Large-scale de novo production of DNA

Abstract

The present invention provides a cell-free process for the production of DNA, the process comprising the steps of a) providing an open linear double stranded DNA molecule de novo synthesized comprising a sequence of interest; b) generating a closed linear DNA template with the open linear double stranded DNA molecule, wherein the closed linear DNA template comprises the sequence of interest; and c) amplifying the closed linear DNA template by Rolling Circle Amplification (RCA). The invention also provides a cell-free process for the production of RNA.

Description

[0001] Large-scale de novo production of DNA

[0002] This application claims the benefit of European Patent Application EP25382287.8 filed on March 26th, 2026.

[0003] Technical Field

[0004] The present invention belongs to the field of processes for de novo production of nucleic acids. In particular, the invention relates to de novo cell-free processes for the large-scale production of nucleic acids. The processes of the invention are particularly useful for large-scale production of therapeutically suitable nucleic acids.

[0005] Background Art

[0006] Microbial fermentation, where genetically engineered microorganisms like Escherichia coli or Saccharomyces cerevisiae are used to produce DNA, is at present the most widely employed method for large-scale production of DNA.

[0007] However, microbial fermentation faces several challenges that limit its efficiency, scalability, and applicability. One major issue of microbial fermentation is the risk of contamination with residual host-DNA and bacterial endotoxins, which can affect the quality of the final DNA product, especially for pharmaceutical and medical applications, where high purity is critical. There are also concerns about the genetic stability of microorganisms during long-term cultivation, as genetic drift or mutations in the microbial population can alter the efficiency of DNA production over time.

[0008] More recently, techniques have been developed for producing DNA in large amounts by enzymatic amplification in the absence of host cells. However, these techniques still rely on the use of DNA templates produced in bacteria, and therefore the risks and limitations associated to bacterial usage still remain, in particular the presence of residual host-DNA and endotoxins.

[0009] Important progress has also been made in the development of de novo synthetic processes for producing DNA, that is, chemical or enzymatic processes that do not need pre-existing precursor templates. However, these methods are often very limited in terms of scalability as they are costly and inefficient for producing large amounts of DNA, which has hindered their industrial application.

[0010] Therefore, in spite of the efforts made so far, there is an evident need for DNA productionprocesses that overcome the limitations of current technologies.

[0011] Summary of Invention

[0012] The present inventors have devised a new process that addresses the aforementioned challenges by specifically combining de novo synthesis of DNA with cell-free enzymatic processing and amplification.

[0013] The specific combination of steps of the process of the invention unexpectedly allows the large-scale production of high-quality DNA in an entirely cell-free setting, circumventing the risks and limitations of current cell-based and synthetic methods. By being entirely cell-free, the process of the invention completely avoids the risk of contamination with bacterial DNA and endotoxins, which has become a growing concern in the pharmaceutical and biotechnological industries. At the same time, the generation of an intermediate closed linear DNA template used for Rolling Circle Amplification (RCA) allows for large-scale production, at low costs, of DNA with high-quality and purity, which makes it highly suitable for medical applications, such as gene therapy. Importantly, the absence of amplification steps (e.g. PCR) during the synthesis of the DNA template significantly reduces the risk of sequence error introduction and propagation during the downstream amplification, thereby enhancing the overall reliability of the method.

[0014] In view of the above, it is evident that the cost-effective and safe DNA production process provided by present invention constitutes a great step forward in the field of DNA synthesis technologies.

[0015] Thus, in a first aspect, the invention provides a cell-free process for the production of DNA, the process comprising the steps of a) providing an open linear double stranded DNA molecule de novo synthesized comprising a sequence of interest; b) generating a closed linear DNA template with the open linear double stranded DNA molecule, wherein the closed linear DNA template comprises the sequence of interest; and c) amplifying the closed linear DNA template by Rolling Circle Amplification (RCA).

[0016] In a second aspect, the invention provides a cell-free process for the production of RNA, the process comprising the step of producing DNA by a cell-free process as defined in the first aspect and transcribing the DNA to RNA.

[0017] Brief Description of Drawings

[0018] Fig. 1, related to Example 1, shows the agarose gel electrophoresis (AGE) from theindicated samples obtained on the experiments. RCA products, after digestion, produced the expected band corresponding to the sequence of interest.

[0019] Fig. 2, related to Example 1, shows the agarose gel electrophoresis (AGE) of the final clDNA product.

[0020] Fig. 3, related to Example 2, shows the agarose gel electrophoresis (AGE) of the final open DNA produced according to the process of the invention using Adaptor I (A), Adaptor II (B, two left columns), and Adaptor III (B, two right columns) for the production of the intermediate clDNA.

[0021] Fig. 4, related to Example 2, shows the agarose gel electrophoresis (AGE) of the final clDNAs produced according to the process of the invention and containing Adaptor I.

[0022] Fig. 5, related to Example 3, shows the agarose gel electrophoresis (AGE) of the final open DNA produced according to the process of the invention using the synthetic DNA of sequence SEQ ID NO: 17 (A) or the synthetic DNA of sequence SEQ ID NO: 18 (B).

[0023] Fig. 6, related to Example 3, shows the agarose gel electrophoresis (AGE) of the final clDNAs produced according to the process of the invention with the protelomerase TelA.

[0024] Detailed description of the invention

[0025] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0026] As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the” also include the plural of the noun.

[0027] As above indicated, the present invention provides a cell-free process for the production of DNA, the process comprising the steps of a) providing an open linear double stranded DNA molecule de novo synthesized comprising a sequence of interest; b) generating a closed linear DNA template with the open linear double stranded DNA molecule, wherein the closed linear DNA template comprises the sequence of interest; and c) amplifying the closed linear DNA template by Rolling Circle Amplification (RCA).As used herein, “cell-free process for the production of DNA” refers to a process for the production of DNA where all the steps are carried out in vitro in cell-free conditions (i.e. , without involving any cells), including steps a) to c) and any previous and subsequent production steps.

[0028] The term “DNA” refers to a polymer of deoxyribonucleotides. The term “RNA” refers to a polymer of ribonucleotides. The DNA and RNA may be single- or double-stranded, optionally incorporating synthetic, non-natural, or modified nucleotides capable of incorporation into DNA or RNA. When sequences of polynucleotides are indicated, they refer to informative molecules and only one strand is shown. Nonetheless, the skilled person in the art will understand that they can be in the form of single or double stranded nucleic acid polymers.

[0029] The term “open linear double stranded DNA” refers to any linear DNA molecule that comprises two complementary strands with free 5’ and 3’ ends. The term “closed linear DNA” or “clDNA” or "IcDNA" refers to a single stranded covalently closed DNA molecule that forms a “dumbbell” or “doggy-bone” shaped structure under conditions allowing nucleotide hybridization. Therefore, although the clDNA is formed by a closed single stranded DNA molecule, the formation of the “dumbbell” structure by the hybridization of two complementary sequences within the same molecule generates a structure consisting on a linear double-stranded middle segment closed at both ends (e.g. by single stranded loops). Therefore, a clDNA has no free 5’ or 3’ ends. A clDNA can be generated, for example, by attaching hairpin DNA adaptors — for instance, by the action of a ligase — to both ends of an open linear double stranded DNA. Also, a clDNA can be generated by the action of a protelomerase on an open linear double stranded DNA that comprises at least two protelomerase target sequences, thereby generating a clDNA closed at both ends with a portion of a protelomerase target sequence. A "partially closed DNA product" refers to a DNA molecule consisting on a linear double-stranded middle segment closed at one single end (e.g. by a single stranded loop).

[0030] The term "de novo synthesized DNA molecule" refers to a DNA molecule produced by a process entirely cell-free and that does not require a pre-existing precursor template i.e., by a template-less process. Methods for de novo synthesis of DNA molecules are well known in the art and belong to the common general knowledge of the skilled person. They include, for example, template-independent enzymatic synthesis (e.g., TdT method) or chemical synthesis (e.g., phosphoramidite synthesis).

[0031] The term “Rolling Circle Amplification” or “RCA” refers to nucleic acid amplificationreactions involving the amplification of covalently closed DNA molecules, such as clDNA, with a strand-displacement polymerase, wherein the polymerase performs the extension of a new strand around the closed DNA molecule. The polymerase displaces the hybridized copy and continues polynucleotide extension around the template to produce concatameric DNA comprising tandem units of the amplified DNA template. These linear single stranded products serve as the basis of sequence amplification reactions, resulting in formation of concatameric double stranded DNA products. There are thus multiple copies of each amplified single unit DNA in the concatameric double stranded DNA products The skilled in the art knows, making use of their general knowledge and / or the instructions of the manufacturer, how to adjust the conditions of the amplification step depending on the enzymes and the characteristics of the template to be amplified.

[0032] Depending on how the template clDNA is generated, the concatameric DNA will contain different sequences flanking each amplified DNA sequence of interest. For example, in the concatameric DNA each repeated sequence of interest may be flanked by restriction sites, protelomerase target sequences, or a combination thereof.

[0033] In one embodiment, the process comprises the steps of a) providing an open linear double stranded DNA molecule de novo synthesized comprising a sequence of interest; b) generating a closed linear DNA template with the open linear double stranded DNA molecule of step a), wherein the closed linear DNA template comprises the sequence of interest; and c) amplifying the closed linear DNA template of step b) by Rolling Circle Amplification (RCA).

[0034] In one embodiment, the closed linear DNA template generated in step b) is a de novo closed linear DNA template. In another embodiment, the process is a de novo cell-free process for the production of DNA. In another embodiment, the process does not comprise a DNA polymerase extension step previous to step c).

[0035] In one embodiment, the open linear double stranded DNA molecule de novo synthesized is a synthetic open linear double stranded DNA molecule.

[0036] In one embodiment, the sequence of interest comprises an expression cassette. The term “expression cassette” refers to a sequence comprising one or more promoter or enhancer elements and a gene or other coding sequence which encodes, for example, an mRNA, miRNA, siRNA, InRNA, protein (e.g. enzyme), polypeptide, or peptide of interest. The expression cassette may further comprise other elements that regulate the expression of the coding sequence, such as a transcription termination site.

[0037] In one embodiment, the sequence of interest comprises an expression cassette flankedby inverted terminal repeats (ITRs). The ITRs can be at any suitable distance from the expression cassette. As used herein, the term “terminal repeat” or “TR” includes any viral terminal repeat or synthetic sequence that comprises at least one minimal required origin of replication and a region comprising a palindrome hairpin structure. A Rep-binding sequence (“RBS”) (also referred to as RBE (Rep-binding element)) and a terminal resolution site (“TRS”) together constitute a “minimal required origin of replication” and thus the TR comprises at least one RBS and at least one TRS. TRs that are the inverse complement of one another within a given stretch of polynucleotide sequence are typically each referred to as an “inverted terminal repeat” or “ITR”. In the context of a virus, ITRs mediate replication, virus packaging, integration and provirus rescue.

[0038] In one embodiment, step b) comprises attaching hairpin DNA adaptors to the open linear double stranded DNA molecule to generate the closed linear DNA template comprising the sequence of interest. The skilled person would appreciate that, in order to generate a closed linear DNA, the adaptors must be attached to both ends of the open double stranded DNA. The adaptors at either end can be the same adaptor or different adaptors.

[0039] As used herein, “hairpin DNA adaptor” refers to a DNA molecule with a structure consisting on a linear double-stranded segment closed at one end. The hairpin DNA adaptor may form a single stranded loop when it contains a non-complementary region, whereas in other cases it may not form a single stranded loop when it does not contain a non-complementary sequence.

[0040] In one embodiment, the sequence of the hairpin DNA adaptors is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0041] In one embodiment, the hairpin DNA adaptors comprise at least one modified nucleotide.

[0042] A “modified nucleotide” is any nucleotide (e.g., adenosine, guanosine, cytidine, and thymidine) that has been chemically modified — by modification of the base, the sugar or the phosphate group — or that incorporates a non-natural moiety in its structure. Thus, the modified nucleotide may be naturally or non-naturally occurring depending on the modification. Without limitations, modified nucleotides include of 2-amino-deoxyadenosine, 5-methyl-deoxycytidine, thiophosphate nucleotide, LNA nucleotide, Inosine, 8-oxo-deoxyAdenosine and 5-fluoro-deoxyuracil, and L-DNA nucleotide.

[0043] In one embodiment, the attaching hairpin DNA adaptors comprises contacting the open linear double stranded DNA molecule with the hairpin DNA adaptors together with a ligase under suitable conditions.The skilled in the art knows that some open linear double stranded DNA molecules may have overhangs (sticky ends) while others do not (blunt ends). Both types of open linear double stranded DNA molecules can be used in the method of the invention. The skilled person knows that a hairpin DNA adaptor with sticky ends can be directly attached to an open linear double stranded DNA with sticky ends (sticky-end ligation). An open linear double stranded DNA with blunt ends can also be dA-tailed by a process of adding a terminal 3’deoxy adenosine nucleotide, for instance using Taq polymerase, and then ligated to a hairpin DNA adaptor comprising an overhanging T. Also, an open linear double stranded DNA can be digested with a restriction enzyme to generate overhangs that can be attached to a hairpin DNA adaptor with overhangs.

[0044] Thus, in a particular embodiment, the attaching hairpin DNA adaptors to the open linear double stranded DNA molecule comprises contacting the open linear double stranded DNA molecule with at least one restriction enzyme to generate an open linear double stranded DNA fragment and attaching the hairpin DNA adaptors to the open linear double stranded DNA fragment. The skilled person would appreciate that, in this embodiment, the open linear double stranded DNA molecule comprises at least two restriction sites flanking the sequence of interest. The skilled person would appreciate that the digestion and attachment (i.e. , ligation) can be carried out sequentially or simultaneously in a single reaction volume. Also, the amplification can be carried out directly after the digestion / ligation, or after an intermediate purification step.

[0045] In another particular embodiment, the attaching hairpin DNA adaptors to the open linear double stranded DNA molecule comprises dA-tail ing an open linear double stranded DNA molecule with blunt ends, particularly with a Taq polymerase, to generate an open linear double stranded DNA molecule comprising 3’-dA overhangs, and attaching the hairpin DNA adaptors to the open linear double stranded DNA molecule comprising 3’-dA overhangs, particularly wherein the hairpin DNA adaptors comprise 5’-dT overhangs. The term “dA-tailing” refers to the addition of adenine nucleotides to the 3' ends of DNA strands.

[0046] In another embodiment, the open linear double stranded DNA molecule and the hairpin DNA adaptors comprise overhangs, and step b) comprises directly attaching the hairpin DNA adaptors to the open linear double stranded DNA molecule to generate the closed linear DNA template comprising the sequence of interest.

[0047] The use of restriction enzymes, Taq polymerases, protelomerases, and ligases (for attaching) is routinary in the field of molecular biology, therefore the skilled in the art wouldknow how to adjust the conditions of the reaction depending on the enzymes used, and which restriction enzyme should be used depending on the restriction site to be targeted.

[0048] In one embodiment, the step b) comprises contacting the open linear double stranded DNA molecule with at least one protelomerase, to generate the closed linear DNA template comprising the sequence of interest. The skilled person would appreciate that, in this embodiment, the open linear double stranded DNA molecule comprises at least two protelomerase target sites flanking the sequence of interest.

[0049] As used herein, “protelomerase” is any polypeptide capable of cleaving and rejoining a template comprising a protelomerase target site in order to produce a covalently closed linear DNA molecule. Thus, the protelomerase has DNA cleavage and ligation functions. Enzymes having protelomerase-type activity have also been described as telomere resolvases (for example in Borrelia burgdorferi). If this DNA contains a protelomerase target site, the enzyme can cut the DNA at this site and ligate the ends to create a linear double stranded covalently closed DNA molecule. The ability of a given polypeptide to catalyze the production of closed linear DNA from a template comprising a protelomerase target site can be determined using any suitable assay described in the art.

[0050] Examples of suitable protelomerases for use in the process of the invention include those from bacteriophages such as phiHAP-1 from Halomonas aquamarina, PY54 from Yersinia enterolytica, phiKO2 from Klebsiella oxytoca, VP882 from Vibrio sp., Vp58.5 from Vibrio parahaemolyticus, N15 (also known as TelN) from Escherichia coli, and TelA from Agrobacterium tumefaciens, or variants of any thereof.

[0051] In one embodiment, the protelomerase comprises or consists of a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical, to SEQ ID NO: 7. In a more particular embodiment, the protelomerase comprises or consists of SEQ ID NO: 7. In another embodiment, the protelomerase comprises or consists of a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical, to SEQ ID NO: 19. In a more particular embodiment, the protelomerase comprises or consists of SEQ ID NO: 19.

[0052] In the present invention the term "identical" or "identity" refers to the percentage of positions that are identical in the two sequences when the sequences are optimally aligned. If, in the optimal alignment, a position in a first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the sequences exhibit identity with respect to that position. The percentage of identity determines the number of identical amino acids or nucleotides over a defined length in a given alignment. Thus, the level of identity between two sequences or ("percent sequenceidentity") is measured as a ratio of the number of identical positions shared by the sequences with respect to the number of positions compared (i.e. , percent sequence identity = (number of identical positions / total number of positions compared) x 100). A gap, i.e., a position in an alignment where an amino acid or nucleotide is present in one sequence but not in the other, is regarded as a position with non-identical amino acid or nucleotide and is counted as a compared position.

[0053] A number of mathematical algorithms for rapidly obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. For purposes of the present invention, the sequence identity between amino acid sequences is preferably determined using algorithms based on global alignment, such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), preferably implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277); or the BLAST Global Alignment tool (Altschul et al., “Basic local alignment search tool”, 1990, J. Mol. Biol, v. 215, pages 403-410), using default settings. Local alignment also can be used when the sequences being compared are substantially the same length.

[0054] A “protelomerase target sequence” is any DNA sequence whose presence in a DNA molecule allows for its conversion into a closed linear DNA molecule by the enzymatic activity of a protelomerase. In other words, the protelomerase target sequence is required for the cleavage and re-ligation of double stranded DNA by protelomerase to form covalently closed linear DNA. Typically, a protelomerase target sequence comprises any perfect palindromic sequence i.e. any double-stranded DNA sequence having two-fold rotational symmetry, also described herein as a perfect inverted repeat. In a particular embodiment, the protelomerase target sequence is of sequence SEQ ID NO: 9, SEQ ID NO: 20, or SEQ ID NO. 21.

[0055] In one embodiment, the open linear double stranded DNA molecule comprises at least two protelomerase target sites flanking the sequence of interest, at least two restriction sites flanking the sequence of interest, or both. In a more particular embodiment, the open linear double stranded DNA molecule comprises at least two restriction sites flanking the sequence of interest and at least two protelomerase target sites flanking the at least two restriction sites and the sequence of interest. In another embodiment, the closed linear DNA template comprises at least two restriction sites flanking the sequence of interest.

[0056] In one embodiment, the process is a large-scale cell-free DNA production process. In one embodiment, the process is a large-scale de novo cell-free DNA production process. Inanother embodiment, the process produces at least 1 mg, at least 10 mg, at least 50 mg, at least 100 mg, at least 1 g, at least 10 g, at least 100 g, at least 500 g, or at least 1 kg of DNA.

[0057] In one embodiment, the step a) comprises de novo synthesizing an open linear double stranded DNA molecule comprising a sequence of interest. That is, the open linear double stranded DNA molecule is synthesized in a template-free process that does not involve a DNA polymerase extension step.

[0058] In one embodiment, the open linear double stranded DNA molecule de novo synthesized provided in step a) is synthesized by a process comprising chemical and / or enzymatical de novo (i.e. template-free) synthesizing at least two oligonucleotides and assembling of the open linear double stranded DNA molecule. Methods for de novo synthesis and assembly are well known in the art and belong to the common general knowledge of the skilled person. In a particular embodiment, the de novo synthesis is chemical synthesis, template-free enzymatic synthesis, or both. In another particular embodiment, the assembly is Gibson assembly or Polymerase cycling assembly (PCA).

[0059] In one embodiment, the open linear double stranded DNA molecule is at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10000, at least 15000, or at least 25000, nucleotides in length. In another embodiment, the open linear double stranded DNA molecule is of a length from 100 to 50,000 nucleotides, from 500 to 25,000 nucleotides, from 1,000 to 20,000 nucleotides, or from 5,000 to 15,000 nucleotides.

[0060] In one embodiment, the amplification in step c) is primed with a primase / polymerase enzyme and / or with primers. In one particular embodiment, the primase / polymerase enzyme comprises a sequence at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0061] The term “primase / polymerase enzyme” refers to a DNA-directed primase / polymerase enzyme, such as the enzymes from the archaeo-eukaryotic primase (AEP) superfamily. These enzymes present the capacity of starting DNA chains with dNTPs. Enzymes from this superfamily that can be used in the invention are, for example, Thermus thermophilus primase / polymerase (TthPrimPol) or human primase / polymerase (hsPrimPol, CCDC111, FLJ33167, EukPrim2 or hPrimPoll). “Thermus thermophilus primase / polymerase” or“TthPrimPol” refers to the primase / polymerase of the bacteria Thermus thermophilus of sequence SEQ ID NO: 1. The nucleotide and protein sequences are available in the NCBI Entrez database as NC_005835 and WP_01 1173100.1, respectively.

[0062] In one embodiment, the amplification in step c) is carried out with a strand-displacement DNA polymerase. The term “strand-displacement DNA polymerase” refers to a DNA polymerase that performs a 3' end elongation reaction while removing a double-stranded portion of template DNA. Strand displacement DNA polymerases that can be used in the present invention may not be particularly limited, as long as they have such a stranddisplacement activity, such as phi29 DNA polymerase and Bst DNA polymerase.

[0063] Depending on the selected polymerase, the skilled in the art would know that the reaction conditions for a 3' end elongation reaction may be adequately set. For example, when phi29 DNA polymerase is used, a reaction may be performed at an optimum temperature for the reaction from 25°C to 35 °C.

[0064] Thus, in a particular embodiment, the strand displacement DNA polymerase is selected from the group consisting of phi29 DNA polymerase, Bst DNA polymerase, Bea (exo-) DNA polymerase, Klenow fragment of Escherichia coli DNA polymerase I, Vent (Exo-) DNA polymerase, DeepVent (Exo-) DNA polymerase, and KOD DNA polymerase. In a more particular embodiment, the strand displacement DNA polymerase is phi29 DNA polymerase. In another particular embodiment, phi29 DNA polymerase comprises or consists of a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical, to SEQ ID NO: 8. In a more particular embodiment, the phi29 DNA polymerase comprises or consists of SEQ ID NO: 8. In an even more particular embodiment, the strand displacement DNA polymerase is a chimeric protein comprising a phi29 DNA polymerase. The skilled in the art knows how to obtain chimeric DNA polymerases with improved characteristics, for example, as disclosed in WQ2011000997.

[0065] In one embodiment, the amplification in step c) generates concatameric DNA comprising a plurality of repeats of the sequence of interest. In one embodiment, the amplification in step c) generates double stranded concatameric DNA comprising a plurality of repeats of the sequence of interest. In one embodiment, the amplification in step c) generates concatameric DNA comprising a plurality of repeats of the sequence of interest flanked by restriction sites, protelomerase target sites, or both.

[0066] In one embodiment, the amplification in step c) generates concatameric DNA comprising a plurality of repeats of the sequence of interest, and wherein the process further comprises the step d) generating a plurality of open linear double stranded DNA products, a plurality of partially closed DNA products, or a plurality of closed linear DNA products, comprising the sequence of interest. In one embodiment, the amplification in step c)generates concatameric DNA comprising a plurality of repeats of the sequence of interest, and wherein the process further comprises the step d) generating with the concatameric DNA of step c) a plurality of open linear double stranded DNA products, a plurality of partially closed DNA products, or a plurality of closed linear DNA products, comprising the sequence of interest. The skilled person would appreciate that depending on the sequences flanking the sequence of interest in the concatamers, they can be processed by different techniques to generate the final DNA products. For example, if they comprise restriction sites flanking the sequence of interests, they can be processed by restriction enzymes to generate a plurality of open linear double stranded DNA products, which can optionally be later converted into closed linear DNA products or partially closed DNA products by attaching (e.g. ligating) hairpin adaptors to one or both ends of the open linear products. The digestion and ligation can be carried out sequentially or simultaneously. Also, if the concatamers comprise protelomerase target sites, they can be processed by a protelomerase to generate a plurality of closed linear DNA products. The skilled person knows that the concatamers can be processed directly after amplification or they can be first purified using the appropriate techniques.

[0067] In one embodiment, the amplification of step c) is carried out in the presence of at least one type of modified nucleotide.

[0068] In one embodiment, the step d) comprises contacting the concatameric DNA with a protelomerase thereby generating a plurality of closed linear double stranded DNA products. In another embodiment, the concatameric DNA comprises protelomerase target sites between each repeat of the sequence of interest, and step d) comprises contacting the concatameric DNA with a protelomerase thereby generating a plurality of closed linear double stranded DNA products.

[0069] In one embodiment, step d) comprises contacting the concatameric DNA with at least one restriction enzyme thereby generating a plurality of open linear double stranded DNA products and, optionally, attaching hairpin DNA adaptors to the plurality of open linear double stranded DNA products to generate a plurality of closed linear DNA products or a plurality of partially closed DNA products . In another embodiment, the concatameric DNA comprises restriction sites between each repeat of the sequence of interest and step d) comprises contacting the concatameric DNA with at least one restriction enzyme thereby generating a plurality of open linear double stranded DNA products and, optionally, attaching hairpin DNA adaptors to the plurality of open linear double stranded DNA products to generate a plurality of closed linear DNA products or a plurality of partially closed DNA products.In one embodiment, the process further comprises the step e) of purifying the plurality of open linear double stranded DNA products or the plurality of closed linear double stranded DNA products. In one embodiment, the process further comprises the step e) of purifying the plurality of open linear double stranded DNA products or the plurality of closed linear double stranded DNA products, of step d). Any known method known by the skilled person and suitable for purifying nucleic acids, in particular open linear double stranded DNA molecules or closed linear DNA molecules, can be used.

[0070] As above indicated, in a second aspect the invention provides a cell-free process for the production of RNA, the process comprising the step of producing DNA by a cell-free process as defined in the first aspect transcribing the DNA to RNA. All the embodiments above provided for the process of the first aspect are also meant to apply to the process of this second aspect.

[0071] The skilled person would know how to transcribe the DNA to RNA using any cell-free transcription methods that belongs of the common general knowledge, for example, by in vitro transcription with an RNA polymerase.

[0072] In one embodiment, the RNA is messenger RNA (mRNA).

[0073] As used herein, the terms “messenger RNA” or "mRNA" or "transcript" refers to any RNA polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, or ex vivo. Typically, an mRNA is single-stranded and comprises a 5'-cap structure, a 5'IITR, an ORF, a 3'IITR and a 3' tailing sequence.

[0074] Sequences disclosed in the present application are shown in Table 1 below:

[0075] Table 1

[0076] SEQ ID Name Sequence

[0077] NO:

[0078] 1 TthPrimPol MRPIEHALSYAAQGYGVLPLRPGGKEPLGKLVPHGLKN ASRDPATLEAWWRSCPRCGVGILPGPEVLVLDFDDPEA WEGLRQEHPALEAAPRQRTPKGGRHVFLRLPEGVRLSA SVRAIPGVDLRGMGRAYVVAAPTRLKDGRTYTWEAPLT PPEELPPVPQALLLKLLPPPPPPRPSWGAVGTASPKRLQ ALLQAYAAQVARTPEGQRH LTLI RYAVAAGGLI PHGLDP REAEEVLVAAAMSAGLPEWEARDAVRWGLGVGASRPL VLESSSKPPEPRTYRARVYARM RRWV

[0079] 2 TthPrimPol2 MRPIEHVLGYARLGYAVLPLLPGEKRPHPRLVPHGLKEA

[0080] SRDPATLEAWWRSCPQAGAGILPAPEVLVLDFDDPEAW

[0081]

[0082] ERLKEEHPTLLEAPRARTPRGGVHVYLRLPPEAVGRLSASVRAIPGVDLRGLGRSYLVAPPTTLPTGAYVWEVPLRRP EELPPVPAPLLARLLPPPPPPREVWTPVEGVSPKRLQAL LQAYAAQVAHTPEGQRHNTLIRYAVAAGGLLPHGLDPR EAEEALVAAAMSAGLPEAEARAAARWGLEVGASRPLAL EPSLAPSGPRTYRARVYARMRRWA

[0083] SynPrimPoU RAPHSSPLEAALGYARLGYPVLPLLPGEKRPHPRLVPHG LKEASQDPATLEAWWRSCPEAGVGILAPAGVLVLDFDD PTAWEGLRQEHPALEAAPRQRTPKGGVHVFLRLPEGVR LSASVRAIPGVDLRGMGRAYVVAAPTRLKDGRTYSWEV PLVRPAELPPVPEALLARLLPPPPPPPREVWTPVEGASP KRLRALLEAYAHRVAATPPGTRHNTLIRYAVAAGGLIPH GLDPREAEEALVAAALSAGLPEREARAAVRWGLEVGRS RPLDLESSLPPRLPWTYRGRVYARVRRWA

[0084] SynPrimPol2 AHSTSPLEAALGYARMGYPVLPVAPGSKRPHPRLVPHG LKEASTDPATIEAWWRSCPEANVGILAPEGVLVLDLDDP TAWEGLRQLHPALLAAPRARTPRGGVHLYLRLPPGVRL SASVRALPGVDLRGMGRAYVVAAPSRLKDGRTYAWVV PLRRPSELPPVPQALLARLLAPPPPPAARPVSTPVEGAS PKRLRALLEAACHRVAHTPPGQRHNTLIRHAVAVGGLIP HGLDQEEAEEALVAAALSAGLPEREARAAVRWGLEAGA SRPLVLKPSLPPRLRWTIRRRVRARGRRHG

[0085] SynPrimPol3 ALSTSPLEAALGYARMGYPVLPVAPGSKRPHPRLVRHG LKEATTDPALI EAWWSSRPEAN VGI LPPAGVLVLDLDSP AGWAGLRQLHPALLAAPRARTPGGGVHLYFRLPPGVRL SASVRVLPGVDLRGMGRAYVVAPPSRHADGRTYEWVV PLRRPSELPPVPQALLALLLAPPPPPAARPVSTPVASAR PKAYRAALEAECDRVARTPPGQRH NTLI RHAVALGGLI P RGLDQEEVEGALLAAALRAGLPEREARAAIRWGLEAGA SRPLALKPRLPPRLRWTIRRRARARGRRHG

[0086] SynPrimPol4 ALSTSPLEAALAYARMGYPVLPVAPGSKRPHPRLVRHG LKEATTDPAQIEAWWSRRPEANVGILTPSGVLVLDLDSP DGWAGLRQLHPALLPAPKVRTPGGGVHLYFRLPPGVRL RASVRVLPGIDLRGMGRAYVVAPPSRHAHGRTYEWVVP LRAPSELPPVPQALLALLLAPPPPPAARPVSPPVASARP DAYRAALEAECDRVARTPPGQRHNTLIRHAVALGGLVP RGLDEEEVEGALLAAALRAGLAEREARATIRWGLEAGAS RPLDLRPRLPPRLRWRIRRRARARGRRHG

[0087] TelN MSKVKIGELINTLVNEVEAIDASDRPQGDKTKRIKAAAAR YKNALFNDKRKFRGKGLQKRITANTFNAYMSRARKRFD DKLHHSFDKNINKLSEKYPLYSEELSSWLSMPTANIRQH MSSLQSKLKEIMPLAEELSNVRIGSKGSDAKIARLIKKYP DWSFALSDLNSDDWKERRDYLYKLFQQGSALLEELHQL KVNHEVLYHLQLSPAERTSIQQRWADVLREKKRNVVVID YPTYMQSIYDILNNPATLFSLNTRSGMAPLAFALAAVSGR RMIEIMFQGEFAVSGKYTVNFSGQAKKRSEDKSVTRTIY TLCEAKLFVELLTELRSCSAASDFDEVVKGYGKDDTRSE NGRINAILAKAFNPWVKSFFGDDRRVYKDSRAIYARIAYE MFFRVDPRWKNVDEDVFFMEILGHDDENTQLHYKQFKL ANFSRTWRPEVGDENTRLVALQKLDDEMPGFARGDAG VRLHETVKQLVEQDPSAKITNSTLRAFKFSPTMISRYLEF AADALGQFVGENGQWQLKI ETPAI VLPDEESVETI DEPD DESQDDELDEDEIELDEGGGDEPTEEEGPEEHQPTALK PVFKPAKN NGDGTYKI EFEYDGKHYAWSGPADSPMAA MRSAWETYYS MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSE

[0088]

[0089] phi29YKIGNSLDEFM AWVLKVQADLYFH N LKFDGAFI I N WLER NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKI HTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVG YKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLK GFKDIITTKKFKKVFPTLSLGLDKEVRYAYRGGFTWLND RFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGK YVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNE YLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKF KATTGLFKDFI DKWTYI KTTSEGAI KQLAKLM LNSLYGKF ASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGV FITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDI VDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKL VEGSPDDYTDI KFSVKCAGMTDKI KKEVTFEN FKVGFSR KMKPKPVQVPGGVVLVDDTFTIK

[0090] 9 TelN target TATCAGCACACAATTGCCCATTATACGCGCGTATAATG GACTATTGTGTGCTGATA

[0091] sequence

[0092] 10 Hairpin DNA AGGGCTAACCA(5FU)T(5FU)AGGTTAG

[0093] adaptor

[0094] 14 Hairpin DNA AGGGGAAGAGCATATGCTCTTC

[0095] adaptor

[0096] 15 Hairpin DNA AGGGCGAGACCGCGCGGTCTCG

[0097] adaptor

[0098] 16 Hairpin DNA AGGGCGAGACCATATGGTCTCG

[0099] adaptor

[0100] 19 TelA MPASKRKTKTPVLVERIDHFVDQVKEAMKSDDTLRNRKI RDLWDAEVRYHFDNGRTEKTLELYIMKYRNALKAEFGV KSTPLAICNMKKLRERLNTYIARADYTKTGVATSIVEKIER AEFNTAGRKPTVLLRIADFISAMNGMGTKEEMQSLWNA EIGTMKGRAQTTIISYITKYRNAIREAFGDDHPMLKIATGD AAMYDDARRVKMEKIARKHGALITFENYRQVLKICADKLL SADPLMIGIGLIGMTGRRPYEVFTQAEFSPAPYGKGVSK WSLLFNGQAKTKQGEGTKFGITYEI PVLARSETI LAAYRR LRESGQGKLWHGMSIDDFSSETRLLLRDTVFNLFEDLW PKEELPKPYGLRHLYAEVAFHNFAPPHVTKNSYFAAILG HNNNDLETSLSYMTYTLPEDRDDALARAKRINERTLQQ MATIAPVSRKA

[0101] 20 TelA target AATAACAATATCATGATATTGTTATT

[0102] sequence

[0103] (minimal)

[0104] 21 TelA target GCGATCGATCATAATAACAATATCATGATATTGTTATT

[0105] GTAATCGATCGC

[0106]

[0107] sequence

[0108] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps.

[0109] Furthermore, the word “comprise” encompasses the case of “consisting of”. Additionalobjects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

[0110] Example 1

[0111] 1. Materials and reagents

[0112] Enzymes, buffers and reagents used in the examples are listed in Table 1 below.

[0113] Table 1

[0114] Material Manufacturer Reference Batch number Format dNTPs Thermofisher R0186 2676081 100 mM Scientific

[0115] Pyrophosphatase Genscript N / A U3659HD220- 1370 lls / rn 1 / P5HE001 L DNA Primase Cocoon N / A 238-220211-01 290 lls / rn (DPAS) L Phi29 (CPS) Thermo 230 lls / rn Scientific L

[0116] NaOH Merck 1.37020.100 1 M

[0117] 0

[0118] Protelomerase Genscript N / A 2500 U / MI (TelN)

[0119] ExoIl I Genscript N / A U284WPAUG0- 5000 U / MI 3 / P03JB001

[0120] ExoT5 Genscript N / A 8000 U / MI 0

[0121] Bsal Genscript N / A 1500 U / mL 0

[0122] ATP Merck A7699

[0123] PEG 4000 Fisher 15424099 J61495.AK 50 %

[0124] Scientific

[0125] Oligonucleotide Genscript N / A 100 uM

[0126]

[0127] (Hairpin DNAadaptor) (SEQ ID

[0128]

[0129] NO: 10)

[0130] Buffers:

[0131] • 20x TelN buffer: 400 mM Tris-HCI, 200mM (NH4)2SO4, 200mM KCI, 20mM MgSO4, pH 8.8.

[0132] • 2x RCA buffer: 100 mM Tris-Base, 100 mM Tris-HCI, 100 mM KCI, 20 mM MgCI2, 2 mM DTT, pH 8.

[0133] • 1M NaOH

[0134] • 10X Ligation buffer: 400 mM TrisHCI, 213.71 mM MgCI2, 100 mM DTT.

[0135] • 10X Exonuclease buffer: 500 mM Potassium Acetate, 200 mM Tris-Acetate, 100 mM Magnesium Acetate Tetrahydrate, 10 mM DTT.

[0136] 2. Methods and Results

[0137] 2.1. Protelomerase (TelN) Digestion and clean up

[0138] To validate that high amounts of high-quality DNA can be directly produced de novo in completely cell-free conditions, a de novo synthesized open linear double-stranded DNA of SEQ ID NO: 11 produced by Ribbon Bio was digested with TelN in order to close the ends and generate a closed linear DNA (clDNA). The digestion was carried out by incubating 100 ug of DNA with 1 unit of TelN per 1 ug of DNA in TelN buffer (20 mM Tris-HCI, 10mM (NH4)2SO4, 10mM KCI, 1mM MgSO4, pH 8.8.) at 30 °C for 1 hour.

[0139] Part of the sample was purified by GeneJet PCR purification column (Fisher Scientific, 10578360) following manufacturer’s instruction, and the rest was cleaned up with exonuclease III and exonuclease T5 following manufacturer’s instruction, with 500 units of each exonuclease per mg of DNA at 37 °C for 1 hour. The exonucleases cleaned up the non-closed DNA and produced a purer sample of clDNA.

[0140] 2.2. RCA from clDNA

[0141] The clDNA samples after cleaning with exonucleases were used as templates for Rolling Circle Amplification (RCA). Briefly, 0.5 ug of each sample were used at 1 ug / mL concentration on RCA and this was denatured with 1 M of NaOH for 5 min. Denaturation was neutralized with RCA Buffer (50 mM Tris-Base, 50 mM Tris-HCI, 50 mM KCI, 10 mM MgCI2, 1 mM DTT, pH 8) and then, the rest of the reagents and enzymes were added: 5 mM dNTPs, 2 units / mL of DPAS and CPS, 0.5 units / mL of Pyrophosphatase. Incubation was performed at 30 °C overnight.After overnight incubation, the RCA amplification product (concatemers) was quantified. RCA amplification product concentration was determined via precipitation with isopropanol. Briefly, the precipitation was carried out by mixing 50ul of isopropanol with 5ul of sodium acetate pH 5.5 (Thermo Fisher Scientific, AM9740). Then, 50ul of the sample were added. Next, the mixture was centrifuged at 12000 xg for 20 minutes. Pellet was homogenized with 50ul of H2O. Concentrations were measured in nanodrop, which showed that both samples produced around 1000 ng / uL concatemeric DNA concentration.

[0142] Bsal was then added to the RCA amplification products to digest the concatemers and generate double stranded DNA fragments, each containing the sequence of interest. The digestions were carried out by incubating for 1 hour at 37 °C 250 ug of the RCA products (concatemers) with 2 unit of Bsal per ug of concatamers.

[0143] Digestions were analyzed by Agarose Gel Electrophoresis (AGE) following manufacturer’s instructions. As shown in Figure 1, digestions of concatemers generated by the amplification of clDNAs produced the band of the gene of interest as it was expected. In addition, digestion from cleaned up DNA produced less impurities than the non-treated one.

[0144] Finally, the open linear double stranded DNA fragments were converted into clDNAs by ligating hairpin DNA adaptors (SEQ ID NO: 10) to both ends. The ligation was carried out by incubating 25 ug of DNA with 0.5 mM ATP, 1:50 ratio of DNA template: oligonucleotide adapter, 1 unit of T4 ligase per ug of DNA and 2.5% PEG in ligation buffer (40 mM TrisHCI, 21.37 mM MgCI2, 10 mM DTT) at 37°C for 1 hour. Exo III was added at 0.012 units per ug of DNA and digestion was incubated for 3 hours. Final clDNA were purified by GeneJet PCR Purification column (Fisher Scientific, 10578360), as shown in Figure 2.

[0145] These results clearly demonstrate that de novo synthesized open linear double stranded DNA can be used to generate a clDNA template, which can be later amplified to obtain large amounts of high-quality synthetic DNA in completely cell-free conditions.

[0146] Example 2

[0147] A de novo synthesized double-stranded linear DNA of sequence SEQ ID NO: 12 or 13 was digested with Bsal (Thermofisher, ER0292) or Esp3l (Thermofisher, FD0454), respectively, to produce the overhangs compatibles with the corresponding adaptors — for the DNA of SEQ ID NO: 12, Adaptor I of sequence AGGGGAAGAGCATATGCTCTTC (SEQ ID NO: 14); and for the DNA of SEQ ID NO: 13, either Adaptor II of sequence AGGGCGAGACCGCGCGGTCTCG (SEQ ID NO: 15) or Adaptor III of sequenceAGGGCGAGACCGCGCGGTCTCG (SEQ ID NO: 16).

[0148] Then the digestion product (0.01 mg / mL) was ligated to the corresponding adaptor (1 M) in the presence of the DNA ligase T4 (1000U / mg; Thermofisher, EL0014), ATP (10mM), and ligation buffer (50 mM Tris-HCI, pH 7.5, 10 mM MgCI2, 1 mM ATP, 10 mM DTT), by incubating for 1 hour at 37 °C at 500 rpm. The ligation reaction was then cleaned by exonuclease III and exonuclease T5 as above indicated.

[0149] The resulting intermediate clDNA was then amplified by RCA. The amplification was carried out by mixing the clDNA (0.1 ug / mL) with dNTPs (5 mM), pyrophosphatase (ThermoScientific) (0.548 Us / mL), primase / polymerase (SEQ ID NO: 3) (2 Us / mL), and phi29 DNA polymerase (ThermoScientific) (2 Us / mL), in RCA buffer (50 mM Tris-HCI, 10 mM MgCI2, 10 mM (NH4)2SO4, 4 mM DTT. pH 7.5), for 3 hours at 30 °C. Final DNA concentration was measured as indicated above obtaining a value of 300 ng / uL

[0150] In order to generate open DNA as a final product, the amplification product (i.e. concatemeric DNA) was digested with BspQI (NEB, R0712S) for 1 hour at 40 °C with agitation (500 rpm), purified and analyzed by AGE as above indicated. As shown in Figure 3, the observed bands in several replica reactions were sharp, well-defined, and of the expected size, with minimal smearing or degradation, clearly indicating that the process of the invention allows the generation of large quantities of high-quality open DNA with all the adaptors tested.

[0151] Alternatively, in order to generate the clDNA as a final product, the concatemers were digested with Bsal. . Then the digestion product (0.100 mg / mL) was ligated to the corresponding adaptor (1pM) in the presence of the DNA ligase T4 (1000U / mg;

[0152] Thermofisher, EL0014), ATP (10mM), and ligation buffer (50 mM Tris-HCI, pH 7.5, 10 mM MgCI2, 1 mM ATP, 10 mM DTT), by incubating for 1 hour at 37 °C at 500 rpm. The ligation reaction was then cleaned by exonuclease III and exonuclease T5 and analyzed by AGE as above indicated. Again, as shown in Figure 4, the well-defined bands of the expected size demonstrated that the process of the invention allows the generation of large quantities of high-quality clDNA.

[0153] Example 3

[0154] A de novo synthesized open linear double-stranded DNA of SEQ ID NO: 17 or 18 was processed with TelA (NEB, M0621S) in order to close the ends and generate a closed linear DNA (clDNA). The digestion was carried out by incubating the DNA (0.01 mg / mL) with TelA (3000U / mg) in reaction buffer (200 mM Tris-HCI, 100 mM (NH4)2SO4, 100 mMKCI, 20 Mm, 2 mM MgSO4and 0.001% Tween20. pH 8.8) at 42 °C for 1 hour.

[0155] The reaction product (i.e. intermediate clDNA) was cleaned up with exonuclease III and exonuclease T5 and amplified by RCA as indicated in Example 2. Briefly, the amplification was carried out by mixing the clDNA template at 0.1 ug / mL with dNTPs (5 mM), pyrophosphatase (ThermoScientific) (0.548 Us / mL), primase / polymerase (SEQ ID NO: 3) (2 Us / mL), and phi29 DNA polymerase (ThermoScientific) (2 Us / mL), in RCA buffer (50 mM Tris-HCI, 10 mM MgCI2, 10 mM (NH4)2SO4, 4 mM DTT. pH 7.5). The amplification reaction was incubated for 3 hours at 30 °C in a thermomixer. A DNA concentration of 300 ng / uL was as measured by I PA precipitation followed by nanophotometer analysis.

[0156] In order to generate an open DNA as a final product, the amplification product was digested with BspQI, purified and analyzed by AGE as indicated above. As shown in Figure 5, the observed bands were sharp, well-defined, and of the expected size, with minimal smearing or degradation, clearly indicating that the protelomerase TelA can be used in the process of the invention to generate large quantities of high-quality open DNA in cell-free conditions.

[0157] Alternatively, in order to generate a clDNA as a final product, the concatemers were digested with TelA (NEB, M0621S) in order to close the ends and generate a clDNA. The digestion was carried out by incubating the DNA (0.100 mg / mL) with TelA (3000U / mg) in reaction buffer (200 mM Tris-HCI, 100 mM (NH^SCL, 100 mM KCI, 20 Mm, 2 mM MgSCL and 0.001% Tween20. pH 8.8) at 42 °C for 1 hour, purified and analyzed by AGE as indicated above. Again, as shown in Figure 6, the well-defined bands of the expected size demonstrated that the protelomerase TelA can be used in the process of the invention allows to generate large quantities of high-quality clDNA.

[0158] Citation List

[0159] WO2011000997

Claims

Claims1. A cell-free process for the production of DNA, the process comprising the steps of: a) providing an open linear double stranded DNA molecule de novo synthesized comprising a sequence of interest;b) generating a closed linear DNA template with the open linear double stranded DNA molecule, wherein the closed linear DNA template comprises the sequence of interest; andc) amplifying the closed linear DNA template by Rolling Circle Amplification (RCA).

2. The process according to claim 1, wherein the step b) comprises attaching hairpin DNA adaptors to the open linear double stranded DNA molecule or contacting the open linear double stranded DNA molecule with at least one protelomerase, to generate the closed linear DNA template comprising the sequence of interest.

3. The process according to claim 2, wherein the attaching hairpin DNA adaptors to the open linear double stranded DNA molecule comprises:- contacting the open linear double stranded DNA molecule with at least one restriction enzyme to generate an open linear double stranded DNA fragment and attaching the hairpin DNA adaptors to the open linear double stranded DNA fragment; or- dA-tailing the open linear double stranded DNA molecule and attaching the hairpin DNA adaptors to the dA-tailed open linear double stranded DNA molecule.

4. The process according to any one of claims 1-3, wherein the process is a large-scale cell-free DNA production process.

5. The process according to any one of claims 1-4, wherein the open linear double stranded DNA molecule comprises at least two protelomerase target sites flanking the sequence of interest, at least two restriction sites flanking the sequence of interest, or both.

6. The process according to any one of claims 1-5, wherein the open linear double stranded DNA molecule de novo synthesized provided in step a) is synthesized by a process comprising chemical and / or enzymatical de novo synthesizing at least two oligonucleotides and assembling of the open linear double stranded DNA molecule.

7. The process according to any one of claims 1-6, wherein the open linear double stranded DNA molecule is of a length from 100 to 50,000 nucleotides, from 500 to 25,000 nucleotides, from 1,000 to 20,000 nucleotides, or from 5,000 to 15,000 nucleotides.

8. The process according to any one of claims 1-7, wherein the protelomerase comprises a sequence at least 70% identical to SEQ ID NO: 7 or SEQ ID NO: 19.

9. The process according to any one of claims 1-8, wherein the amplification in step c) is primed with primers or with a primase / polymerase enzyme; particularly, wherein the primase / polymerase enzyme comprises a sequence at least 70% identical to a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

10. The process according to any one of claims 1-9, wherein the amplification in step c) is carried out with a strand-displacement DNA polymerase; particularly, a phi29 polymerase.

11. The process according to any one of claims 1-10, wherein the amplification in step c) generates concatameric DNA comprising a plurality of repeats of the sequence of interest, and wherein the process further comprises the step d) generating a plurality of open linear double stranded DNA products or a plurality of closed linear DNA products, comprising the sequence of interest.

12. The process according to claim 11, wherein the process further comprises the step e) purifying the plurality of open linear double stranded DNA products or the plurality of closed linear DNA products.

13. The process according to any one of claims 11-12, wherein step d) comprises contacting the concatameric DNA with a protelomerase thereby generating a plurality of closed linear DNA products.

14. The process according to any one of claims 11-12, wherein step d) comprises contacting the concatameric DNA with at least one restriction enzyme thereby generating a plurality of open linear double stranded DNA products and, optionally, attaching hairpin DNA adaptors to the plurality of open linear double stranded DNA products to generate a plurality of closed linear DNA products.

15. A cell-free process for the production of RNA, the process comprising the step of producing DNA by a cell-free process as defined in any one of claims 1-14 and transcribing the DNA to RNA.

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