Improved reaction mixture for in vitro messenger ribonucleic acid transcription

Abstract

The current invention relates to a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription comprising: ribonucleotide triphosphates (rNTPs) comprising adenosine triphosphates (ATPs), cytidine triphosphates (CTPs), uridine triphosphates (UTPs), guanosine triphosphates (GTPs), an RNA polymerase and magnesium (Mg2+), wherein a molar concentration of the magnesium in said reaction mixture is at least above about 35 mM and lower or equal to about 50 mM. The invention further relates to use of said reaction mixture, a method for producing a mRNA by incubating a DNA template in said reaction mixture and a kit comprising said reaction mixture.

Description

[0001] IMPROVED REACTION MIXTURE FOR IN VITRO MESSENGER RIBONUCLEIC ACID TRANSCRIPTION FIELD OF THE INVENTION The present invention relates to an improved reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription. The invention further relates to use of said reaction mixture, a method for producing a mRNA by incubating a DNA template in said reaction mixture and a kit comprising said reaction mixture. BACKGROUND Applications of synthetic mRNA have grown and become considerably diversified in recent years. In particular, the use of RNA-based vaccines against infectious diseases has emerged over the past decade because of advances in RNA production and formulation. RNA vaccines can trigger stronger and more potent cellular and humoral immune response than plasmid DNA (pDNA) and avoid any potential risk of host cell genome integration. In line with this, rapid-response RNA vaccine production platform technologies are being developed to combat viral epidemics and pandemics. Cost-yield optimization of these platforms is necessary to obtain affordable, rapid-response, high-quality RNA vaccine production. Messenger RNA (mRNA) synthesis by in vitro transcription (IVT) is a cell-free process to produce RNA by enzymatic reactions. In addition, it allows for co- transcriptional capping by incorporating cap analogs at the 5'-end of the transcripts. Capping of the RNA structure plays a crucial role in a variety of cellular processes which include translation initiation, splicing, intracellular transport and turnover. The IVT reaction can be scaled to produce high amounts of mRNA to meet the high demands of RNA vaccine production. Scaling up the IVT reaction, further necessitates cost-yield optimization, especially in relation to expensive input reagents such as enzymes and capping analogs. US20190085368 describes manufacturing methods for production of RNA transcripts. However, the concentration of the reaction mixture components is not optimized, resulting in a suboptimal RNA yield. US20190085368 further mentions co-transcriptional capping using very high amounts of cap analog and resulting in a ˜80% capping efficiency. This high abundance of uncapped species is undesirable when developing therapeutic RNA. Since only capped mRNA is translated into protein, the presence of a high abundance of uncapped species (being 20%) is problematic as efficacy (protein production / mg RNA) is reduced by 20% and 20% of the final drug substance is an inert impurity, decreasing process productivity. Increasing capping efficiency is important for cost-yield optimization. In fact, the major cost driver in RNA production is the 5′ cap analog purchase price. Furthermore, the produced RNA needs to be purified in order to remove contaminants of the IVT reaction, further contributing to the overall high cost of RNA production. US2023183769 describes an IVT method with co-transcriptional capping. However, in US2023183769 nucleoside triphosphates are added at high concentrations, thereby increasing the capping efficiency, but limiting the overall mRNA yield. The present invention targets at solving at least one of the above-mentioned disadvantages. SUMMARY OF THE INVENTION The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription according to claim 1. More particular, the invention relates to a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription comprising: ribonucleotide triphosphates (rNTPs) comprising adenosine triphosphates (ATPs), cytidine triphosphates (CTPs), uridine triphosphates (UTPs), guanosine triphosphates (GTPs), an RNA polymerase and magnesium (Mg2+), wherein a molar concentration of the magnesium in said reaction mixture is at least above about 35 mM and lower or equal to about 50 mM. The inventors surprisingly found that said specific concentration range is optimized not only for optimizing the mRNA yield and improving the cost / yield ratio of the IVT reaction (especially when performing co-transcriptional capping), but also for decreasing the level of contaminants present in the obtained IVT reaction product, thereby reducing the cost related to purification of the produced mRNA. As such, the current invention, which allows for optimization of the IVT reaction, also minimizes complex and expensive purification steps. In the current invention, the upstream IVT process consistently delivers a high yield of capped mRNA transcripts and low impurity levels, requiring minimal downstream processing with minimal product losses. Furthermore, the optimized reaction mixture is validated for multiple constructs having different sizes. Preferred embodiments of the reaction mixture are shown in any of the claims 2 to 11. A preferred embodiment relates to a reaction mixture comprising a molar ratio of total NTPs to magnesium which is less than 0.85. Related thereto, in a second aspect, the present invention relates to a use according to claim 12. More particular, the use as described herein relates to the use of aforementioned reaction mixture for improving capping efficiency during a co- transcriptional capping reaction. Present invention provides optimal reaction conditions allowing to reduce the amount of cap analog necessary for performing efficient co-transcriptional capping. In a third aspect, the present invention relates to a method according to claim 13. More particular, the method as described herein relates to a method for producing a capped messenger ribonucleic acid (mRNA) comprising: incubating a DNA template in aforementioned reaction mixture, performing an in vitro transcription (IVT) and co-transcriptional capping reaction of said DNA template, and thereby producing the capped mRNA. Preferred embodiments of the method are shown in any of the claims 14 to 26. In a last aspect, the invention relates to a kit according to claim 27. More particular, the kit as described herein relates to a kit for use in the production of capped RNA, wherein said kit comprises aforementioned reaction mixture for performing an in vitro RNA transcription and co-transcriptional capping reaction, DNase for removal of template DNA molecules, and a metal chelator for terminating the IVT reaction. A preferred embodiments of the kit is shown in claim 28. DESCRIPTION OF FIGURES Figure 1 shows process performance of the IVT reaction (Figure 1A: IVT yield (RNA concentration in µg / µl), Figure 1B: capping efficiency (%) and Figure 1C: dsRNA contamination (ng dsRNA / mg mRNA)) using an IVT reaction mixture according to an embodiment of the invention. Figures 2-7 show process performance of the IVT reaction (yield, capping efficiency dsRNA contamination and residual pDNA) and RNA product parameters after purification (yield, integrity and residual protein) using an IVT reaction mixture according to an embodiment of the invention. Bars represent prediction intervals, which allow to predict in what range a future individual observation will fall. Figure 2 shows the yield (RNA concentration in mg / ml) as measured for 245 IVT reactions (including 45 different constructs) when using an optimized IVT reaction mixture according to an embodiment of the current invention. Figure 2B shows the yield (RNA concentration in mg / ml) in function of constructs having a different size (nt=nucleotides). Figure 3 shows the capping efficiency (%) as measured for 111 IVT reactions (including 19 different constructs) when using an optimized IVT reaction mixture according to an embodiment of the current invention. Figure 3B shows the capping efficiency (%) for constructs having a different size (nt=nucleotides). Figure 4 shows the dsRNA content (ng / mg RNA) as measured for 83 IVT reactions using unmodified UTP (including 36 different constructs) and 36 IVT reactions using modified UTP (including 31 different constructs) when using an optimized IVT reaction mixture according to an embodiment of the current invention. Figure 4B shows the dsRNA content (ng / mg RNA) for constructs having a different size (nt=nucleotides). Figure 5 shows the residual plasmid DNA (pDNA, ng / mg) content as measured for 39 IVT reactions (including 6 different constructs comprising an Ampicillin resistance gene, AMPR when using an optimized IVT reaction mixture according to an embodiment of the current invention. Figure 5B shows the residual pDNA content for constructs having a different size (nt=nucleotides). Figure 6 shows RNA purity and integrity assessment for 6 constructs (Figure 6A-F) as measured by capillary electrophoresis, showing low impurity levels when using an optimized IVT reaction mixture according to an embodiment of the current invention. Figure 7 shows (A) the purification yield for 28 IVT reactions (including 3 different constructs) post-purification compared to pre-purification (%), (B) RNA integrity for 22 IVT reactions (including 3 different constructs) post-purification compared to pre- purification (ratio) and (C) residual protein content (protein / RNA (%)) after purification for 19 purification reactions (including 2 different constructs) of mRNA prepared using an IVT reaction mixture according to an embodiment of the current invention. Figure 8 shows a comparison of IVT process performance using IVT reaction mixtures having a different magnesium concentration, including an IVT reaction mixture according to an embodiment of the invention having a molar concentration of 45 mM. Figure 9 shows the visual appearance after IVT incubation of 3 IVT reaction mixtures having a different magnesium concentration, including an IVT reaction mixture according to an embodiment of the invention having a molar concentration of 45 mM. The IVT reactions were performed in triplicate (Figures 9A-C). Figure 10 shows the visual appearance after DNase incubation of the 3 IVT reaction mixtures having a different magnesium concentration, including an IVT reaction mixture according to an embodiment of the invention having a molar concentration of 45 mM. The IVT reactions were performed in triplicate (Figures 10A-C). Figure 11 shows the visual appearance after EDTA addition of the 3 IVT reaction mixtures having a different magnesium concentration, including an IVT reaction mixture according to an embodiment of the invention having a molar concentration of 45 mM. The IVT reactions were performed in triplicate (Figures 11A-C). DETAILED DESCRIPTION OF THE INVENTION The present invention concerns an improved reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription. The invention further relates to use of said reaction mixture, a method for producing a mRNA by incubating a DNA template in said reaction mixture and a kit comprising said reaction mixture. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. As used herein, the following terms have the following meanings: “A”, “an”, and “the” as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a compartment” refers to one or more than one compartment. “About” as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier “about” refers is itself also specifically disclosed. “Comprise”, “comprising”, and “comprises” and “comprised of” as used herein are synonymous with “include”, “including”, “includes” or “contain”, “containing”, “contains” and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. Whereas the terms “one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any ≥3, ≥4, ≥5, ≥6 or ≥7 etc. of said members, and up to all said members. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination. Nucleotides (comprising ribonucleotides and deoxyribonucleotides) are the basic building blocks of DNA and RNA. Ribonucleotides themselves are basic monomeric building blocks for RNA. Deoxyribonucleotides, formed by reducing ribonucleotides with the enzyme ribonucleotide reductase (RNR), are essential building blocks for DNA. The general structure of a ribonucleotide consists of a phosphate group, a ribose sugar group, and a nucleobase, in which the nucleobase can either be adenine, guanine, cytosine, or uracil. Without the phosphate group, the composition of the nucleobase and sugar is known as a nucleoside. "Ribonucleotide" relates to a nucleotide with a hydroxyl group at the 2'-position of a β- D-ribofuranosyl group. Successive nucleotides are linked together via phosphodiester bonds. In the context of the present invention, the term "RNA" relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. In particular, the term can refer to double stranded RNA (dsRNA), single stranded RNA (ssRNA), isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non- standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA. According to the present invention, the term "RNA" includes and preferably relates to "mRNA" which means "messenger RNA" and relates to a "transcript" which may be produced using DNA as template and encodes a peptide or protein. mRNA typically comprises a 5' untranslated region (5’ -UTR), a protein or peptide coding region and a 3' untranslated region (3'-UTR). mRNA has a limited halftime in cells and in vitro. The term ‘modified mRNA molecules’ means mRNA molecules that contain one or more modified nucleosides (termed "modified nucleic acids"), which have useful properties such as the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced. These modified nucleic acids enhance the efficiency of protein production, intracellular retention of nucleic acids, and viability of contacted cells, as well as possess reduced immunogenicity. An exemplary suitable modified nucleoside may for example by N1-methyl pseudouridine or pseudouridine. For the sake of clarity, a mRNA encompasses any coding RNA molecule, which may be translated by an eukaryotic host into a protein. In a particular embodiment, RNA obtained in said in vitro transcription reaction may be capped and uncapped RNA, modified and unmodified RNA, or RNA with and without poly(A) tail, or any combination thereof. According to the invention, an RNA molecule, such as a messenger RNA (or mRNA), comprises the following types: uncapped unmodified RNA without poly(A)tail, uncapped unmodified RNA with poly(A)tail, uncapped modified RNA without poly(A)tail, uncapped modified RNA with poly(A)tail, capped unmodified RNA without poly(A)tail, capped unmodified RNA with poly(A)tail, capped modified RNA without poly(A)tail, capped modified RNA with poly(A)tail. In the context of the present invention, the term “capped RNA” is to be understood as an RNA molecule of which the 5' end is linked to a guanosine or a modified guanosine, preferably a 7-methylguanosine (N7-methyl guanosine or m7G), connected to a 5' to 5' triphosphate linkage or analog. "Capping" of the RNA structure plays a crucial role in a variety of cellular processes which include translation initiation, splicing, intracellular transport and turnover. In vitro synthesis of capped mRNAs is performed by bacteriophage RNA polymerase (T7, SP6 or T3)- mediated in vitro transcription that co-transcriptionally incorporate cap analogs at the 5'-end of the transcripts. Alternatively, post-transcriptional enzymatic capping may also be used to add a 5’CAP to the IVT produced RNA molecules. In the context of the present invention, the term “uncapped RNA” is to be understood as any RNA molecule that does not comprise a cap as defined in the definition “capped RNA”. Thus, in a particular embodiment, “uncapped mRNA” may refer to an mRNA of which the 5' end is not linked to a 7-methylguanosine, through a 5' to 5' triphosphate linkage, or an analog as previously defined. In the context of the present invention, the term “modified RNA" is to be understood as an RNA molecule which contains at least one modified nucleotide, nucleoside or base, such as a modified purine or a modified pyrimidine. A modified nucleoside or base can be any nucleoside or base that is not A, U, C or G (respectively Adenosine, Uridine, Cytidine or Guanosine for nucleosides; and Adenine, Uracil, Cytosine or Guanine when referring solely to the sugar moiety). In the context of the present invention, the term “unmodified RNA” is to be understood as any RNA molecule that does not comprise a modification as defined in the definition “modified RNA”. As used herein, the term “poly(A) tail” is to be understood as a moiety comprising multiple adenosine monophosphates and is well known in the art. A poly(A) tail is generally produced during a step called polyadenylation that is one of the post- translation modifications which generally occur during the production of mature messenger RNAs; such poly(A) tail contributes to the stability and the half-life of said mRNAs, and can be of variable length. In particular, a poly(A) tail may be equal or longer than 10 adenosine nucleotides, which includes equal or longer than 20 adenosine nucleotides, which includes equal or longer than 100 adenosine nucleotides, and for example about 120 adenosine nucleotides. In the context of the present invention, the term “without poly(A) tail” is to be understood as any RNA molecule that does not comprise a poly(A) tail as described in the definition “poly(A) tail”. In the sense of the invention, the terms “modified and unmodified” are considered distinctly from “capped and uncapped”, as the latter specifically relates to the base at the 5'-end of a RNA molecule, and also distinctly from “with poly(A)tail and without poly(A)tail”. Description Applications of synthetic mRNA have grown and become considerably diversified in recent years. Examples include the generation of vaccines and therapeutics, and CRISPR / Cas9 genome editing applications. In particular, the use of RNA-based vaccines against infectious diseases has emerged over the past decade because of advances in RNA production and formulation. RNA vaccines can trigger stronger and more potent cellular and humoral immune response than plasmid DNA (pDNA) and avoid any potential risk of host cell genome integration. In line with this, rapid- response RNA vaccine production platform technologies, are being developed to combat viral epidemics and pandemics. Cost-yield optimization of these platforms is necessary to obtain affordable, rapid-response, high-quality RNA vaccine production. RNA can be efficiently synthesized in vitro (by in vitro transcription, IVT) with prokaryotic phage polymerases, such as T7, T3 and SP6. The in vitro transcription methodology is known to the skilled person and comprises besides an appropriate RNA polymerase, a purified linear DNA template containing a promoter, ribonucleotide triphosphates, spermidine and a buffer system that includes dithiothreitol (DTT) and magnesium ions. The cap and poly(A) tail structures characteristic of mature mRNA can be added during or after the synthesis by enzymatic reactions with capping enzymes and Poly(A) Polymerase, respectively. Magnesium ions are known to influence the catalytic activity of the RNA polymerase and to induce RNA degradation. The current disclosure found an optimum in magnesium concentration to optimize RNA yield, minimize production costs, while still maintaining consistent product quality even under inherent process fluctuations. The optimum in magnesium concentration results from a complex interplay of different factors which will be discussed in more detail below, such as enzyme saturation, magnesium-facilitated RNA degradation, and precipitation out of solution through magnesium pyrophosphate. The present invention relates to an improved reaction mixture having a specific concentration range of magnesium for in vitro messenger ribonucleic acid (mRNA) transcription with an optimized cost / yield ratio. The invention further relates to the use of said reaction mixture, a method for producing a mRNA by incubating a DNA template in said reaction mixture and a kit comprising said reaction mixture. In summary, the invention relates to: A reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription comprising: ribonucleotide triphosphates (rNTPs) comprising adenosine triphosphates (ATPs), cytidine triphosphates (CTPs), uridine triphosphates (UTPs), guanosine triphosphates (GTPs), an RNA polymerase and magnesium (Mg2+), wherein a molar concentration of the magnesium in said reaction mixture is at least above about 35 mM and lower or equal to about 50 mM. In an embodiment of the reaction mixture, the total molar concentration the NTPs in the reaction mixture is between 16 and 40 mM. In an embodiment, said reaction mixture comprises a molar ratio of total NTPs to magnesium which is less than 0.85. In an embodiment, the reaction mixture further comprises a cap analog for co- transcriptional capping, wherein a molar concentration of said cap analog in the reaction mixture is between about 1 and about 6 mM. In an embodiment, the reaction mixture further comprises a less than 1 molar ratio of total NTPs plus said cap analog to said magnesium. In an embodiment, a concentration of RNA polymerase in the reaction mixture is between 4 and 8 kU / ml. In an embodiment, said reaction mixture has a pH of less than 8. In a further aspect, the invention relates to use of aforementioned reaction mixture for improving a capping efficiency during a co-transcriptional capping reaction. In a further aspect, the invention relates to a method for producing a messenger ribonucleic acid (mRNA) comprising: incubating a DNA template in aforementioned reaction mixture, performing an in vitro transcription (IVT) reaction of said DNA template, and thereby producing the mRNA. In an embodiment of aforementioned method, said DNA template has a concentration of less than about 50 µg / ml. In an embodiment of aforementioned method, the method further comprises removing template DNA molecules by adding DNase, wherein said DNase concentration is added at a concentration reaction below 20 U / µg of DNA. In a further aspect, the invention relates to a kit for use in the production of RNA, wherein said kit comprises aforementioned reaction mixture for performing an in vitro RNA transcription, DNase for removal of template DNA molecules, and a metal chelator for terminating the IVT reaction. In an embodiment, said kit further comprises a reaction mixture for purification of the RNA. In a first aspect, the disclosure provides a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription comprising: ribonucleotide triphosphates (rNTPs) comprising adenosine triphosphates (ATPs), cytidine triphosphates (CTPs), uridine triphosphates (UTPs), guanosine triphosphates (GTPs), an RNA polymerase and magnesium (Mg2+), wherein a molar concentration of the magnesium in said reaction mixture is at least above about 35 mM and lower or equal to about 50 mM. In a preferred embodiment, the reaction mixture further comprises a cap analog for co-transcriptional capping. In an embodiment of the reaction mixture, as disclosed herein, the total molar concentration the rNTPs in the reaction mixture is between 16 mM and 40 mM. Preferably, the total molar concentration of the rNTPs in the reaction mixture is between 16 mM and 39 mM, between 16 mM and 38 mM, between 16 mM and 37 mM, between 16 mM and 36 mM, between 16 mM and 35 mM, between 16 mM and 34 mM, between 16 mM and 33 mM, between 16 mM and 32 mM, between 16 mM and 31 mM, between 16 mM and 30 mM, between 16 mM and 29 mM, between 16 mM and 28 mM, between 16 mM and 27 mM, between 16 mM and 26 mM, between 16 mM and 25 mM, between 16 mM and 24 mM, between 16 mM and 23 mM, between 16 mM and 22 mM, between 16 mM and 21 mM, between 16 mM and 20 mM, between 16 mM and 19 mM, between 16 mM and 18 mM, or between 16 mM and 17 mM and all ranges and subranges therebetween. Alternatively, the total molar concentration of the rNTPs in the reaction mixture is between 17 mM and 40 mM, between 18 mM and 40 mM, between 19 mM and 40 mM, between 20 mM and 40 mM, between 21 mM and 40 mM, between 22 mM and 40 mM, between 23 mM and 40 mM, between 24 mM and 40 mM, between 25 mM and 40 mM, between 26 mM and 40 mM, between 27 mM and 40 mM, between 28 mM and 40 mM, between 29 mM and 40 mM, between 30 mM and 40 mM, between 31 mM and 40 mM, between 32 mM and 40 mM, between 33 mM and 40 mM, between 34 mM and 40 mM, between 35 mM and 40 mM, between 36 mM and 40 mM, between 37 mM and 40 mM, between 38 mM and 40 mM, or between 39 mM and 40 mM and all ranges and subranges therebetween. In yet another alternative embodiment, the total molar concentration of the rNTPs in the reaction mixture is between 17 mM and 39 mM, between 18 mM and 38 mM, between 19 mM and 37 mM, between 20 mM and 36 mM, between 21 mM and 35 mM, between 22 mM and 34 mM, between 23 mM and 33 mM, between 24 mM and 32 mM, between 25 mM and 31 mM, between 26 mM and 30 mM, or between 27 mM and 29 mM and all ranges and subranges therebetween. Preferably, the individual rNTPs (ATP, CTP, UTP, and GTP) are present in equimolar ratios in the reaction mixture. However, it would be obvious to the skilled person that the ratios of the individual rNTPs can vary without departing from the scope of the invention. In an embodiment, the reaction mixture comprises a molar ratio of total rNTPs to said magnesium which is less than 0.85. Alternatively, the reaction mixture comprises a less than 1 molar ratio of total rNTPs to said magnesium. More preferably, the reaction mixture comprises a less than 0.90, more preferably less than 0.85 molar ratio of total rNTPs to said magnesium. In a preferred embodiment, the reaction mixture comprises a molar ratio of total rNTPs to said magnesium between 0.50 and 1, more preferably between 0.50 and 0.90, more preferably between 0.50 and 0.85, and all ranges and subranges therebetween. As used herein and unless otherwise specified, the term “magnesium” (abbreviated as “Mg” or “Mg2+”) is to be understood as a chemical element essential to the basic nucleic acid chemistry of all cells of all known living organisms. In the present invention, magnesium can be in any salt form comprising magnesium chloride and magnesium acetate. More than 300 enzymes require magnesium ions for their catalytic action, including enzymes using or synthesizing ATP and those that use other nucleotides to synthesize DNA and / or RNA. According to the invention, Mg2+ ions are provided by any of the described magnesium forms and are needed to catalyze the reactions driven by for example RNA polymerases such as T3, T7, SP6, the pyrophosphatase and the DNase I. Accordingly, this component needs to be provided throughout the whole reaction and has a specific function for the enzymes and hence influences IVT yield. As described above, magnesium is a cofactor of the nucleic acid polymerization catalyzed by DNA and RNA polymerases. The positions of two magnesium ions are conserved in the active site of all presently crystallized polymerases and it has been proposed that these two ions are essential for catalysis. As such, the magnesium ion concentration has an influence on the elongation mode of the RNA polymerase enzyme and hence the RNA yield. In line with this, the current disclosure shows that a higher magnesium concentration results on average in a higher amplification factor (meaning a higher RNA yield). Conventional concentrations of approximately 19 mM of magnesium are used during in vitro transcription reactions. One important drawback when using such a magnesium concentration is the presence or generation of certain by-products of the in vitro synthesis process, including double- stranded RNA (dsRNA), that trigger cellular immune responses. Patent application WO2022248565 discloses the reduction of dsRNA by-product formation during in vitro transcription (IVT) with an elevated concentration of magnesium in the reaction. In particular, WO2022248565 describes a method for reducing double stranded RNA (dsRNA) formation during in vitro transcription in the presence of at least about 35 mM of magnesium - compared to conventional concentrations of approximately 19 mM of magnesium. The main advantage of this method to produce IVT RNAs is a reduction of 50-70% of total dsRNA while the yield and integrity of the produced RNA is not compromised. As such, by providing a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription, wherein a molar concentration of the magnesium in said reaction mixture is at least above about 35 mM, the magnesium concentration is high enough to saturate the RNA polymerase (allowing transcription to proceed) and reduce the formation of dsRNA by-products. However, it is known that too high concentrations of magnesium can be inhibitory and can degrade RNA by catalyzing in-line attack of the 2’-oxygen on the backbone phosphate. Furthermore, the inventors have found that a too high concentration of magnesium leads to a cloudy appearance of the IVT reaction product. This appearance seems to be caused by the excessive precipitation of insoluble magnesium pyrophosphate (Mg2PPi) (see Example 3, Figures 9-11). T7 RNA polymerase needs Mg2+ ions for binding to the DNA template and the Mg2+ ion also forms complexes with NTPs prior to entering the active site of the T7 RNA polymerase enzyme. When the T7 RNA polymerase binds to the DNA template, this complex binds to the magnesium-NTPs complex and pyrophosphate is released. As such, the pyrophosphate anion (PPi) is a product of transcription, with one PPi molecule generated for every nucleotide triphosphate that is incorporated into the growing RNA chain. As a result, PPi produced during enzymatic nucleic acid synthesis can react with Mg2+ in the reaction buffer to form insoluble magnesium pyrophosphate (Mg2PPi). Such a precipitate can be a disadvantage when performing analytical methods to assess the RNA quality. Furthermore, without wishing to be bound by theory, it is known that RNA and inorganic pyrophosphate, can self-assemble to form composite microsponge structures composed of nanocrystalline magnesium pyrophosphate sheets (Mg2P2O7●3.5H2O) with RNA adsorbed to their surfaces. The microsponge particles contain high loadings of RNA (15-21 wt.%). Said precipitates and microsponge particles remove RNA from the IVT reaction mixture solution, thereby reducing the RNA yield of the reaction. Furthermore, said precipitates and microsponge particles can be lost during further purification steps, again reducing the RNA yield of the reaction. Mg2PPi precipitation can strongly inhibit the transcription reaction. This can be due to a lack of free Mg2+ in solution, thereby reducing the rate of the transcription reaction. But, without wishing to be bound by theory, Mg2PPi precipitation can also strongly inhibit the transcription reaction by means of a physical interaction between the RNA polymerase and the Mg2PPi precipitation at the moment of the transition. In the transcription reaction, PPi4− ion is released from the incorporated MgNTP2− through the dephosphorylation by RNA polymerase. Therefore, if the Mg2PPi precipitation occurs immediately after the PPi4- ion is released, the precipitation that occurs close to the RNA polymerase may inhibit the transcription reaction by physical interaction. In this case, the transcription reaction may stop even if the free Mg2+ concentration is sufficient. Furthermore, as described above, the magnesium pyrophosphate can also co-assemble with RNA into nanostructured particles, thereby precipitating RNA out of solution and reducing the yield of RNA production. In an embodiment, the reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription further comprises the enzyme pyrophosphatase. In an embodiment, said IVT reaction is performed in the presence of pyrophosphatase. In another specific embodiment of the reaction mixture or the method of the present invention, the concentration of pyrophosphatases is about and between 0.01 U / ml to about 40 U / ml, preferably about and between 0.1 U / ml and about 20 U / ml, more preferably about and between 1 U / ml and about 10 U / ml. In some embodiments, said concentration of pyrophosphatase may be at least about 0.01 U / ml. As used herein, the term “pyrophosphatase”, also known as diphosphatase, is to be understood as acid anhydride hydrolases that act upon diphosphate bonds. The term preferably relates to inorganic pyrophosphatase which catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate. Inorganic pyrophosphate is released when a nucleoside triphosphate is incorporated / polymerized into the growing chain. Pyrophosphate is an inhibitor of RNA polymerization and therefore, removal leads to an increase in RNA yield in IVT. Mg ions are necessary for catalytic activity of crystalline pyrophosphatase. In a further aspect, pyrophosphatase may also be selected from the list comprising tobacco acid pyrophosphatase, which catalyses the hydrolysis of a phosphoric ester, various organic pyrophosphatases, which act upon organic molecules with the pyrophosphate group (but excluding triphosphatases that act on the final bond), thiamine pyrophosphatase. Furthermore, the inventors of the present invention have unexpectedly found that too high concentrations of magnesium lead to a reduced capping efficiency. As such, the magnesium concentration should be lower or equal to 50 mM in order to efficiently and cost-effectively perform a co-transcriptional capping reaction. However, a too low magnesium concentration (i.e. meaning 35 mM or below) leads to a reduced reaction yield and an increased formation of dsRNA (an unwanted side product of the IVT reaction). By providing a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription, wherein a molar concentration of the magnesium in said reaction mixture is lower or equal to about 50 mM, a high capping efficiency can be maintained, even when lower concentrations of cap analog are present in the reaction mixture. As such, by limiting the magnesium concentration, the concentration of cap analog can be reduced, resulting in a more cost-efficient IVT reaction. In fact, the major cost driver in RNA production is the 5′ cap analog purchase price. In a preferred embodiment a molar concentration of said cap analog in the reaction mixture is between about 1 and about 6 mM. In a preferred embodiment a molar concentration of said cap analog in the reaction mixture is below 6.0 mM, more preferably below 5.5 mM, more preferably below 5.0 mM, more preferably below 4.5 mM, more preferably below 4.0 mM. In a preferred embodiment, said reaction mixture comprises a molar ratio of cap analog to total rNTPs which is less than 0.125. In a preferred embodiment, said reaction mixture comprises a molar ratio of cap analog to total rNTPs which is less than 0.100, such as a molar ratio between 0.050 and 0.090. In a preferred embodiment, said reaction mixture comprises a molar ratio of cap analog to total rNTPs between 0.050 and 0.125, more preferably between 0.060 and 0.110, more preferably between 0.070 and 0.100, more preferably between 0.080 and 0.090. As used herein, “cap analogs” are caps which are biologically equivalent to a 7- methylguanosine (m7G), and comprise traditional analogs such as G(5’)ppp(5’)G, m7G(5’)ppp(5’)G or m2,2,7G(5’)ppp(5’)G, but also Anti-Reverse Cap Analog (ARCA) 3'-O-Me-m7G(5')ppp(5')G, Unmethylated Cap Analog G(5')ppp(5')G, Methylated Cap Analog for A+1 sites m7G(5')ppp(5')A; Unmethylated Cap Analog for A+1 sites G(5')ppp(5')A. Anti-Reverse Cap Analog (ARCA) is a modified cap analog in which the 3' OH group (closer to m7G) is replaced with -OCH3that forces ARCA incorporation in the correct orientation and subsequently results in a translatable mRNA population. In a preferred embodiment, said cap analog is a trinucleotide with a 5’-m7G joined by a 5-5´triphosphate linkage to an AG sequence. The adenine has a methyl group on the 2´-O position, such as illustrated in formula I below: Formula I In order to use such a cap analog in an in vitro transcription reaction the template must contain an AG in place of a GG following the T7 promoter in the initiation sequence. The incorporation of this trinucleotide in the beginning of a transcript results in a Cap-1 structure, meaning that the 2' hydroxyl of the ribose in the penultimate nucleotide with respect to the cap nucleotide is methylated. The reaction mixture further comprises ribonucleotide triphosphates (rNTPs) comprising adenosine triphosphates (ATPs), cytidine triphosphates (CTPs), uridine triphosphates (UTPs), guanosine triphosphates (GTPs). RNA synthesis can also be carried out with a mixture of modified nucleotides in place of the regular mixture of A, G, C and U triphosphates. For expression applications, the modified nucleotides of choice are the naturally occurring 5´-methylcytidine and / or pseudouridine in the place of C and U, respectively. These have been demonstrated to confer desirable properties to the mRNA, such as increased mRNA stability, increased translation, and reduced immune response in the key applications of protein replacement and stem-cell differentiation. In some embodiments, the RNA molecules comprise at least one chemical modification comprising backbone modification, sugar modification, or base modification. In this context, a modified RNA molecule comprises nucleotide modifications, e.g. backbone modifications, sugar modifications or base modifications. A sugar modification in connection with the present disclosure is a chemical modification of the sugar of the nucleotides of the RNA molecule. Furthermore, a base modification in connection with the present disclosure is a chemical modification of the base moiety of the nucleotides of the RNA molecule. In this context, nucleotide modifications are selected from nucleotide modifications that are applicable for transcription and / or translation. In further embodiments, the modified RNA comprises nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, α- thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl- pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5- hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α- thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza- guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino- purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio- adenosine, 8-azido-adenosine, 7-deaza-adenosine, and a combination thereof. The molar concentration of each rNTP is of importance. In particular, the ratio of magnesium to total rNTPs (including cap analog) is a critical parameter influencing efficient IVT through the catalytic activity of T7 polymerase. Magnesium is a cofactor of the nucleic acid polymerization catalyzed by DNA and RNA polymerases. The positions of two magnesium ions are conserved in the active site of all presently crystallized polymerases and it has been proposed that these two ions are essential for catalysis. In the following, [rNTP] stands for the (equal) concentration of each type of rNTP introduced in the sample, and [rNTP]totstands for the sum of all [rNTP] (being 4 × [rNTP] if the concentration is the same for each rNTP) plus [cap analog], also described as “total rNTPs plus cap analog molar concentration”. In a preferred embodiment, the reaction mixture comprises a less than 1 molar ratio of total rNTPs plus cap analog to said magnesium. More preferably, the reaction mixture comprises a less than 0.90, more preferably less than 0.85 molar ratio of total rNTPs plus cap analog to said magnesium. In a preferred embodiment, the reaction mixture comprises a molar ratio of total rNTPs plus cap analog to said magnesium between 0.50 and 1, more preferably between 0.50 and 0.90, more preferably between 0.50 and 0.85. When a too low concentration of rNTPs is used, it limits RNA production. One explanation for this phenomenon is that if [Mg2+]tot < [rNTP]tot, the free magnesium concentration ([Mg2+]free) will be very low; in such conditions, the elongation velocity is expected to be drastically reduced, a scenario that has been observed with HIV-1 reverse transcriptase, which is closely related to T7 RNA polymerase. In solution, one magnesium ion, Mg2+, can react with one nucleotide, NTP4− , to form MgNTP2− with a dissociation constant, Kd, of ∼30 μM at 37°C. A second magnesium ion can react with the MgNTP2− complex, but with a much larger Kd of ∼25 mM. The other species have either a large Kd or are minority species in our buffer. It can therefore be assumed that in the transcription mix, due to the low Kd, essentially each nucleotide reacts with one magnesium ion to form MgNTP2− complexes, provided that [Mg2+]tot > [rNTP]tot. It can then be assumed that one of the two ions in the catalytic site is brought in by the incoming rNTP, and the second ion is provided from the pool of free magnesium ions left in solution. This suggests that if [Mg2+]tot< [rNTP]tot, the free magnesium concentration ([Mg2+]free) will be very low and the elongation velocity is expected to be drastically reduced. On the other hand, high concentrations of rNTPs become inhibitory. One explanation for this phenomenon is that when hydrogen ions are released from the rNTP during the formation of the magnesium-rNTP complex, the pH of the reaction is reduced which blocks the binding between the T7 RNA polymerase and the DNA. This indicates that the higher the rNTP and magnesium concentration, the more hydrogen ions are released and the fall in pH is greater, subsequently inhibiting enzyme activity. Therefore, the optimal amount of rNTPs must be used in relation to the amount of magnesium added initially in order to synthesize high yields of RNA. Further examination of interaction between Mg2+ and rNTPs indicated that an optimal IVT reaction mixture comprises an Mg2+ concentration above about 35 mM and lower or equal to about 50 mM and a total rNTPs plus cap analog molar concentration ([rNTP]tot) between 22 and 32 mM. Further increasing the concentration of each rNTP had a deleterious effect on IVT, causing RNA yield to plateau. These data suggest the optimal balance of Mg2+ and total rNTPs plus cap analog needed for effective IVT requires a molar ratio between total rNTPs plus cap analog and Mg2+ between 1.00:1.10 and 1.00:1.85, preferably between 1.00:1.20 and 1.00:1.80, more preferably between 1.00:1.30 and 1.00:1.80, more preferably between 1.00:1.40 and 1.00:1.80, more preferably between 1.00:1.50 and 1.00:1.80, more preferably between 1.00:1.60 and 1.00:1.80, more preferably between 1.00:1.70 and 1.00:1.75, such as 1: 1.73. As such, the Mg2+ molar concentration is preferably always higher than the total rNTPs plus cap analog molar concentration ([rNTP]tot). In an embodiment, the invention relates to a reaction mixture for in vitro transcription and co-transcriptional capping of RNA comprising: a cap analog, ribonucleotide triphosphates (rNTPs), an RNA polymerase and magnesium (Mg2+), said reaction mixture comprising: (a) a total molar rNTP concentration; (b) a molar concentration of cap analog; (c) a molar concentration of Mg2+ that is at least 20% greater than the sum of the total molar rNTP concentration and the molar cap analog concentration. In a further embodiment, the sum of the total molar rNTP concentration and the molar cap analog concentration([rNTP]tot) is preferably between 22 and 32 mM. In a preferred embodiment, the molar concentration of Mg2+ is at most 85% greater than the sum of the total molar rNTP concentration and the molar cap analog concentration. As such, by providing a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription, wherein a molar concentration of the magnesium in said reaction mixture is at least above about 35 mM and lower or equal to about 50 mM, the magnesium concentration was high enough to saturate the RNA polymerase (allowing transcription to proceed), reduce the generation of certain by-products of the in vitro synthesis process, including double-stranded RNA (dsRNA) (that trigger cellular immune responses), whilst being low enough to minimize unwanted precipitation formation of magnesium pyrophosphate (Mg2PPi), optimize capping efficiency and reduce Mg-facilitated RNA degradation. The current disclosure found an optimum in Mg concentration to optimize RNA yield, minimize production costs, while still maintaining consistent product quality even under inherent process fluctuations. As such, the current disclosure allows for cost- yield optimization and contributes amongst other things towards automation of rapid-response, high-quality RNA vaccine production. In an aspect, the invention relates to use of above-mentioned reaction mixture comprising a cap analog for co-transcriptional capping, for improving yield of capped RNA molecules. In an embodiment, the invention relates to use of above-mentioned reaction mixture comprising cap analog for co-transcriptional capping, for improving yield of capped RNA molecules, even when the molar concentration of cap analog is reduced compared to conventional reaction mixtures for co-transcriptional capping. “Conventional reaction mixtures” relates to reaction mixtures comprising more than 6 mM of cap analog. In a particular embodiment, the yield after the IVT reaction is not compromised or preferably increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, or even more when compared to normal circumstances. In particular, the concentration range of magnesium as disclosed in the current invention was found to significantly optimize the yield of RNA from the IVT reaction. In an embodiment, the optimized IVT reaction mixture produces high-quality mRNAs having a yield of at least 3 µg RNA per µl IVT reaction product (3 mg per ml), preferably at least 4 µg RNA / µl, more preferably at least 5 µg RNA / µl, more preferably at least 6 µg RNA / µl, more preferably at least 7 µg RNA / µl, more preferably at least 8 µg RNA / µl, more preferably at least 9 µg RNA / µl, more preferably at least 10 µg RNA / µl (wherein the concentration is measured after the IVT reaction, but before any further processing steps such as EDTA treatment and purification has occurred). The RNA concentration can be measured by any technique known from the state of the art, for instance by measuring ultraviolet absorbance at 260 nm and 280 nm. Calculation of the RNA concentration is based on the absorbance at 260 nm. Furthermore, RNA purity is judged as the 260 nm / 280 nm ratio and a low ratio indicates contamination by protein. ln a specific embodiment, the integrity of RNA after the IVT reaction is not compromised or preferably increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even higher, when compared to normal circumstances. The integrity of the RNA can be measured by any suitable means such as by capillary electrophoresis peak profiles, which may be obtained on a bioanalyzer. “Normal circumstances” as used herein relate to IVT reactions using conventional reaction mixtures, such conventional reaction mixtures not having a molar concentration of magnesium at least above about 35 mM and lower or equal to about 50 mM. In an embodiment, said molar concentration of the magnesium is about 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42, mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM or 50 mM. In a particular embodiment, magnesium is in a form selected from the group comprising magnesium chloride, magnesium acetate, magnesium sulfate, magnesium hydroxide, magnesium oxide, magnesium gluconate, magnesium malate, magnesium orotate, magnesium glycinate, magnesium ascorbate, magnesium citrate, magnesium borate, magnesium salicylate, magnesium bromide, magnesium stearate, magnesium carbonate, or any combination thereof. In yet a further embodiment, magnesium can be in any salt form comprising magnesium chloride (MgCl2) and magnesium acetate (MgOAc2). In particular, during in vitro transcription (IVT) reaction magnesium is in the form of magnesium chloride. The reaction mixture comprises a DNA-dependent RNA polymerase. Said RNA polymerase can be any appropriate RNA polymerase such as a bacteriophage RNA polymerase (for instance lambda, T4, T7, SP6, and SP8 RNA polymerase). In a preferred embodiment, the reaction mixture comprises T7 RNA polymerase. Overall, greater amounts of enzyme appear to proportionally increase transcript production. The amount of RNA polymerase affects the amount of RNA produced and the rate at which it is produced. The transcription reaction reaches completion much sooner when higher concentrations of RNA polymerase are present. Nonetheless, exceeding a certain concentration of RNA polymerase does not produce substantially greater yields and excess RNA polymerase has been reported to inhibit large-scale transcriptions. In an embodiment, a concentration of RNA polymerase (RNAP) in the reaction mixture is between 4 and 8 kU / ml. In an embodiment, a concentration of RNA polymerase in the reaction mixture is 4 kU / ml, 5 kU / ml, 6 kU / ml, 7 kU / ml or 8 kU / ml. In an embodiment, a concentration of RNA polymerase in the reaction mixture is 4 kU / ml, 5 kU / ml, 6 kU / ml, 7 kU / ml or 8 kU / ml or any value in between. In an embodiment, a concentration of RNA polymerase in the reaction mixture is between 4.5 and 7.5 kU / ml, more preferably between 5.0 and 7.0 kU / ml, more preferably between 5.5 kU / ml and 6.5 kU / ml, such as 6 kU / ml. Previous studies have observed that monovalent salt ions, such as sodium and chloride, can have a strong inhibitory effect on RNAP activity. Chloride ions have been reported to compete with the DNA template for anion-binding sites on the polymerase, increasing the Michaelis constant (KM) between the polymerase and the promoter. Conversely, spermidine and related polycations have been reported to improve transcription reaction yields. Spermidine is usually considered a critical component for in vitro transcription reactions. Spermidine is a polyamine that potentially interacts with inhibitory polyanions and has been shown to enable the polymerase to dissociate from the DNA template and initiate new chain synthesis. In addition, spermidine has been reported to prevent the inhibition of RNA synthesis by exogenous RNA and to improve the overall efficiency of T7 RNAP transcription in vitro. A moderate amount of spermidine, around 2 to 5 mM, appears to improve overall RNA yield but becomes inhibitory at 10 mM. As such, in an embodiment, the reaction mixture comprises a molar concentration of spermidine between about 1 and about 10 mM. In an embodiment, the reaction mixture comprises a molar concentration of spermidine of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM. In an embodiment, the reaction mixture comprises a molar concentration of spermidine of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM or any value in between. In an embodiment, the reaction mixture comprises a molar concentration of spermidine between 1 and 5 mM, more preferably between 1 and 3 mM, such as 2 mM. In an embodiment, the reaction mixture further comprises dithiothreitol (DTT), a reducing agent that protects against protein oxidation. In an embodiment, a molar concentration of DTT in the reaction mixture is between about 10 and about 100 mM. In an embodiment, a molar concentration of DTT in the reaction mixture is between about 10 and about 20 mM, between about 20 and about 30 mM, between about 30 and about 40 mM, between about 40 and about 50 mM, between about 50 and about 60 mM, between about 60 and about 70 mM, between about 70 and about 80 mM, between about 80 and about 90 mM or between about 90 and about 100 mM. In an embodiment, a molar concentration of DTT in the reaction mixture is 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM. In an embodiment, the reaction mixture further comprises a Ribonuclease (RNase) inhibitor, for protecting the transcribed RNA from degradation by RNases. RNase inhibitors are recombinant enzymes used to inhibit RNase activity. In an embodiment, the reaction mixture comprises between about 100 and about 1000 Units (U) / ml RNase inhibitor. In an embodiment, the reaction mixtures comprises between about 100 and about 200 Units (U) / ml RNase inhibitor, between about 200 and about 300 Units (U) / ml RNase inhibitor, between about 300 and about 400 Units (U) / ml RNase inhibitor, between about 400 and about 500 Units (U) / ml RNase inhibitor, between about 500 and about 600 Units (U) / ml RNase inhibitor, between about 600 and about 700 Units (U) / ml RNase inhibitor, between about 700 and about 800 Units (U) / ml RNase inhibitor, between about 800 and about 900 Units (U) / ml RNase inhibitor or between about 900 and about 1000 Units (U) / ml RNase inhibitor. In an embodiment, the reaction mixture comprises 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 Units (U) / ml RNase inhibitor. One unit is defined as the amount of protein to inhibit 5 ng RNase A. The pH during the IVT reaction must be maintained because it otherwise decreases with the incorporation of ribonucleotides. Furthermore, RNA is unstable at alkaline pH and shows enhanced stability of the RNA phosphodiester bond at acidic pH. RNA hydrolysis is a reaction in which a phosphodiester bond in the sugar-phosphate backbone of RNA is broken, cleaving the RNA molecule. RNA is susceptible to this base-catalyzed hydrolysis because the ribose sugar in RNA has a hydroxyl group at the 2’ position. This feature makes RNA chemically unstable compared to DNA, which does not have this 2’-OH group and thus is not susceptible to base-catalyzed hydrolysis. As such, it is of importance to include a buffer system in the reaction mixture. Common buffer systems used in RNA in vitro transcription include 4-(2- hydroxy-ethyl)-1-piperazineethanesulfonic acid (HEPES) and tris(hydroxymethyl)amino-methane (Tris). In a preferred embodiment, the reaction mixture further comprises a buffering system, such as a HEPES or a Tris buffer. In a preferred embodiment, said buffer has a pH below 8, such as 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 or 6. In a preferred embodiment, said buffer has a pH below 7, such as a pH of 6.5. This is in contrast with IVT reaction processes disclosed in the prior art (US20190085368 for instance discloses a pH between 7- 8.5). The inventors discovered that a pH below 7, more preferably a pH of 6.5 represents an optimal pH, displaying an increased stability of the produced RNA (less hydrolysis and less enzymatic degradation of the RNA by RNases).On the other hand, a pH below 6 leads to a reduced capping efficiency and a reduced RNA polymerase activity (leading to a reduced RNA yield). In an embodiment, said reaction mixture has a pH of less than 8, such as 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 or 6. In a preferred embodiment, said reaction mixture has a pH below 7, such as a pH of 6.5. In a further aspect, the current invention relates to a method for producing a messenger ribonucleic acid (mRNA) comprising incubating a DNA template in a reaction mixture as mentioned above, performing an in vitro transcription (IVT) reaction of said DNA template, and thereby producing the mRNA. The invention also relates to a method for producing a capped messenger ribonucleic acid (mRNA) comprising: incubating a DNA template in a reaction mixture as described above, performing an in vitro transcription (IVT) and co-transcriptional capping reaction of said DNA template, and thereby producing the capped mRNA. In a preferred embodiment of the method of the current invention, said incubation time of the DNA template (the time of the IVT and capping reaction) is less than 240 minutes, preferably less than 210 minutes, more preferably less than 180 minutes, more preferably less than 150 minutes, more preferably less than 120 minutes, more preferably less than 90 minutes, such as 75 minutes. This is in contrast with prior art document US20190085368, where an in vitro transcription reaction was allowed to proceed for 4 hours. In a preferred embodiment of the method of the current invention, said incubation time of the DNA template (the time of the IVT and co-transcriptional capping reaction) is between 45 minutes and 240 minutes, preferably between 45 minutes and 180 minutes, more preferably between 45 minutes and 90 minutes, such as between 60 minutes and 90 minutes, for instance 75 minutes. In an embodiment, said incubation time of the DNA template (the time of the IVT and co-transcriptional capping reaction) is 45 minutes, 46 minutes, 47 minutes, 48 minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, 75 minutes, 76 minutes, 77 minutes, 78 minutes, 79 minutes, 80 minutes, 81 minutes, 82 minutes, 83 minutes, 84 minutes, 85 minutes, 86 minutes, 87 minutes, 88 minutes, 89 minutes, 90 minutes or any value in between. Reducing the time of the IVT and co-transcriptional capping reaction, increases cost- yield optimization. In a preferred embodiment of the method of the current invention, said incubation temperature of the DNA template (the temperature of the IVT and co-transcriptional capping reaction) is higher than 37°C, more preferably higher than 38°C, more preferably higher than 39°C, more preferably higher than 40°C, more preferably higher than 41°C, more preferably higher than 42°C, more preferably higher than 43°C, more preferably higher than 44°C, such as 45°C. In a preferred embodiment of the method of the current invention, said incubation temperature of the DNA template (the temperature of the IVT and co-transcriptional capping reaction) is between 25°C and 55 °C, more preferably between 37°C and 50°C, more preferably between 40°C and 50°C, such as 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C or 50°C. In a preferred embodiment of the method of the current invention, said incubation temperature of the DNA template (the temperature of the IVT and co-transcriptional capping reaction) is 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C or any value in between. The DNA template provides the sequence to be transcribed downstream of an RNA polymerase promoter. There are two strategies for generating transcription templates: PCR amplification and linearization of plasmid with a restriction enzyme. Which one to choose will depend on the downstream application. In general, if multiple sequences are to be made and transcribed in parallel, PCR amplification is recommended as it generates many templates quickly. On the other hand, if large amounts of one or a few templates are required, plasmid DNA is recommended, because of the relative ease of producing large quantities of high quality, fully characterized plasmids. There are different versions of plasmids available that allow for propagation of homopolymeric A-tails of defined length. In an embodiment, the DNA template is obtained by PCR amplification. In another embodiment, the DNA template is obtained by linearization of plasmid with a restriction enzyme. In an embodiment, the cap analog is an initiating capped oligonucleotide primer comprising following formula II or a salt form thereof:

[0002] Formula II Wherein each of B1 through is independently a natural, modified or unnatural nucleoside base; M is 0 or 1; L is 0 or 1; q1 is 1 and each of q2 through q9 is independently 0 or 1; R1 is H or methyl; R2 and R3 are independently H, OH, alkyl, O-alkyl, halogen, a linker or a detectable marker; each of X1 through X13 is independently O or S; each of Y1 through Y13 is independently OH, SH, BH3, aryl, alkyl, O-alkyl or O-aryl; each of Z0 through Z22 is independently O, S, NH, CH2, C(halogen)2 or CH(halogen); and each of R4 through are independently H, OH, OMe or a detectable marker. In an embodiment, the cap analog is a initiating capped oligonucleotide primer comprising a structure of formula III or a salt form thereof: Formula III wherein B1and B2are independently a natural, a modified, or an unnatural nucleoside base; and R1, R2, and R3 are independently OH or O-methyl. In an embodiment, when using a cap analog as described in Formula III, the DNA template comprises a promoter region comprising a transcriptional start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and wherein the initiating capped oligonucleotide primer is hybridized to the DNA template at least at nucleotide positions +1 and +2. As discussed above, in a preferred embodiment, the cap analog is a trinucleotide as described in Formula I with a 5’-m7G joined by a 5-5´triphosphate linkage to an AG sequence, wherein the adenine has a methyl group on the 2´-O position. In order to use such a cap analog in an in vitro transcription reaction the template must contain an AG in place of a GG following the T7 promoter in the initiation sequence. As such, in an embodiment, the template contains an AG in place of a GG following the T7 promoter in the initiation sequence. In an embodiment, the cap analog is a trinucleotide as described in Formula IV:

[0003] Formula IV In an embodiment, the cap analog is a compound of formula V: Formula V In an embodiment, the cap analog is a compound of formula VI:

[0004] Formula VI In an embodiment, the cap analog is a compound of formula VII or a salt form thereof: Formula VII wherein: R1, R3, R4are selected from the group consisting of: H, CH3, alkyl, where R substituents with different numbers may be the same or different, Base1is selected from the group consisting of: wherein R5is selected from the group consisting of: H, CH3, alkyl, alkenyl, alkynyl, alkylaryl, X1, X3 are selected from the group consisting of: O, S, Se, where X substituents with different numbers can be the same or different, X2, X4are selected from the group consisting of: O, S, Se, BH3, where X substituents with different numbers can be the same or different, X5 is selected from the group consisting of: O, CH2, CF2, CCl2. In a further embodiment at least one of the substituents among of: X1, X2, X3, X4and X5is different from O. The reaction mixture of the current invention can be used for producing any type or RNA, including both short and long RNA molecules (such as self-amplifying RNAs), i.e. it works equally well on small and long templates. In an embodiment, the method further comprises removing template DNA molecules by adding DNase, wherein said DNase is added at a concentration below 20U / µg of DNA. Residual DNA is one of the critical criteria to assess the quality of the IVT reaction product. The current invention allows to reduce the amount of DNase necessary to remove template DNA molecules at the end of the IVT reaction and as a consequence will reduce the cost related thereto. In an embodiment, said DNase is added at a concentration of 19U / µg of DNA, 18U / µg of DNA, 17U / µg of DNA, 16U / µg of DNA, 15U / µg of DNA, 14U / µg of DNA, 13U / µg of DNA, 12U / µg of DNA, 11U / µg of DNA, 10U / µg of DNA, 9U / µg of DNA, 8U / µg of DNA, 7U / µg of DNA, 6U / µg of DNA, 5U / µg of DNA, 4U / µg of DNA, 3U / µg of DNA, 2U / µg of DNA or 1U / µg of DNA. In a preferred embodiment, said DNA template has a concentration between 20 and 200 µg / ml. In a preferred embodiment, said DNA template has a concentration of less than about 200 µg / ml. The inventors observed that a too high DNA concentration can lead to a decreased RNA integrity. Higher DNA starting concentrations increase the impurity and the IVT reaction speed (leading to a reduced integrity and higher degree of impurity). In a preferred embodiment, said DNA template has a concentration of less than about 100 µg / ml, preferably less than 50 µg / ml. In an embodiment, said DNA template has a concentration of 50 µg / ml, 49 µg / ml, 48 µg / ml, 47 µg / ml, 46 µg / ml, 45 µg / ml, 44 µg / ml, 43 µg / ml, 42 µg / ml, 41 µg / ml, 40 µg / ml, 39 µg / ml, 38 µg / ml, 37 µg / ml, 36 µg / ml, 35 µg / ml, 34 µg / ml, 33 µg / ml, 32 µg / ml, 31 µg / ml, 30 µg / ml, 29 µg / ml, 28 µg / ml, 27 µg / ml, 26 µg / ml, 25 µg / ml, 24 µg / ml, 23 µg / ml, 22 µg / ml, 21 µg / ml, 20 µg / ml, 19 µg / ml, 18 µg / ml, 17 µg / ml, 16 µg / ml, 15 µg / ml, 14 µg / ml, 13 µg / ml, 12 µg / ml, 11 µg / ml, 10 µg / ml, 9 µg / ml, 8 µg / ml, 7 µg / ml, 6 µg / ml, 5 µg / ml, 4 µg / ml, 3 µg / ml, 2 µg / ml, 1 µg / ml or less than 1 µg / ml. In an embodiment, said DNA template has a concentration of 50 µg / ml, 49 µg / ml, 48 µg / ml, 47 µg / ml, 46 µg / ml, 45 µg / ml, 44 µg / ml, 43 µg / ml, 42 µg / ml, 41 µg / ml, 40 µg / ml, 39 µg / ml, 38 µg / ml, 37 µg / ml, 36 µg / ml, 35 µg / ml, 34 µg / ml, 33 µg / ml, 32 µg / ml, 31 µg / ml, 30 µg / ml, 29 µg / ml, 28 µg / ml, 27 µg / ml, 26 µg / ml, 25 µg / ml, 24 µg / ml, 23 µg / ml, 22 µg / ml, 21 µg / ml, 20 µg / ml, 19 µg / ml, 18 µg / ml, 17 µg / ml, 16 µg / ml, 15 µg / ml, 14 µg / ml, 13 µg / ml, 12 µg / ml, 11 µg / ml, 10 µg / ml, 9 µg / ml, 8 µg / ml, 7 µg / ml, 6 µg / ml, 5 µg / ml, 4 µg / ml, 3 µg / ml, 2 µg / ml, 1 µg / ml or less than 1 µg / ml or any value in between. In a preferred embodiment, said DNA template has a concentration between 20 µg / ml and 80 µg / ml, more preferably between 20 µg / ml and 70 µg / ml, more preferably between 30 µg / ml and 60 µg / ml, more preferably between 30 µg / ml and 50 µg / ml, such as 40 µg / ml. The current invention allows to reduce the amount of DNA template necessary to obtain a sufficiently high yield. By reducing the amount of DNA template, the cost related thereto can be reduced. “Amplification factor” as used herein, refers to the ratio of mRNA produced in a certain in vitro transcription reaction to DNA template incubated in / added to the mixture of said specific in vitro transcription reaction. Said ratio could for instance be expressed as a weight ratio (w / w) or as a molar ratio. When co-transcriptional capping occurs during said in vitro transcription reaction, said amount of mRNA produced refers to both the capped and uncapped mRNA species. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture is more than 50, preferably more than 60, more preferably more than 70, more preferably more than 80, more preferably more than 90, more preferably more than 100. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture is between 50 and 500, such as between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 250, between 250 and 300, between 300 and 350, between 350 and 400, between 400 and 450 or between 450 and 500. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture is between 50 and 250, such as 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or any value in between. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture, wherein said DNA template is incubated for a period of less than 240 minutes, is more than 50, preferably more than 60, more preferably more than 70, more preferably more than 80, more preferably more than 90, more preferably more than 100. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture, wherein said DNA template is incubated for a period of less than 210 minutes, is more than 50, preferably more than 60, more preferably more than 70, more preferably more than 80, more preferably more than 90, more preferably more than 100. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture, wherein said DNA template is incubated for a period of less than 180 minutes, is more than 50, preferably more than 60, more preferably more than 70, more preferably more than 80, more preferably more than 90, more preferably more than 100. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture, wherein said DNA template is incubated for a period of less than 150 minutes, is more than 50, preferably more than 60, more preferably more than 70, more preferably more than 80, more preferably more than 90, more preferably more than 100. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture, wherein said DNA template is incubated for a period of less than 120 minutes, is more than 50, preferably more than 60, more preferably more than 70, more preferably more than 80, more preferably more than 90, more preferably more than 100. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture, wherein said DNA template is incubated for a period of less than 90 minutes, is more than 50, preferably more than 60, more preferably more than 70, more preferably more than 80, more preferably more than 90, more preferably more than 100. In a preferred embodiment, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture, wherein said DNA template is incubated for a period of 75 minutes, is more than 50, preferably more than 60, more preferably more than 70, more preferably more than 80, more preferably more than 90, more preferably more than 100. Furthermore, a reduced amount of DNA template will further decrease the amount of DNase needed to remove template DNA molecules at the end of the IVT reaction and will reduce the cost related thereto. In addition, a reduced amount of DNA (and concomitantly DNase) will lead to a higher RNA purification yield. By incubating a DNA template in a reaction mixture as mentioned above, performing an in vitro transcription (IVT) reaction of said DNA template, and thereby producing the mRNA, wherein a molar concentration of the magnesium in said reaction mixture is at least above about 35 mM and lower or equal to about 50 mM, the magnesium concentration was high enough to saturate the RNA polymerase (allowing transcription to proceed), reduce the generation of certain by-products of the in vitro synthesis process, including double-stranded RNA (dsRNA) (that trigger cellular immune responses), whilst being low enough to minimize unwanted precipitation formation of magnesium pyrophosphate (Mg2PPi), improve capping efficiency and reduce Mg-facilitated RNA degradation. In an embodiment, the ratio of DNA template to magnesium concentration is below 0.200, more preferably below 0.100, more preferably below 0.05. In an aspect, the invention relates to use of above-mentioned reaction mixture, for decreasing a level of contaminants during an in vitro transcription reaction of producing RNA molecules (uncapped or capped). In an embodiment, said contaminants comprise dsRNA and / or proteins, such as enzymes. As discussed above, it was particularly found that adding an optimal concentration of magnesium to the IVT reaction preferably about and between above 35 mM to about 50 mM resulted in a reduced formation of dsRNA. The formation of dsRNA was preferably reduced by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% when compared to normal circumstances. In a preferred embodiment, the dsRNA content after the IVT reaction was below 300 ng dsRNA / mg mRNA, preferably below 250 ng dsRNA / mg RNA, more preferably below 200 ng dsRNA / mg mRNA, more preferably below 150 ng dsRNA / mg RNA. In the context of the present invention, the terms ‘reducing’ or alternatively ‘to reduce’ are meant to be to ‘lessen’, to ‘decrease’, to ‘minimize’, or to ‘diminish’ the formation of dsRNA. Accordingly, where a sample would under normal circumstances contain a particular amount of dsRNA after in vitro transcription, the term ‘reducing’ means that said amount of dsRNA is lower when subjecting said sample to the method of the present invention. In particular, the amount of dsRNA is preferably reduced by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, when compared to normal circumstances. In the context of the present invention, the term ‘the formation’ is meant to be ‘the emergence’, ‘the development’, ‘the origination’, or ‘the generation’ of dsRNA in said in vitro transcription reaction. Specifically, molecules obtained after in vitro transcription typically comprise dsRNA, while the presence of elevated magnesium in the reaction results in a reduced formation of such dsRNA. In the context of the present invention, the term “double stranded RNA” or “dsRNA” is meant to be any RNA molecule with sufficient internal homology to form significant secondary structures such as hairpins due to hybridization of internal complementary sequences with one another via Watson-Crick base pairing of nucleotide bases within the complementary sequences. Significant secondary structures generally involve stretches of homology greater than approximately nine bases, but the exact length depends to some extent on context and on whether such secondary structures impart any biological function to the molecule. In particular, molecules obtained after in vitro transcription typically comprise dsRNA with two separate complementary strands and may vary in size for example from 20 nucleotides to 200 nucleotides or even more than 500 nucleotides. In the context of the present invention, dsRNA is formed as a byproduct identified in IVT reactions which can arise from T7 RNA-dependent RNA polymerase activity. In particular, three main types of byproduct in the IVT reaction may result in formation of dsRNA molecules. The first is formed by 3’-extension of the run-off products annealing to complementary sequences in the body of the run-off transcript either in cis (by folding back on the same RNA molecule) or trans (annealing to a second RNA molecule) to form extended duplexes. The second type of dsRNA molecules is formed by hybridization of an antisense RNA molecule to the run-off transcript. The antisense RNA molecules have been reported to be formed in a promoter- and run-off transcript-independent manner. Alternatively, a promoter-independent transcription of full-length anti-sense RNA has been also reported as a novel mechanism of dsRNA generation in T7 RNA polymerase-driven IVT reaction. A third form of dsRNA results from random pairing of abortive transcripts, either in cis (i.e. within the same molecule) or in trans (between two different molecules). According to the invention, dsRNA encompasses any kind of the described RNA byproducts in an IVT reaction. Methods for detecting dsRNA rely essentially on immunological approaches such as immunofluorescence, ELISA, immunoblot as well as antibody-independent methods such as nucleic acid fluorescent in situ hybridization (FISH) or cellulose-based dsRNA isolation have also been used for dsRNA detection. As used herein and as described in the examples, immunological methods such as anti dsRNA J2 antibody immunoblotting, use antibodies as structural probes that specifically recognize the A-helix structure adopted by dsRNA. Commercially available J2 anti-dsRNA lgG2a (and to a lesser extent the lgG2a K1 and IgM K2 mAb or 9D5 mAb) have become the golden standards in dsRNA detection. Furthermore, intact mass spectrometry can be used to quantify the abundance and lengths of different 3’-end- extended dsRNA species. Furthermore, it was particularly found that adding an optimal concentration of magnesium to the IVT reaction preferably about and between above 35 mM to about 50 mM, resulted in an optimized capping efficiency at a lower molar concentration of a cap analog then conventionally used. As such, in an aspect, the invention relates to use of above-mentioned reaction mixture comprising a cap analog for co-transcriptional capping, for improving a capping efficiency during a co-transcriptional capping reaction. In an embodiment, the optimized IVT reaction mixture produces high-quality capped mRNAs with a capping efficiency of at least 80%, preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90% even when using a molar concentration of cap analog of less than 6 mM. In addition, it was found that adding an optimal concentration of magnesium to the IVT reaction preferably about and between above 35 mM to about 50 mM, resulted in a decreased unwanted precipitation formation of magnesium pyrophosphate (Mg2PPi). The precipitation formation of magnesium pyrophosphate was preferably decreased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% when compared to normal circumstances. In an aspect, the invention relates to use of above-mentioned reaction mixture for decreasing precipitation of magnesium pyrophosphate during an in vitro transcription reaction of producing RNA molecules. In an embodiment, above- mentioned reaction mixture comprises a cap analog for co-transcriptional capping. In an embodiment of the method for producing a mRNA according to the current invention, the pH of said IVT reaction is less than 8. As described above, RNA is unstable at alkaline pH and shows enhanced stability of the RNA phosphodiester bond at acidic pH. In a preferred embodiment, the IVT reaction occurs in the presence of a buffering system, such as a HEPES or a Tris buffer. In a preferred embodiment, said buffer has a pH below 8, such as 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 or 6. In a preferred embodiment, said buffer has a pH below 7, such as a pH of 6.5. This is in contrast with IVT reaction processes disclosed in the prior art (US20190085368 for instance discloses a pH between 7-8.5). The inventors discovered that a pH below 7, more preferably a pH of 6.5, represents an optimal pH, displaying an increased stability of the produced RNA (less hydrolysis and less enzymatic degradation of the RNA by RNases). On the other hand, a pH below 6 leads to a reduced capping efficiency and a reduced RNA polymerase activity (leading to a reduced RNA yield). In an embodiment, said reaction mixture has a pH of less than 8, such as 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1 or 6. In a preferred embodiment, said reaction mixture has a pH below 7, such as a pH of 6.5. In a preferred embodiment, said reaction mixture has a pH between 6.0 and 6.9, such as a pH of 6.5. The advantages of such a pH range are described above. In a specific embodiment of the present invention, said IVT transcription reaction is terminated by addition of a metal chelator such as selected from the list comprising: BAPTA (1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), DFOA (Deferoxamine Mesylate), Dimethoxynitrophenamine (1-(2-Nitro-4,5- dimethoxyphenyl)-1,2-diaminoethane-N,N,N',N'-tetraacetic Acid), EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-bisO- aminoethyl ether)- N,N,N',N'-tetraacetic acid), CDTA (1 ,2-cyclohexylenedinitrilo)tetraacetic acid), DPTA (diethylenetriaminepentaacetic acid), PIH (pyridoxal isonicotinoyl hydrazone), TPEN (N’- Tetrakis(2-pyridylmethyl)ethylenediamine). In a specific embodiment, said IVT transcription reaction is terminated by addition of a metal chelator, such as EDTA. As used herein, the term “EDTA” is to be understood as an aminopolycarboxylic acid acting as a scavenger for metal ions. This results in deactivation of metal-dependent enzymes, either as an assay for their reactivity or to suppress damage to DNA, proteins, and polysaccharides. In addition to metal ion chelation, EDTA also acts as a selective inhibitor against dNTP hydrolyzing enzymes such as Taq polymerase, dUTPase, MutT, etc. In particular, as used in the present invention, EDTA chelates divalent cations such as magnesium and is needed to protect RNA from being degraded during enzyme inactivation. Nuclease activity and in particular RNA nuclease is highly dependent on the concentrations of divalent cation magnesium. In particular, it is known that one molecule of a metal chelator such as EDTA is capable of chelating one metal ion. The addition of metal chelators thus potentially has two benefits. On the one hand, it will stop enzymatic reactions that require the presence of metal ions as a cofactor, and secondly it will chelate metal ions thereby preventing the formation of the aggregate. In yet another specific embodiment, the concentration of said metal chelator is about and between 10 and about 100 mM. In an embodiment, after removing template DNA molecules by adding DNase and a metal chelator for terminating the IVT reaction, the reaction product is purified. Purification of RNA can occur by any method known from the state of the art. In an embodiment, said purification step comprises purification by means of magnetic beads, wherein in a first step the RNA is bound to the beads in the presence of a binding buffer, the beads with the RNA are washed in a second step by means of a wash buffer and the RNA is eluted from the beads by means of an elution buffer. In a preferred embodiment, the beads comprise magnetic beads with functionalized silica surfaces to selectively bind nucleic acids. Purification is typically lengthy, requiring multiple steps, hence decreasing yields. As described above, current invention allows to reduce the amount of DNase necessary. By decreasing the amount of contaminants such as dsRNA and enzymes (such as DNase) in the IVT reaction process, downstream purification of the reaction product is simplified. As such, current invention not only enables a reduction in costs related to the IVT reaction itself (by increasing the yield and capping efficiency and decreasing the amount of DNase necessary), but further allows to reduce the costs associated with purification of the IVT reaction product. In a further aspect, the current invention relates to a kit for use in the production of RNA, wherein said kit comprises a reaction mixture as described above for performing an in vitro RNA transcription, DNase for removal of template DNA molecules, and a metal chelator for terminating the IVT reaction. In an embodiment, all reagents are available as plug-and-play pre-mixes at the correct concentration and ratio. In an embodiment, the reaction mixture consists of 3 separate mixes: a first mix comprising ATP, CTP, UTP, GTP and a cap analog (“NTPs mix”), a second mix comprising RNA polymerase (preferably T7 RNA polymerase), RNase inhibitor and inorganic pyrophosphatase (“enzyme mix”) and a third mix comprising HEPES buffer, MgCl2and spermidine (“buffer mix”). In an embodiment, said kit further comprises a reaction mixture for purification of the RNA. In an embodiment, said purification reaction mixture comprises magnetic beads for binding the RNA and appropriate buffers for binding, washing and eluting the RNA from the beads (a binding buffer, a wash buffer and an elution buffer). In a preferred embodiment, the concentration of the rNTPs, RNA polymerase, magnesium, cap analog respectively is a final concentration presented in the reaction mixture. The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention. The present invention will be now described in more details, referring to examples that are not limitative. EXAMPLES AND / OR DESCRIPTION OF FIGURES The present invention will now be further exemplified with reference to the following examples. The present invention is in no way limited to the given examples or to the embodiments presented in the figures. Example 1. Process performance using an IVT reaction mixture according to an embodiment of the invention. An optimized IVT reaction mixture was used to produce three different capped mRNAs (target 1, 2, and 3 have respectively 1166, 4060, and 4284 nucleotides), each having a DNA template concentration of less than 50 µg / ml. The magnesium concentration in the reaction mixture was at least above about 35 mM and lower or equal to about 50 mM, whereas the total molar concentration of ribonucleotide triphosphates (ATP, CTP, UTP and GTP) plus cap analog for co-transcriptional capping was between 22 and 32 mM. The molar ratio of total NTPs plus cap analog to the magnesium concentration was less than 0.85. The concentration of T7 RNA polymerase in the reaction mixture was between 4 and 8 kU / ml. By providing a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription, wherein a molar concentration of the magnesium in said reaction mixture is at least above about 35 mM, the magnesium concentration is high enough to saturate the RNA polymerase (allowing transcription to proceed) and reduce the formation of dsRNA by-products. Furthermore, by providing a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription, wherein a molar concentration of the magnesium in said reaction mixture is lower or equal to about 50 mM, a high capping efficiency can be maintained, even when lower concentrations of cap analog (below 6 mM) are present in the reaction mixture. Figure 1 shows the process performance of the IVT reactions (A: mRNA yield, B: capping efficiency, C: dsRNA content). As can be seen from figure 1, the IVT reaction mixture produces high-quality mRNAs with yield > 4 μg / μL (Figure 1A) and efficient capping > 90% (Figure 1B). In addition, the double-stranded RNA contents are < 200 ng dsRNA / mg RNA (Figure 1C). The reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription according to the current invention delivers a high yield of capped mRNA transcripts and low impurity levels. By decreasing the amount of contaminants such as dsRNA in the IVT reaction process, downstream purification of the reaction product is simplified. As such, current invention not only enables a reduction in costs related to the IVT reaction itself (by optimizing the yield and capping efficiency), but further allows to reduce the costs associated with purification of the IVT reaction product. Example 2. In vitro transcription process performance for multiple constructs having a different size using an IVT reaction mixture according to an embodiment of the invention. An optimized IVT reaction mixture was used to produce various capped mRNAs, each having a DNA template concentration of less than 50 µg / ml. The magnesium concentration in the reaction mixture was at least above about 35 mM and lower or equal to about 50 mM, whereas the total molar concentration of each of the ribonucleotide triphosphates (ATP, CTP, UTP and GTP) plus cap analog for co- transcriptional capping was between 22 and 32 mM. The molar ratio of total NTPs plus cap analog to the magnesium concentration was less than 0.85. The concentration of T7 RNA polymerase in the reaction mixture was between 4 and 8 kU / ml. After removing template DNA molecules by adding DNase and a metal chelator for terminating the IVT reaction, the reaction product is purified. The purification step comprises purification by means of magnetic beads with functionalized silica surfaces to selectively bind nucleic acids. In a first step the RNA is bound to the beads in the presence of a binding buffer, the beads with the RNA are washed in a second step by means of a wash buffer and the RNA is eluted from the beads by means of an elution buffer. Purification is typically lengthy, requiring multiple steps, hence decreasing yields. As described above, current invention allows to reduce the amount of DNase necessary. By decreasing the amount of contaminants such as dsRNA and enzymes (such as DNase) in the IVT reaction process, downstream purification of the reaction product is simplified. As such, current invention not only enables a reduction in costs related to the IVT reaction itself (by increasing the yield and capping efficiency and decreasing the amount of DNase necessary), but further allows to reduce the costs associated with purification of the IVT reaction product. The IVT reaction mixture produces high-quality mRNAs with high yield (Figure 2A- B) and efficient capping (Figure 3A-B). In addition, the double-stranded RNA content and the residual pDNA content is low showing low impurity levels (Figures 4-6). The reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription according to the current invention delivers a high yield of capped mRNA transcripts and low impurity levels. By decreasing the amount of contaminants such as dsRNA in the IVT reaction process, downstream purification of the reaction product is simplified and product losses and integrity loss is minimized (Figure 7). As such, current invention not only enables a reduction in costs related to the IVT reaction itself (by increasing the yield and capping efficiency), but further allows to reduce the costs associated with purification of the IVT reaction product. Figure 2 shows the yield (RNA concentration in mg / ml) as measured for 245 IVT reactions (including 45 different constructs). The mean yield is 5.6 mg / ml, having a 95% prediction interval between 4.0 and 7.2 mg / ml (Figure 2A). Figure 2B shows the yield (RNA concentration in mg / ml) for constructs having a different size. Figure 3 shows the capping efficiency (%) as measured for 111 IVT reactions (including 19 different constructs). The mean capping efficiency is 94.8%, having a 95% prediction interval between 87.5 and 97.9% (Figure 3A). Figure 3B shows the capping efficiency (%) for constructs having a different size. This is much higher than described in prior art documents such as US20190085368. US20190085368 is focused on enzymatic capping, but mentions co-transcriptional capping using very high amounts of cap analog, where the cap analog: GTP molar ratio in the reaction is 4:1. US20190085368 describes that this typically results in ˜80% capping efficiency. This high abundance of uncapped species is undesirable when developing therapeutic RNA. Since only capped mRNA is translated into protein, the presence of a high abundance of uncapped species (being 20%) is problematic as efficacy (protein production / mg RNA) is reduced by 20% and 20% of the final drug substance is an inert impurity, decreasing process productivity. Increasing capping efficiency is important for cost-yield optimization. Figure 4 shows the dsRNA content (ng / mg RNA) as measured for 83 IVT reactions using UTP (including 36 different constructs) and 36 IVT reactions using modified UTP (N1-methyl pseudouridine, including 31 different constructs). All data obtained for the IVT reactions using modified UTP (mUTP) was below the Limit Of Quantification (LOQ) of the assay. In Figure 4A, this means that all mUTP results are actually corresponding to the Lower LOQ (LLOQ) of the particular assay run. This means that the dsRNA level for IVT reactions using modified UTP (having a mean dsRNA content of 127.2 ng / mg RNA, with a 95% prediction interval between 38.9 and 397.4 ng / mg RNA) is a conservative estimate as in fact, the levels may be far below the LLOQ). The mean dsRNA content when using unmodified UTP is 237.7 ng / mg RNA, having a 95% prediction interval between 75.8 and 727.0 ng / mg RNA (Figure 4A). Figure 4B shows the dsRNA content (ng / mg RNA) of IVT reactions using unmodified UTP for constructs having a different size. As all data obtained for the IVT reactions using modified UTP was below the Limit Of Quantification (LOQ) of the assay, figure 4B only displays results for the IVT reactions using unmodified UTP. Figure 5 shows the residual plasmid DNA (pDNA) content as measured for 39 IVT reactions (including 6 different constructs comprising an Ampicillin resistance gene, AMPR). The mean residual pDNA content was 1.22 ng / mg, having a 95% prediction interval between 0.03 and 29.5 ng / mg (Figure 5A). Figure 5B shows the residual pDNA content for constructs having a different size. Figure 6 shows RNA purity and integrity assessment for 6 constructs (Figure 6A-F) as measured by capillary electrophoresis, showing low impurity levels when using the optimized IVT reaction mixture according to an embodiment of the current invention. Figure 7A shows the purification yield for 28 IVT reactions (including 3 different constructs) post-purification compared to pre-purification (%) of mRNA prepared using an IVT reaction mixture according to an embodiment of the current invention. Figure 7B shows RNA integrity for 22 IVT reactions (including 3 different constructs) post-purification compared to pre-purification (ratio) of mRNA prepared using an IVT reaction mixture according to an embodiment of the current invention. Figure 7C shows residual protein content (protein / RNA (%)) after purification for 19 purification reactions (including 2 different constructs) (C) of mRNA prepared using an IVT reaction mixture according to an embodiment of the current invention. Example 3. Comparison of IVT process performance using IVT reaction mixtures having a different magnesium concentration, including an IVT reaction mixture according to an embodiment of the invention. IVT reactions including co-transcriptional capping using 3 IVT reaction mixtures having a different magnesium concentration were performed and their performance was evaluated. The concentration of all the components in the reaction mixture was kept constant, except for the concentration of magnesium. Reaction mixture A has a concentration of 30 mM MgCl2, reaction mixture B has a concentration of 45 mM MgCl2, reaction mixture C has a concentration of 55 mM MgCl2. Reaction mixture B thus representing an IVT reaction mixture according to an embodiment of the invention and reaction mixture A and C having a lower and higher magnesium concentration, respectively. The reactions were performed in triplicate. The results show that there is a higher yield (RNA in mg / ml) and a significant decrease in dsRNA formation (in ng / mg RNA) when using a higher MgCl2 concentration (see Figure 8). However, a higher MgCl2 concentration also results in a decreased capping efficiency (%) and a loss in integrity of the produced mRNA (%) (see Figure 8). In addition, the inventors have found that a too high concentration of magnesium leads to a cloudy appearance of the IVT reaction product (see Figures 9-11). This appearance seems to be caused by the excessive precipitation of insoluble magnesium pyrophosphate (Mg2PPi) even after addition of EDTA. PPi produced during enzymatic nucleic acid synthesis can react with Mg2+ in the reaction buffer to form insoluble magnesium pyrophosphate (Mg2PPi). Such a precipitate can be a disadvantage when performing analytical methods to assess the RNA quality. Furthermore, it is known that RNA and inorganic pyrophosphate can self-assemble to form composite microsponge structures composed of nanocrystalline magnesium pyrophosphate sheets (Mg2P2O7●3.5H2O) with RNA adsorbed to their surfaces. The microsponge particles contain high loadings of RNA (15-21 wt.%). Said precipitates and microsponge particles remove RNA from the IVT reaction mixture solution, thereby reducing the RNA yield of the reaction. Furthermore, said precipitates and microsponge particles can be lost during further purification steps, again reducing the RNA yield of the reaction. In addition, Mg2PPi precipitation can strongly inhibit the transcription reaction. This can be due to a lack of free Mg2+ in solution, thereby reducing the rate of the transcription reaction. But, without wishing to be bound by theory, Mg2PPi precipitation can also strongly inhibit the transcription reaction by means of a physical interaction between the RNA polymerase and the Mg2PPi precipitation at the moment of the transition. In this case, the transcription reaction may stop even if the free Mg2+ concentration is sufficient. Furthermore, as described above, the magnesium pyrophosphate can also co-assemble with RNA into nanostructured particles, thereby precipitating RNA out of solution and reducing the yield of RNA production. Figure 9 shows the visual appearance after IVT incubation of the 3 IVT reaction mixtures having a different magnesium concentration. The tubes comprising the 3 different IVT reaction mixtures are displayed besides each other (the left tube contains 30 mM MgCl2, the middle tube contains 45 mM MgCl2, the right tube contains 55 mM MgCl2). As described above the IVT reactions were performed in triplicate (Figures 9A-C). Figure 10 shows the visual appearance after DNase incubation of the 3 IVT reaction mixtures having a different magnesium concentration. The tubes comprising the 3 different IVT reaction mixtures are displayed besides each other (the left tube contains 30 mM MgCl2, the middle tube contains 45 mM MgCl2, the right tube contains 55 mM MgCl2). As described above the IVT reactions were performed in triplicate (Figures 10A-C). Figure 11 shows the visual appearance after EDTA addition of the 3 IVT reaction mixtures having a different magnesium concentration. The tubes comprising the 3 different IVT reaction mixtures are displayed besides each other (the left tube contains 30 mM MgCl2, the middle tube contains 45 mM MgCl2, the right tube contains 55 mM MgCl2). As can be evidenced from Figures 9-11, the right tube containing 55 mM MgCl2 has a cloudy appearance. As such, by providing a reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription according to an embodiment of the invention, the magnesium concentration is high enough to saturate the RNA polymerase (allowing transcription to proceed) and reduce the formation of dsRNA by-products, whilst simultaneously minimizing the amount of precipitation and achieving a sufficiently high capping efficiency (even when lower concentrations of cap analog are present in the reaction mixture) and integrity of the produced mRNA. Example 4: comparison of an IVT reaction according to an embodiment of the current invention and a prior art IVT reaction. US20190085368 describes manufacturing methods for production of RNA transcripts. However, the concentration of the reaction mixture components is not optimized, resulting in a suboptimal RNA yield. Example 3 of US20190085368 for instance describes a 1 mL transcription reaction utilizing 250 μg of plasmid DNA template, 7.5 mM of each nucleoside triphosphate (NTP) and 7000 U (7 kU / ml) of T7 RNA polymerase. The in vitro transcription reaction in US20190085368 was allowed to proceed for 4 hours at 37° C under constant mixing and the total reaction yield was 5.4 mg RNA transcript. As such, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture (the amplification factor) is very low. More specifically, the amplification factor in US20190085368 is only 21.6 (production of 5.4 mg RNA when 0.250 mg DNA template was used), and this despite high concentrations of NTPs, DNA template and RNA polymerase and a long incubation time of 4 hours. The IVT reaction mixture of the current invention is optimized. When using a Mg2+ concentration at least above about 35 mM and lower or equal to about 50 mM, combined with a molar ratio of total NTPs plus cap analog to said magnesium of less than 0.85, the inventors discovered that a high amplification factor could be obtained. It is known that when a too low concentration of rNTPs is used, it limits RNA production. On the other hand, high concentrations of rNTPs become inhibitory. One explanation for this phenomenon is that when hydrogen ions are released from the rNTP during the formation of the magnesium-rNTP complex, the pH of the reaction is reduced which blocks the binding between the T7 RNA polymerase and the DNA. This indicates that the higher the rNTP and magnesium concentration, the more hydrogen ions are released and the fall in pH is greater, subsequently inhibiting enzyme activity. Therefore, the optimal amount of rNTPs must be used in relation to the amount of magnesium added initially in order to synthesize high yields of RNA. One explanation for this phenomenon is that if [Mg2+]tot < [rNTP]tot, the free magnesium concentration ([Mg2+]free) will be very low; in such conditions, the elongation velocity of T7 RNA polymerase is expected to be drastically reduced. When a 1 mL transcription reaction was performed using the reaction mixture of the current invention (a Mg2+ concentration of at least above about 35 mM and lower or equal to about 50 mM, combined with a molar ratio of total NTPs plus cap analog to said magnesium of less than 0.85) and utilizing only 40 μg of plasmid DNA template and 6000 U of T7 RNA polymerase for 75 minutes at a temperature between 40°C- 50°C, the total reaction yield was 5.6 mg RNA transcript. As such, the weight ratio of mRNA produced to DNA template incubated in the reaction mixture of the current invention (the amplification factor) is significantly higher than the one described in US20190085368. More specifically, the amplification factor when using a reaction mixture according to the current invention is 140 (production of 5.6 mg RNA when only 0.040 mg DNA template was used), and this despite lower concentrations of NTPs, DNA template and RNA polymerase and a shorter incubation time than described in US20190085368. These results clearly indicate that the optimized reaction mixture of the current invention shows a significant improvement over the prior art. The present invention is in no way limited to the embodiments described in the examples and / or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

CLAIMS 1. A reaction mixture for in vitro messenger ribonucleic acid (mRNA) transcription comprising: - ribonucleotide triphosphates (rNTPs) comprising adenosine triphosphates (ATPs), cytidine triphosphates (CTPs), uridine triphosphates (UTPs), guanosine triphosphates (GTPs), an RNA polymerase and magnesium (Mg2+), wherein a molar concentration of the magnesium in said reaction mixture is at least above about 35 mM and lower or equal to about 50 mM.

2. The reaction mixture according to claim 1, wherein the total molar concentration the rNTPs in the reaction mixture is between 16 and 40 mM.

3. The reaction mixture according to claims 1 or 2, wherein said reaction mixture comprises a molar ratio of total rNTPs to magnesium which is less than 0.

85.

4. The reaction mixture according to any of the previous claims, further comprising a cap analog for co-transcriptional capping, wherein a molar concentration of said cap analog in the reaction mixture is between about 1.0 and about 6.0 mM.

5. The reaction mixture according to claim 4, wherein a molar concentration of said cap analog in the reaction mixture is below about 4.0 mM.

6. The reaction mixture according to any of the previous claims 4 to 5, wherein said reaction mixture comprises a molar ratio of cap analog to total rNTPs which is less than 0.

125.

7. The reaction mixture according to any of the previous claims 4 to 5, wherein said reaction mixture comprises a molar ratio of cap analog to total rNTPs which is less than 0.100.

8. The reaction mixture according to any of the previous claims, wherein a concentration of RNA polymerase in the reaction mixture is between 4 and 8 kU / ml.

9. The reaction mixture according to any of the previous claims, wherein a concentration of RNA polymerase in the reaction mixture is below 7 kU / ml.

10. The reaction mixture according to any of the previous claims, wherein said reaction mixture has a pH of less than 8.

11. The reaction mixture according to any of the previous claims, wherein said reaction mixture has a pH of less than 7.

0.

12. Use of a reaction mixture according to any of the previous claims 4-11, for improving a capping efficiency during a co-transcriptional capping reaction.

13. A method for producing a capped messenger ribonucleic acid (mRNA) comprising: incubating a DNA template in a reaction mixture according to any of the previous claims 1-11, performing an in vitro transcription (IVT) and co-transcriptional capping reaction of said DNA template, and thereby producing the capped mRNA.

14. The method according to claim 13, wherein said DNA template has a concentration of less than about 200 µg / ml.

15. The method according to claim 13, wherein said DNA template has a concentration of less than about 100 µg / ml. 16.The method according to claim 13, wherein said DNA template has a concentration of less than about 50 µg / ml.

17. The method according to any of the previous claims 13-16, wherein the weight ratio of capped mRNA produced to DNA template incubated in the reaction mixture is more than 50.The method according to any of the previous claims 13-17, wherein the weight ratio of capped mRNA produced to DNA template incubated in the reaction mixture is more than 100. The method according to any of the previous claims 13-18, wherein said IVT and co-transcriptional capping reaction of said DNA template is performed for less than 240 minutes. The method according to any of the previous claims 13-19, wherein said IVT and co-transcriptional capping reaction of said DNA template is performed for less than 210 minutes. The method according to any of the previous claims 13-20, wherein said IVT and co-transcriptional capping reaction of said DNA template is performed for less than 180 minutes. The method according to any of the previous claims 13-21, wherein said IVT and co-transcriptional capping reaction of said DNA template is performed for less than 150 minutes. The method according to any of the previous claims 13-22, wherein said IVT and co-transcriptional capping reaction of said DNA template is performed for less than 120 minutes. The method according to any of the previous claims 13-23, wherein said IVT and co-transcriptional capping reaction of said DNA template is performed for less than 90 minutes. The method according to any of the previous claims 13-24, wherein said IVT and co-transcriptional capping reaction of said DNA template is performed at a temperature between 40°C and 50°C. The method according to any of the previous claims 13-25, further comprising removing template DNA molecules by adding DNase, wherein said DNase concentration is added at a concentration reaction below 20 U / µg of DNA.

27. A kit for use in the production of capped RNA, wherein said kit comprises a reaction mixture according to any of the previous claims 1-11 for performing an in vitro RNA transcription and co-transcriptional capping reaction, DNase for removal of template DNA molecules, and a metal chelator for terminating the IVT reaction.

28. Kit according to claim 27, wherein said kit further comprises a reaction mixture for purification of the RNA.

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