactants, with the proviso that a small amount of cationic species may be present for DNA synthesis that requires chemical denaturation. Surprisingly, the use of higher concentrations of nucleotide salts in the process of the present invention allows the use of these minimal buffer substances.

[0186] A "buffer-free" system is one in which there is no pH buffer provided or defined in the reaction component mixture, and no added salts or detergents. This "no added buffer" system contains only reaction components required for DNA synthesis only, and contains cationic species provided only for chemical modification (if required). Thus, there are no additional ions added to this system other than those that serve a specific purpose in the DNA synthesis reaction. Counterions provided with the nucleotides (as complexes) serve to stabilize the nucleotides prior to use in the process.

[0187] Application of heat (exposure to 95°C for several minutes) is used to denature double-stranded DNA, although other approaches more suitable for DNA synthesis may be used. Double-stranded DNA can be easily denatured by exposure to high or low pH environments, or environments in which cations are absent or present at very low concentrations, such as in deionized water. Polymerases require the binding of short oligonucleotide primer sequences to single-stranded regions of the DNA template in order to initiate replication of the DNA template. The stability of this interaction, and therefore the efficiency of DNA synthesis, depends on the metal cations, particularly magnesium (Mg), which can be considered as an essential part of the process. 2+ ) ions.

[0188] Enzymatic DNA synthesis may also require the presence of additional divalent metal ions, i.e., divalent cations that are supplied externally to the nucleotide complex. The method is to use a divalent metal ion: magnesium (Mg 2+ ), Manganese (Mn 2+ ), Calcium (Ca 2+ ), Beryllium (Be 2+ ), Zinc (Zn 2+ ), and strontium (Sr 2+) salts. The divalent ions most commonly used in DNA synthesis are magnesium or manganese, as they act as cofactors in DNA synthesis. It should be noted that any suitable anion can be utilized in such salts, but the choice of anion may affect the pH of the reaction mixture and should be appropriately accounted for.

[0189] Detergents may also be included in the reaction mixture in certain embodiments. Examples of suitable detergents include Triton X-100™, Tween 20™, and any derivatives thereof. Stabilizing agents may also be included in the reaction mixture. Any suitable stabilizing agent may be used, particularly bovine serum albumin (BSA) and other stabilizing proteins. Reaction conditions may also be improved by adding agents that unwind DNA and make template denaturation easier. Such agents include, for example, dimethyl sulfoxide (DMSO), formamide, glycerol, and betaine. DNA condensing agents may also be included in the reaction mixture. Such agents include, for example, polyethylene glycol or cationic lipids or cationic polymers.

[0190] However, in certain embodiments, these components may be reduced or eliminated from the reaction mixture, for example in systems where added buffers are minimal or absent. It should be understood that those skilled in the art can use these additional components and conditions based on their general knowledge to modify and optimize the synthesis conditions for the process of the present invention. Similarly, the specific concentrations of certain agents can be selected based on past examples in the art and further optimized based on general knowledge.

[0191] As an example, the suitable reaction buffer used in the RCA-based method in the art is 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 20 mM (NH4)2SO4, 5% glycerol, 0.2 mM BSA, 1 mM dNTP.The preferred reaction buffer used in RCA amplification is usually 30 mM Tris-HCl pH 7.9, 30 mM KCl, 7.5 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, 2 mM dNTP.This buffer is particularly suitable for use with Phi29 DNA polymerase when conventional nucleotides are purchased.

[0192] A suitable reaction buffer for use with the nucleotide complex of the present invention is 60 mM Tris pH 8.0. A more suitable reaction buffer is 30 mM Tris pH 8.0. Alternative conditions include 30 mM Tris HCl, pH 7.9, 5 mM (NH4)2SO4, and 30 mM KCl. Under certain circumstances, enzymatic DNA synthesis may be performed in water ("no added buffer").

[0193] Enzymatic DNA synthesis may also include the use of one or more additional proteins. The template may be amplified in the presence of at least one pyrophosphatase, such as yeast inorganic pyrophosphatase. Two, three, four, or five or more different pyrophosphatases may be used. These enzymes can degrade pyrophosphate generated by polymerase from dNTPs during strand replication. Accumulation of pyrophosphate in the reaction may cause inhibition of DNA polymerase, reducing the rate and efficiency of DNA amplification. Pyrophosphatase can degrade pyrophosphate to non-inhibitory phosphate. One example of a pyrophosphatase suitable for use in the process of the present invention is Saccharomyces cerevisiae pyrophosphatase, which is commercially available from New England Biolabs, Inc.

[0194] In the process of the present invention, any single-stranded binding protein (SSBP) can be used to stabilize single-stranded DNA. SSBP is an essential component of living cells and participates in all processes involving ssDNA, such as DNA replication, repair, and recombination. In these processes, SSBP can bind to transiently formed ssDNA and help stabilize ssDNA structure. One example of a SSBP suitable for use in the process of the present invention is the T4 gene 32 protein, which is commercially available from New England Biolabs, Inc.

[0195] The yield of the reaction relates to the amount of DNA synthesized. The expected yield from the process according to the invention can be more than 3 g / l. The amount of DNA synthesized is preferably more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g / l or more. The preferred amount of DNA synthesized is 5 g / l. 30 mM nucleotide complex can yield 9.74 g / l of DNA. The present invention improves the possible yield from the enzymatic synthesis of DNA. The aim of the present invention is to improve the yield of cell-free enzymatic DNA synthesis processes so that DNA can be synthesized on a large scale in a cost-effective manner. The present invention makes the production / synthesis of DNA economically possible on an industrial scale using enzymatic processes catalyzed by DNA polymerases or polymerases. The process allows efficient incorporation of nucleotides into the DNA product. It is believed that the process of the present invention allows the reaction mixture to be scaled up to several liters, including tens of liters. Improved yield, productivity, or processability may be compared to the same reaction mixture in which all of the nucleotides are supplied as conventional salts with monovalent cation counterions (typically lithium or sodium).

[0196] In one embodiment, the present invention relates to a method for enhancing DNA synthesis. This enhancement can be compared to an identical reaction mixture except that all of the nucleotide complexes used are exclusively monovalent cation counterions or mixtures thereof. The method can involve the use of the novel nucleotide complexes prepared as described herein.

[0197] In one aspect, the invention provides a cell-free method for synthesizing DNA, comprising contacting a DNA template with at least one nucleotidyl transferase in the presence of one or more nucleotide complexes, each of said nucleotide complexes being associated with between 0.2 and 2 divalent cations and between 0 and 1 monovalent cation, preferably less than 0.2 monovalent cations. The monovalent cations are preferably polyatomic monovalent cations, optionally containing a nitrogen atom. Optionally, the nucleotide complexes may also be associated with between 0 and 4 zwitterionic molecules per nucleotide.

[0198] The concentrations of nucleotides referred to herein with respect to DNA synthesis are preferably the starting concentrations of nucleotides at the start of the process, ie the initial concentrations when the reaction mixture is formed.

[0199] The present invention may also relate to a cell-free process for synthesizing DNA, comprising contacting a DNA template with at least one nucleotidyl transferase in the presence of one or more nucleotide complexes at a concentration of more than 30 mM. The present invention provides a cell-free process for the enzymatic synthesis of DNA, comprising the use of nucleotides provided as complexes, each of said complexes being a nucleotide associated with between 0.2 and 1.5 divalent cations and between 0.2 and 1, optionally less than 0.2 polyatomic monovalent cations, preferably the nucleotide complexes are obtained, provided or present at a concentration greater than 30 mM. Optionally, the nucleotide complexes may also be associated with between 0 and 4 zwitterionic molecules per nucleotide.

[0200] The nucleotide complex may be present at a concentration of at least 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, or 120 mM. Optionally, the complex is present at a concentration of 40 mM to 160 mM, 50 mM to 150 mM, 60 mM to 140 mM, 70 mM to 130 mM, or 80 mM to 120 mM, or any range between these values.

[0201] The present invention further provides an enzymatic DNA synthesis carried out under conditions of reduced or even absent additionally provided divalent cations, preferably magnesium, comprising the use of nucleotide complexes, each of said complexes comprising nucleotides associated with between 0.2 and 1.5 divalent cations and between 0 and 1, optionally less than 0.2 monovalent cations. The provision of divalent cations in the nucleotide complexes avoids the further use of divalent cation salts in the process. However, in certain circumstances, the amount of divalent cation salts, such as magnesium, is reduced using the complexes of the present invention.

[0202] As used herein, one skilled in the art will understand that the novel nucleotide complexes, with respect to ionic entities, substantially contain only those recited with respect to the complexes. In other words, the novel nucleotide complexes consist essentially of the recited entities.

[0203] Terms A. A process for the preparation of a nucleotide complex, comprising the step of drying a nucleotide complex comprising a nucleotide associated with between 0.2-1.5 divalent cations and between 0.2-2.5 polyatomic monovalent cations per nucleotide, wherein the polyatomic monovalent cations are volatile under vacuum.

[0204] B. The process of claim A, wherein the polyatomic monovalent cation is the conjugate acid of a base, and the base is the volatile moiety. C. The process of any one of clauses A and B, wherein the deprotonated form of the polyatomic monovalent cation is the volatile moiety.

[0205] D. The process of any one of the preceding clauses, wherein the drying step is carried out at room temperature or with the application of heat. E. The process of any one of the preceding clauses, wherein the drying step is carried out under vacuum.

[0206] F. Drying step: 10 5 Pa~10 -10 2. The process according to any one of the preceding clauses, carried out under a pressure of less than 1 Pa. G. The process of any one of the preceding clauses, wherein substantially all of the polyatomic monovalent cations become volatile and are removed in the drying process.

[0207] H. The process of any one of the preceding clauses, wherein substantially all of the polyatomic monovalent cations form volatile moieties and are removed in the drying process. I. The method of any one of the preceding clauses, wherein when the nucleotide complex is resuspended in water, a decrease in pH results.

[0208] J. The method of any one of the preceding clauses, wherein the divalent cation is selected from any one or more of magnesium, manganese, or calcium. K. The method of any one of the preceding clauses, wherein the divalent cations are present in a 1:1 ratio with the nucleotide.

[0209] L. The process of any one of the preceding clauses, wherein the monovalent cation is rendered volatile under vacuum, and the resulting volatile moiety has a boiling point of less than 100° C. at standard pressure. M. The method of any one of the preceding clauses, wherein the polyatomic monovalent cation comprises a nitrogen atom, optionally comprising a protonated nitrogen atom.

[0210] N. The process of any one of the preceding clauses, wherein the polyatomic monovalent cation is an ammonium salt or an ionic derivative thereof. O. The method of any one of the preceding clauses, wherein the dried nucleotide complex is resuspended in a solvent.

[0211] P. The method of claim G, wherein the nucleotide complex is resuspendable in the absence of a buffer. Q. The process of any one of the preceding clauses, wherein the nucleotide complex is dried to obtain a powder.

[0212] R. A nucleotide complex produced by the process according to any one of the preceding clauses. S. A cell-free process for the enzymatic synthesis of DNA in solution, comprising obtaining a nucleotide complex as described in clause R and adding a nucleotidyl transferase.

[0213] T. The method, nucleotide complex, or cell-free method of any one of the preceding clauses, wherein the nucleotide complex is soluble. U. The cell-free process of clause S or T, wherein the nucleotide complex is obtained at a concentration of at least 80 mM, optionally at least 100 mM.

[0214] V. The cell-free process of any one of clauses S to U, wherein the nucleotide complex and nucleotidyl transferase form a reaction mixture. Wa) template nucleic acid; b) a primer; c) primase, d) denaturing agents, such as sodium hydroxide or ammonium hydroxide; e) buffering agents, e.g. buffer salts; f) pyrophosphatase, and / or g) Magnesium or manganese salts The cell-free process of any one of clauses S to V, wherein additional components are added to the reaction mixture, including but not limited to any one or more of:

[0215] X. The cell-free process of clause W, wherein a magnesium salt or a manganese salt is added to the reaction mixture as a cofactor for the nucleotidyl transferase such that the ratio of the sum of magnesium and / or manganese to nucleotide does not exceed 2:1.

[0216] Y. The cell-free process of any one of clauses S to X, wherein said nucleotidyl transferase is a DNA polymerase, preferably a strand-displacing DNA polymerase. Z. The cell-free process of clause Y, wherein said nucleotidyl transferase is capable of isothermal DNA synthesis.

[0217] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. "And / or," as used herein, should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be considered as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, each as if individually described herein.

[0218] Unless the context indicates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0219] Although the present invention has been described by way of example with reference to certain embodiments, it will be further appreciated by those skilled in the art that the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the present invention as defined in the appended claims.

[0220] The invention will now be described with reference to some non-limiting examples. Working Example material and method material and method reagent The following supplied reagents were used in the examples shown: Solution 1- 200mM dATP:4NH4 + Solution 2- 200mM dCTP:4NH4 + Solution 3- 200mM dGTP:4NH4 + Solution 4- 200mM dTTP:4NH4 + Solution 5- 66mM dATP:2Mg 2+ Solution 6- 59mM dCTP:2Mg 2+ Solution 7- 64mM dGTP:2Mg 2+ Solution 8- 74mM dTTP:2Mg 2+ Phi29 DNA polymerase, stock concentration 1.6g / L or 0.8g / L (prepared in-house) Thermostable pyrophosphatase, stock concentration 2000U / mL (NEB) DNA primer, stock concentration 5mM (Oligofactory) Plasmid template: eGFP CMV as shown in Figure 1, stock concentration 1.393 g / L (produced in-house) - Examples 2 and 3 ProTLx-K 15-10-15-0-15 eGFP B5x4 iAMB2016 DS3763 as shown in Figure 2, stock concentration 595ng / uL (produced in-house) - Examples 4 to 6 Nuclease-free water (Sigma Aldrich) 5M NaOH (Sigma Aldrich) PEG8000 (Applichem) Tris-Base (Thermo Fisher Scientific) Tris-HCl (Sigma Aldrich) NaCl (Sigma Aldrich) Dimethylsulfoxonium-(isobutanoyl)methylide-C7H 14 O2S (hereafter simply referred to as ylide) (Merck Life Science) L-Histidine (Thermo Fisher Scientific) EXAMPLES

[0221] Preparation of a dNTP mix according to the invention: Ammonium (dNTP: 4NH4 + For the complex, the individual dNTPs (solutions 1-4) were mixed 1:1:1:1 to generate a dNTP mixture with a stock concentration of 200 mM. The mix was stored at -20 °C.

[0222] Magnesium mixed complex (dNTP:NH4 + / Mg 2+ For the 200 µL PCR reaction, dNTPs (solutions 1-4 and 5-8) were mixed to result in equimolar amounts of each specific nucleotide (i.e., dATP, dCTP, dGTP, and dTTP). The mixes were prepared and stored at -20 °C.

[0223] [Table 1]

[0224] [Table 2]

[0225] 200mM 4NH4 + For dNTPs, the ammonium nucleotides were mixed to provide equimolar amounts of each nucleotide as detailed in Table 1. The final volume of 4000 μL of mixed dNTPs contained 200 mM 4NH4 + dNTPs were provided.

[0226] 200mM 1Mg 2+ :2NH4 + As for dNTPs, magnesium nucleotides were mixed to give equimolar amounts of each nucleotide. Volumes correspond to 100 mM of each nucleotide in a final volume of 4000 μL. Ammonium: Magnesium (1 Mg 2+ :2NH4 + To make 1Mg dNTPs, a final volume of 6124 μL of magnesium dNTPs was powdered (i.e., 0 μL) in a speedvac at 60° C. The ammonium premixed nucleotides detailed in Table 2 were used with an additional 2000 μL of water to resuspend the powder and bring it to a final volume of 4000 μL, and 1Mg dNTPs were powdered to a final volume of 4000 μL. 2+ :2NH4 + The dNTPs were obtained and stored at -20°C for later use. These steps were necessary because 2Mg dNTPs are insoluble at relatively high concentrations (100mM) but more soluble at relatively low concentrations. Therefore, the soluble nucleotide salts at relatively low concentrations were mixed with ammonium ion-bound dNTPs after concentrating by drying to form the starting material for making new nucleotide complexes or were used as comparisons.

[0227] 200mM 1Mg 2+ For dNTP, the previously prepared ammonium:magnesium (200 mM 1Mg 2+ :2NH4 +) dNTPs were divided into four 1000 μL aliquots in 15 mL Falcon tubes and either reduced to a sticky solid-like pellet (approximately 20 μl) in a speedvac at 60° C. or dried to a powder (0 μl). Each pellet or powder was resuspended in 1000 μL of water and diluted with 200 mM 1Mg 2+ dNTPs were obtained. Examples 2 and 3 were prepared using nucleotide complexes prepared to a pellet of 20 μl.

[0228] 200mM 1Mg 2+ For dNTP, the previously prepared ammonium:magnesium (200 mM 1Mg 2+ :2NH4 + ) dNTPs were divided into 1000 μL aliquots in 2 mL Eppendorf tubes and either reduced to a sticky solid-like pellet (approximately 20 μl) in a speedvac at 60° C. or dried to a powder (0 μl). Each pellet or powder was resuspended in 1000 μL of water and diluted with 200 mM 1Mg 2+ dNTPs were obtained.

[0229] 200mM 1Mg 2+ 1His, 200mM 1Mg 2+ 2His dNTPs, and 200mM 1Mg 2+ For the 2-ylide dNTPs, the previously prepared ammonium:magnesium (200 mM 1Mg 2+ :2NH4 + ) dNTPs were divided into 1000 μL aliquots in 2 mL Eppendorf tubes and reduced to a sticky solid-like pellet (approximately 20 μl) in a speedvac at 60° C. Each pellet was resuspended in 1000 μL of 200 mM L-histidine (Fisher Scientific), 400 mM L-histidine (Fisher Scientific), or 400 mM ylidene phosphate buffer (Fisher Scientific), respectively. 2+ 1His, 200mM 1Mg 2+ 2His dNTPs, and 200mM 1Mg 2+ Two ylide dNTPs were obtained.

[0230] Drying of nucleotide complexes to remove ammonia The above prepared nucleotide complex (1Mg:2NH4) was placed in a speedvac for drying under vacuum. The speedvac used was an Eppendorf Concentrator Plus with a vacuum of 20 mbar, a temperature of 60°C, and a fixed speed of 1400 rpm. All samples were dried for 5-12 h or 5-24 h until the required level of solvent remained.

[0231] pH of dNTP mix The pH of each dNTP mix was measured before and after the drying procedure to document the physical effect of evaporating volatile entities during the drying process. The pH of ammonium-bearing nucleotide salts is shown for comparison.

[0232] [Table 3]

[0233] It can be seen that after drying, the pH of the nucleotide complex has decreased indicating that the ammonia has been released from the nucleotide complex and evaporated. EXAMPLES

[0234] Setting up the DNA amplification reaction Various nucleotides prepared according to Example 1 were tested in DNA amplification reactions to demonstrate whether such complexes can be used with DNA polymerase and to determine the advantages of using such complexes in the preparation of DNA. Two other nucleotide complexes were used as comparisons. One comparison material is the starting material (1Mg:2NH4dNTP) from which the nucleotide complex is prepared according to the present invention. The other comparison material is a nucleotide complex with four ammonium ions (4NH4). The 4NH4 comparison material was used more extensively in the examples presented herein.

[0235] Reactions were set up at 100μl or 200μL scale as follows: denaturation mix was prepared and left at room temperature for 15 minutes while reaction mix was assembled. These were then mixed and DNA polymerase and pyrophosphatase were added. DNA amplification experiments were performed using various dNTPs at various dNTP concentrations. After a suitable period, for example 168 hours, the reaction was stopped and samples were immediately processed as detailed below. Other periods evaluated were 48 hours, 96 hours, 216 hours, and 480 hours.

[0236] For dNTP complexes lacking magnesium, an equimolar amount of magnesium salt as specified below was added to the reaction mix. For dNTP complexes containing magnesium a (i.e., 1 Mg) as described above, no additional magnesium was provided to the reaction mix. Table 4 shows the results of the reaction of dNTP complexes without magnesium (4NH4 + ) and Table 5 shows the experimental protocol reaction set-up for magnesium complexed dNTPs (1Mg 2+ , 1Mg 2+ :2NH4 + , and 1Mg 2+ The reaction setup for 2His is shown.

[0237] Experiments were carried out to determine whether ammonium could be made volatile and removed from the nucleotide complex, resulting in a nucleotide complex that was still functional in DNA synthesis reactions, particularly rolling circle amplification reactions. Materials and methods for RCA are described above. Reactions were allowed to proceed at a temperature of 30° C. for a defined period of time (168 hours) before processing and quantification. The effect of various nucleotide complexes and their concentrations on DNA synthesis. Here we detail the experimental setup for the data shown in Figure 3.

[0238] [Table 4]

[0239] [Table 5]

[0240] Sample Processing Procedure To each aliquot, 800 μl (or 900 μL in later examples - Examples 5 and 6) of water was added for dilution, followed by 500 μL of 25% PEG8000 and 200 μL of 5M NaCl. The solution was mixed by vigorous shaking and vortexing. The DNA was pelleted by centrifugation in a microcentrifuge (14,000×g, 30 min). The supernatant was carefully decanted and the pellet was resuspended in 1800 μL of water (later examples - Examples 5 and 6 use 1000 μL, 2500 μL, or 5000 μL of water) by positive displacement pipetting and vigorous shaking, then rotated overnight. The next day, reaction DNA concentration was quantified from UV absorption measurements in a nanodrop spectrophotometer, then further vortex mixed.

[0241] Data are corrected for a nine-fold increase in reaction volume and concentrations are expressed as g / L of the original volume relative to the dNTP concentration used. result

[0242] [Table 6]

[0243] [Table 7]

[0244] From this data we conclude that initially, reactions containing 20-80 mM dNTPs with four monovalent counterions produce the greatest yields, but as the concentration increases above 80 mM, there is a significant increase in the amount of raw DNA produced for dNTPs with one divalent counterion (in the case of freshly prepared nucleotides, which are assumed to be protonated or associated with a hydronium ion to balance the charge). 2+ The level of dNTP usage is increased by using 4NH4, which yields 24.7 g / L for freshly prepared nucleotide complexes, peaking at a DNA yield of 17.3 g / L. + It is noted that the nucleotide complex containing magnesium ions maintains a consistent efficiency of conversion to DNA over the entire range of dNTP concentrations tested. EXAMPLES

[0245] Example 2 demonstrates that the newly prepared nucleotide complex can be prepared and used for DNA synthesis in the absence of any additional buffer.Therefore, the effect of buffer is examined.Therefore, Example 2 is repeated in the presence of buffer to determine whether such is useful.

[0246] Reactions were set up at a 200 μL scale as follows: the denaturation mix was prepared and left at room temperature for 15 minutes while the reaction mix was assembled. These were then mixed and DNA polymerase and pyrophosphatase were added. DNA amplification experiments were carried out using various dNTPs at various dNTP concentrations with the addition of 30 mM pH 8.0 Tris buffer. After 168 hours, the reaction was stopped and samples were immediately processed as detailed below.

[0247] For dNTP complexes lacking magnesium, an equimolar amount of magnesium salt as specified below was added to the reaction mix. For dNTP complexes containing magnesium a (i.e., 1 Mg) as described above, no additional magnesium was provided to the reaction mix. Table 8 shows the results of the reaction of dNTP complexes without magnesium (4NH4 + ) and Table 9 shows the experimental protocol reaction set-up for magnesium complexed dNTPs (1Mg 2+ , 1Mg 2+ :2NH 4+ ) The reaction setup for

[0248] Effects of various counterion complexes, their concentrations, and the addition of buffer substances on DNA synthesis.

[0249] [Table 8]

[0250] [Table 9]

[0251] Sample Processing Procedure To each aliquot, 800 μL of water was added for dilution, followed by 500 μL of 25% PEG8000 and 200 μL of 5M NaCl. The solution was mixed by vigorous shaking and vortexing. The DNA was pelleted by centrifugation in a microcentrifuge (14,000×g, 30 min). The supernatant was carefully decanted and the pellet was resuspended in 1800 μL of water by positive displacement pipetting and vigorous shaking, then spun overnight. The next day, reaction DNA concentrations were quantified from UV absorption measurements in a nanodrop spectrophotometer, followed by further spun mixing. Data were corrected for a 9-fold increase in reaction volume, and concentrations are expressed as g / L relative to the original volume relative to the dNTP concentrations used.

[0252] result

[0253] [Table 10]

[0254] [Table 11]

[0255] The data suggest that initially, at 20-80 mM dNTP, the highest yields are obtained from nucleotide complexes with four ammonium counterions, but at higher concentrations, there is a significant increase in the amount of raw DNA produced in nucleotide complexes associated with only one divalent counterion (assumed to be protonated or associated with a hydronium ion to balance the charge). 2+ By using standard dNTP: 4NH4, the nucleotide incorporation efficiency remains stable even at 100 mM. + has a peak DNA yield of 19.2 g / L compared to 22.4 g / L when nucleotides prepared according to the new method were used.

[0256] The nucleotide complexes prepared according to the present invention maintain their efficiency of incorporation into DNA over the range of nucleotide concentrations tested. As seen here, the new nucleotide complexes perform less well in a buffered environment, and therefore it is not a necessary component of these reduced counterion complexes. EXAMPLES

[0257] The experimental conditions described in Example 2 were repeated for a different set of nucleotide complexes. dNTP: 1Mg 2+ , dNTP: 1Mg 2+ :2His, dNTP:4NH4 + The effect of various nucleotide complexes and their concentrations on DNA synthesis.

[0258] [Table 12]

[0259] [Table 13]

[0260] Sample processing procedure - same as in Example 2 result

[0261] [Table 14]

[0262] [Table 15]

[0263] [Table 16]

[0264] [Table 17]

[0265] [Table 18]

[0266] From this data we conclude that initially, reactions containing 20-40 mM dNTPs with four monovalent counterions produce the greatest yields after 20 days, but as the concentration increases above 60 mM, dNTPs with one divalent counterion (in the case of freshly prepared nucleotides that are associated with a hydronium ion to balance the charge) are replaced and there is now a significant increase in the amount of raw DNA produced. + produced the highest yield of raw DNA compared to all other dNTPs and conditions tested, with yields peaking at 16.99 g / L and 14.83 g / L DNA yields at 1 mg / mL. 2+ :2His and 4NH4 + For the first 48 hours, the inventors found that concentrations above 60 mM were significantly lower at 1 Mg 2+ It is noted that the :2His dNTP outperforms all other dNTPs. This observation led us to 2+ We conclude that :2His accelerates the initial reaction and results in significant crude yields in the first 48 h of the RCA reaction. EXAMPLES

[0267] Examples 2 and 4 demonstrate that freshly prepared nucleotide complexes can be prepared and used for DNA synthesis. Based on the results observed so far, we have determined that 1 Mg 2+ It was concluded that the initial reaction rate could be accelerated depending on the dNTP complex. Therefore, Example 4 was repeated for 24, 48, and 72 hours.

[0268] Reactions were set up as previously described in Example 4. Reactions were stopped after 24, 48, and 72 hours and samples were immediately processed as detailed below. For dNTP complexes lacking magnesium, an equimolar amount of magnesium salt, as specified below, was added to the reaction mix. For dNTP complexes containing magnesium a (i.e., 1Mg), no additional magnesium was provided to the reaction mix, as previously described in Examples 2 and 4, Tables 12 and 13, respectively. However, the inventors did not provide additional magnesium to the reaction mix by adding an additional 1Mg 2+ dNTP complex, i.e. 1Mg 2+ : 1His and 1Mg 2+ :2, 1Mg at higher dNTP complex concentrations, i.e., 80 mM, 100 mM, and 120 mM 2+ The accelerating effect of dNTP complexes was examined.

[0269] Sample processing procedure - same as in Example 2 Effect of higher concentrations of various nucleotide complexes on DNA synthesis during the first few days of the reaction.

[0270] [Table 19]

[0271] [Table 20]

[0272] [Table 21]

[0273] This data is based on the results of the nucleotide complex prepared according to the present invention, i.e. dNTP:1Mg 2+ 2H3O + Using 80 mM in a long-incubation reaction resulted in a higher yield of raw DNA, 16.43 g / L, whereas the higher concentrations, 100 mM and 120 mM, were not as high as other 1 Mg 2+ dNTP complex (1Mg 2+ : 1His, 1Mg 2+:2His) outperformed four monovalent counterions of dNTPs and one divalent counterion (in the case of freshly prepared nucleotides, which are associated with a hydronium ion to balance the charge) in the first 72 hours of reaction, and 1 Mg 2+ :2His suggests that at higher dNTP concentrations (100 mM and 120 mM), it produces the highest yields of DNA at 17.48 g / L and 15.34 g / L after 72 hours, respectively. 2+ :1His is 1Mg 2+ 2H3O + It soon followed by matching or exceeding the 1Mg 2+ :2His, at the higher concentration, produced the highest yield of DNA after only 48 hours.

[0274] We conclude that complexes with one divalent counterion outperform one divalent counterion alone and four monovalent counterion dNTPs in the first 24 hours, and therefore these complexes accelerate the reaction. 2+ : 1His, 1Mg 2+ :2His is observed.

[0275] 1Mg 2+ : The ylide dNTPs outperform the four monovalent counterion dNTPs and one divalent counterion alone in the first 24 hours at higher dNTP concentrations of 100 mM to 120 mM.

[0276] The inventors believe that the data presented above is consistent with the results obtained with dNTP complexes of one divalent counterion, specifically 1Mg 2+ : 1His, 1Mg 2+ : 2His, and 1Mg 2+ We conclude that this reinforces previous data that the :2 ylide is an accelerant in the first few days of the reaction, thus allowing us to achieve higher yields of DNA production in a shorter period of time. EXAMPLES

[0277] Example 5 shows the use of one bivalent 1Mg dNTP at higher dNTP concentrations. 2+ We demonstrate that dNTP complexes can be used to accelerate the RCA reaction in the first 72 hours to generate higher yields of DNA. Based on the results observed so far, we propose a method for the synthesis of 1 Mg DNA at higher dNTP concentrations. 2+ We conclude that dNTP complexes accelerate DNA production, allowing high DNA yields in 72 hours. Using this conclusion, we 2+ It was decided to examine the ability of the dNTP complex to produce DNA at higher dNTP concentrations. Therefore, Example 5 was repeated at dNTP concentrations of 80 mM to 120 mM and stopped at 120 hours and 240 hours.

[0278] Reactions were set up as previously described in Examples 2 and 4. Reactions were stopped after 120 and 240 hours and samples were immediately processed as detailed below. Magnesium (i.e., 1 Mg) dNTP complexes were used when no additional magnesium was provided to the reaction mix as previously described in Example 4, Table 13.

[0279] Sample processing procedure - same as in Example 2 Effect of various nucleotide complexes at higher concentrations on DNA synthesis.

[0280] [Table 22]

[0281] [Table 23]

[0282] This data is for 1 Mg of the product prepared according to the present invention. 2+ Nucleotide complex, i.e. dNTP:1Mg 2+ :2H3O + , 1Mg2+:1His, 1Mg2+ These results suggest that using :2His at high dNTP concentrations of 80 mM to 120 mM results in higher yields of raw DNA in long-term incubation reactions.

[0283] At higher dNTP concentrations, 1Mg 2+ :2His is the 1Mg equivalent of all other 1Mg 2+ Outperforms dNTP complexes and is 1Mg 2+ :2His produced the highest yields of raw DNA at 120 and 240 hours, 19.75 g / L and 26.93 g / L. 2+ We observed that the 1Mg:2 yridin dNTP complex produced higher raw DNA yields over 120 and 240 hours compared to other 1Mg2+ dNTP complexes at this particular dNTP concentration; 13.08 g / L and 19.54 g / L, respectively. 2+ :2 yridin dNTPs are more efficient than other 1Mg dNTPs at 80 mM dNTP concentration. 2+ It outperforms dNTP complexes.

[0284] The inventors believe that the data presented above is consistent with the results obtained with dNTP complexes of one divalent counterion, specifically 1Mg 2+ : 1His, 1Mg 2+ : 2His, and 1Mg 2+ We conclude that this reinforces previous data that the :2 ylide is an accelerant in the first few days of the reaction, thus allowing us to achieve higher yields of DNA production in a shorter time interval.

[0285] Conclusion: We have demonstrated that 1Mg dNTPs not only accelerates DNA production in the first few hours of the RCA reaction, but also increases the overall raw DNA yield production, especially at higher dNTP concentrations. 2+ We conclude that there are significant advantages to using dNTP complexes.

Claims

1. A nucleotide complex comprising a nucleotide associated with divalent cations present in a ratio of 0.2 to 1.5 divalent cations per nucleotide.

2. A nucleotide complex as described in claim 1, wherein the nucleotide is further associated with zwitterionic molecules present in an amount of four or less zwitterionic molecules per nucleotide.

3. The nucleotide complex of claim 1, which is in solution.

4. The nucleotide complex described in claim 3, wherein the nucleotide complex is present in water.

5. (i) the divalent cation is a divalent metal cation preferably selected from any one or more of calcium, magnesium, or manganese; (ii) the nucleotide complex contains less than 0.5 monovalent cations per nucleotide; and / or (iii) the concentration of additional ions associated with the nucleotide complex is less than 5%, 4%, 3%, 2%, or 1% of the concentration of the nucleotide complex, or is completely absent; The nucleotide complex of claim 1.

6. A nucleotide complex as described in claim 2, wherein the zwitterionic molecule is an amino acid or an ylide.

7. The nucleotide complex described in claim 6, wherein the zwitterionic molecule is selected from any one or more of histidine, lysine, arginine, or dimethylsulfoxonium-(isobutanoyl)methylide.

8. A nucleotide complex as described in claim 1, wherein the nucleotide complex comprises a nucleotide complex containing a nucleotide associated with one magnesium ion per nucleotide.

9. The nucleotide complex of claim 2, wherein the nucleotide complex comprises: (i) a nucleotide complex comprising a nucleotide associated with one magnesium ion and at least one histidine molecule per nucleotide; or (ii) a nucleotide complex comprising a nucleotide associated with one magnesium ion per nucleotide and at least one methylsulfoxonium-(isobutanoyl)methylide molecule; A nucleotide complex comprising:

10. A nucleotide complex consisting essentially of the nucleotide complex of claim 1.

11. The nucleotide complex of claim 1, which is a deoxynucleoside triphosphate (dNTP).

12. 10. A cell-free method for enzymatic synthesis of DNA in solution, comprising obtaining the nucleotide complex of claim 1 and adding a nucleotidyl transferase.

13. 13. The cell-free process of claim 12, wherein the nucleotide complex is obtained at a concentration of 40 mM to 160 mM.

14. 13. The cell-free process of claim 12, wherein the concentration of the nucleotide complex is maintained at a concentration of 40 mM to 160 mM throughout the synthesis of the DNA.

15. (a) a template nucleic acid; (b) a primer; (c) primase, (d) a denaturing agent, such as sodium hydroxide or ammonium hydroxide; (e) buffering agents, such as buffer salts; (f) pyrophosphatase, and / or (g) magnesium salts or manganese salts 13. The cell-free process of claim 12, wherein additional components are added to the cell-free process, including but not limited to any one or more of:

16. 16. The cell-free process of claim 15, wherein a magnesium salt or a manganese salt is added to the reaction mixture as a cofactor for the nucleotidyl transferase such that the ratio of the sum of magnesium and / or manganese to nucleotide does not exceed 2:

1.

17. 13. The cell-free method of claim 12, wherein the nucleotidyl transferase is a DNA polymerase and the synthesis is an isothermal DNA synthesis.

18. The cell-free method described in claim 17, wherein the DNA polymerase is a strand-displacing DNA polymerase.

19. 1. A cell-free process for enzymatically producing at least 15 g / L of DNA in 40 to 55 hours, comprising the use of a nucleotidyl transferase and a nucleotide complex comprising a nucleotide associated with about one magnesium or manganese ion and, optionally, at least one zwitterionic molecule that is histidine, lysine, arginine, or dimethylsulfoxonium-(isobutanoyl)methylide.

20. The cell-free method of claim 12, carried out in water.

21. 1. A process for preparing a nucleotide conjugate, comprising: (i) providing a starting nucleotide complex comprising a nucleotide associated with a multiatomic monovalent cation, the multiatomic portion of the cation being volatilizable; (ii) applying any one or more of heat, vacuum, and / or pH change to the starting nucleotide complex to allow a proportion of the multi-atom moiety to evaporate; (iii) before, during, or after step (ii), mixing the starting nucleotide complex with a second nucleotide complex, wherein the second nucleotide complex comprises a nucleotide associated with a divalent cation; A manufacturing method including:

22. 22. The method of claim 21, wherein the starting nucleotide complex comprises nucleotides associated with monovalent cations present in a ratio of between 0.2 and 4 polyatomic monovalent cations per nucleotide.

23. 22. The method of claim 21, wherein the second nucleotide complex comprises a nucleotide associated with divalent cations present in a ratio of between 0.5 and 4 divalent cations per nucleotide.

24. (iv) mixing the starting nucleotide mixture with a zwitterionic molecule before, during, or after step (ii); 22. The method of claim 21, comprising:

25. The method of claim 21, wherein step (ii) involves the application of heat and / or vacuum.

26. 22. The process of claim 21, wherein the proportion of polyatomic moieties that are vaporized is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the polyatomic moieties present in the starting material.

27. 22. The method of claim 21, wherein substantially all of the polyatomic moieties are vaporized.

28. 22. The method of claim 21, wherein the polyatomic monovalent cation is the conjugate acid of a base, and the base is the volatile polyatomic moiety.

29. 22. The method of claim 21, wherein the deprotonated form of the polyatomic monovalent cation is the volatile polyatomic moiety.

30. 30. The process of claim 29, wherein the resulting volatile polyatomic moiety has a boiling point of less than 100°C at standard pressure.

31. 22. The method of claim 21, wherein the polyatomic monovalent cation is an ammonium ion or an ionic derivative thereof.

32. 22. The method of claim 21, wherein the prepared nucleotide complex is resuspended in a solvent.

33. The method of claim 32, wherein the solvent is water.

34. 33. The method of claim 32, wherein the nucleotide complex is resuspendable in the absence of a buffer.

35. 22. The method of claim 21, wherein the nucleotide complex is a powder.

36. 25. The method of claim 24, wherein the zwitterionic molecule is any one or more of histidine, lysine, arginine, or dimethylsulfoxonium-(isobutanoyl)methylide.

37. 22. The method of claim 21, wherein the divalent cation is calcium, magnesium, or manganese.

38. 22. The method of claim 21 , wherein step (ii) comprises applying heat to the nucleotide complex such that the temperature of the complex is greater than 40° C., greater than 50° C., greater than 60° C., greater than 70° C., greater than 80° C., greater than 90° C., or greater than 100° C.

39. Step (ii) is 10 ５ 22. The method of claim 21, comprising applying a vacuum created at a pressure between 10 Pa and 100 Pa.

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