Linear DNA with increased resistance to exonucleases and methods for its production

Description

TECHNICAL FIELD

[0001] The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product with enhanced resistance to nuclease digestion are provided. The methods comprise, (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase target sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences.BACKGROUND

[0002] DNA is susceptible to degradation by nucleases which are naturally occurring enzymes within organisms and which have a vital role in the regulation of many cellular processes, while also protecting against foreign DNA species. Enzymatic DNA degradation can render gene therapies ineffective and is a substantial consideration when developing gene therapies or DNA vaccines.

[0003] Considerable efforts have been made to extend the effective molecular lifetime of nucleic acids by increasing resistance of the nucleic acid molecules to both extracellular and intracellular nucleases.

[0004] Resistance to nuclease digestion can be accomplished by using closed DNA molecules, such as plasmids or minicircles. However, plasmids and minicircles have limited utility in vivo due to their frequent contamination with toxic agents derived from cell components, fidelity issues that alter the sequence of interest, and presence of different species (supercoiled, linear and open circular).

[0005] Alternatively, resistance to nuclease digestion may be accomplished by producing closed linear DNA molecules. For example, WO2010 / 086626 A1 describes a method for producing a closed linear DNA by utilizing a protelomerase, relying on a complete protelomerase target sequence being present at each end of a DNA sequence of interest. This may generate a desired closed DNA product but also a closed product that is unwanted (e.g. a closed plasmid backbone that is resistant to, e.g., exonuclease digestion), and difficult to separate from the desired product. It also lacks flexibility to produce closed linear DNA products with different closed ends, or linear DNA product that are partially closed.

[0006] Thus, a need exists for a more flexible method for producing a linear DNA product with an enhanced resistance to nuclease (e.g. exonuclease) digestion.DESCRIPTION

[0007] The invention provides a method for producing a linear deoxyribonucleic acid (DNA) product (e.g. a closed linear DNA product) with enhanced resistance to nuclease digestion. The invention is based on the addition of adaptor molecules to a double-stranded DNA molecule and the action of protelomerase to close open ends of DNA at a protelomerase target sequence. The method of the invention relies on the addition of the adaptor molecules, an endonuclease and a protelomerase to the double-stranded DNA molecule comprising a truncated protelomerase target sequence, which may be carried out in a single reaction volume (or single contiguous aqueous volume), or in sequential reactions. Thus, the method for producing a linear DNA product comprises the steps: digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase target sequence is non-functional; appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

[0008] The invention relies on the action of protelomerase, an enzyme that cleaves DNA at a protelomerase target sequence and ligates the cleaved ends together to form covalently closed ends. For example, one protelomerase target sequence, TelN, consists of 56-bp target site consists of a central telO palindrome of 22 base pairs and two 14-base pair flanking sequences (telR and telL) comprising inverted repeats. telO is separated from these repeats by 3 base pairs on each side (SEQ ID NO:1). In general, a protelomerase target sequence is a palindromic sequence, i.e. a double stranded DNA sequence that includes an inverted repeat and is symmetrical around a central point, the cleavage point. Different protelomerase enzymes recognise different sequences, as well known in the art. Examples of protelomerases are TelN protelomerase from E. coli phage N15, Klebsiella phage Phi K02 protelomerase, Yersinia phage PY54 protelomerase, Vibrio phage VP882 protelomerase, Borrelia burgdorferi protelomerase along with their respective target sequences.

[0009] Preferably, the linear DNA product has enhanced resistance to exonuclease (e.g. exonuclease I, exonuclease III and / or exonuclease VIII) digestion. The linear DNA product may be a closed linear DNA product. The linear DNA product may comprise nuclease-resistant nucleotides (i.e. protected nucleotides), such as phosphorothioated nucleotides. The linear DNA product may be a partially closed linear DNA product. The partially closed linear DNA product may comprise nuclease-resistant nucleotides (i.e. protected nucleotides), such as phosphorothioated nucleotides. The linear DNA product may comprise a cassette. The cassette may comprise a coding sequence.

[0010] The invention provides a method of producing a linear DNA product, wherein the method comprises the steps of: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase target sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

[0011] The first and second adaptor molecules may be identical molecules or they may be different molecules. The first adaptor molecule comprises a truncated protelomerase target sequence, which is non-functional, that when appended to the double-stranded DNA molecule forms a functional protelomerase sequence with the truncated protelomerase target sequence at the first end of the digested double-stranded DNA molecule, which truncated protelomerase target sequence is also non-functional. The second adaptor molecule may comprise a truncated protelomerase target sequence, which is non-functional, that when appended to the double-stranded DNA molecule forms a functional protelomerase sequence with the truncated protelomerase target sequence at the first end of the digested double-stranded DNA molecule, which truncated protelomerase target sequence is also non-functional. The second adaptor molecule may comprise a hairpin. The second adaptor molecule may be a double stranded nucleic acid molecule comprising one or more nuclease-resistant nucleotides. The first adaptor molecule may comprise a truncated protelomerase target sequence and the second adaptor molecule may be a double-stranded linear nucleic acid molecule comprising one or more nuclease-resistant nucleotides. The first adaptor molecule may comprise a truncated protelomerase target sequence and the second adaptor molecule may comprise a hairpin. Thus, the linear DNA product produced by the methods described herein is resistant to nuclease (e.g. exonuclease) digestion.

[0012] The double-stranded DNA molecule comprises a truncated protelomerase sequence; the truncated protelomerase target sequence may consist of SEQ ID NO:s 4, 5, 6 or 20. The truncated protelomerase sequence may consist of SEQ ID NO: 4. The truncated protelomerase sequence may consist of SEQ ID NO: 5. The truncated protelomerase sequence may consist of SEQ ID NO: 6. The truncated protelomerase sequence may consist of SEQ ID NO: 20.

[0013] The first adaptor molecule and / or the second adaptor molecule may comprise a sequence selected from SEQ ID NO:8, 9, 10, 11, 12 or 13. The first adaptor and / or the second adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 8 and 9, SEQ ID NO:s 10 and 11 or SEQ ID NO:s 12 or 13.

[0014] The truncated protelomerase sequence may consist of SEQ ID NO:23, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. The truncated protelomerase sequence may consist of SEQ ID NO: 23. The truncated protelomerase sequence may consist of SEQ ID NO: 30. The truncated protelomerase sequence may consist of SEQ ID NO: 31. The truncated protelomerase sequence may consist of SEQ ID NO: 32.

[0015] The first adaptor molecule and / or the second adaptor molecule may comprise a sequence selected from SEQ ID NO:24, 25, 26, 27, 28 or 29. The first adaptor and / or the second adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 24 and 25, SEQ ID NO:s 26 and 27 or SEQ ID NO:s 28 or 29.

[0016] Steps a) to c) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0017] The step of appending a first adaptor molecule and a second adaptor molecule to the first and second ends of the digested DNA molecule is preferably performed in the presence of a ligase. Thus, the invention provides a method of producing a linear DNA product, wherein the method comprises the steps of: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase target sequence is non-functional; (b) ligating a first adaptor molecule to the first end of the digested double-stranded DNA molecule and ligating a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences.

[0018] Steps a) to c) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0019] The appending (or linking or closing) of the first adaptor molecule and / or the second adaptor molecule may be performed by hybridization or ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule may be hybridized to the first end of the linear double-stranded region. The second adaptor molecule may be hybridized to the second end of the linear double-stranded region. The first adaptor molecule may be ligated to the first end of the linear double-stranded region. The second adaptor molecule may be ligated to the second end of the linear double-stranded region. The appending of the first adaptor molecule and the second adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule may be hybridized and ligated to the first end of the linear double-stranded region. The second adaptor molecule may be hybridized and ligated to the second end of the linear double-stranded region. The appending may be performed via a linker or spacer molecule which facilitates joining of the adaptor molecule to the first and / or second end of the linear double-stranded region.

[0020] The method may further comprise, before step (a) (i.e. the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule), a step of amplification of a DNA template molecule to produce the double-stranded DNA molecule. Thus, the invention provides a method for producing a linear DNA product, the method comprises: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences.

[0021] Steps b) to d) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0022] The amplification may be an in vitro or in vivo amplification. Preferably, the amplification is an in vitro amplification. For example, the amplification may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the amplification is rolling circle amplification.

[0023] Thus, the invention provides a method for producing a linear deoxyribonucleic acid (DNA) product, the method comprises: (a) rolling circle amplification of a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences.

[0024] Steps b) to d) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0025] The method may further comprise (after the step of amplification and before the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule) a step of heat-deactivation. Thus, the invention provides a method for producing a linear DNA product, the method comprises: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat deactivation of the reaction of step a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences.

[0026] Steps c) to e) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0027] Preferably, amplification is rolling-circle amplification.

[0028] The step of heat-deactivation may be performed under conditions sufficient to inactive the reagents used during the amplification reaction. The step of heat-deactivation may be performed at a temperature of at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. The step of heat-deactivation may be performed for at least 1 min, at least 3 mins, at least 5 mins, at least 10 mins, at least 15 mins, or at least 20 mins.

[0029] The inventors of the present application have surprisingly discovered that large concatemeric products of the rolling circle amplification reaction can be used to produce the DNA products described herein. This is surprising as the product of the rolling circle amplification has high viscosity and typically has to undergo purification steps before it can be utilized for downstream applications.

[0030] In the method described herein, after the step of amplification, the steps of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product may be performed without purifying the product of the amplification reaction. That is to say that the steps of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA productmay be performed directly after the step of amplification. The steps of digesting digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product may be performed directly after the step of heat-deactivation.

[0031] In the method of the invention, the steps of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product may be performed in a single contiguous aqueous volume.

[0032] The steps of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule and appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule may be carried out in a single contiguous aqueous volume, followed by the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product.

[0033] The step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be carried out, followed by the steps of appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product in a single contiguous aqueous volume.

[0034] In the method of the invention, each step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product may be performed sequentially in separate reactions.

[0035] The inventors of the present application have discovered a method which requires a very few steps to produce the DNA product described herein. The methods described herein are very time efficient. This is, in part, due to the fact that a step of purification is not required after the amplification reaction. Optionally, the product of the amplification reaction may be heat-deactivated. Surprising, the method described herein where the steps of: (a) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences is performed directly after the step of amplification (i.e. without the step of purification) produces higher yields of the DNA product described herein when compared to a method in which the two steps are separated by purification of the amplification product.

[0036] The method may further comprise, after the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, a step of purification of the linear DNA product.

[0037] The method may further comprise, after the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, a step of nuclease digestion. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III and / or T5 exonuclease digestion. The step of nuclease digestion may take place before or after the step of purification. The step of nuclease digestion may allow for removal of any double-stranded DNA molecules and / or adaptor molecules which have not been used to produce linear DNA products.

[0038] Thus, the method for producing a linear DNA product may comprise the steps: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences. (d) incubating the linear DNA product with a nuclease (e.g. an exonuclease).

[0039] The method for producing a linear DNA product may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences; and (e) incubating the linear DNA product with a nuclease (e.g. an exonuclease).

[0040] The step of incubating the linear DNA product with a nuclease (e.g., an exonuclease) may be carried out in the same reaction volume as the step of incubating with a protelomerase, i.e., without purifying the linear DNA product prior to the step of incubating the linear DNA product with a nuclease, e.g., an exonuclease.

[0041] The method for producing a linear DNA product may comprise the steps: a. amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; b. digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; c. appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; d. incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences; e. purification of the linear DNA product; and f. incubating the product of step (d) with a nuclease (e.g. an exonuclease).

[0042] The method for producing a linear DNA product may comprise the steps: a. amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; b. digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; c. appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; d. incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences; e. incubating the single contiguous aqueous volume with a nuclease (e.g. an exonuclease); and f. purification of the linear DNA product.

[0043] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF51, BstMAI, BstV11, BstV2I, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCl, Eam11041, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269I, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0044] Type IIS restriction endonucleases cleave the double-stranded DNA molecule outside of the recognition sequence (i.e. an endonuclease target sequence), which means that the recognition sequence (i.e. endonuclease target sequence) is not included in the linear DNA product.

[0045] The present inventors have surprisingly discovered methods for production of a linear DNA product of enhanced resistance to nuclease digestion. Specifically, the linear DNA product produced by the methods described herein has enhanced resistance to exonuclease digestion (e.g. exonuclease III digestion). The enhanced resistance to exonuclease digestion extends the life of the linear DNA product in a cell (i.e. the linear DNA product has enhanced resistance to intracellular exonucleases) and in a cell-free system (i.e. the linear DNA product has enhanced resistance to extracellular exonucleases). The present inventors have developed a method which relies on the addition of adaptor molecules (i.e. a first adaptor molecule and a second adaptor molecule) to both ends of a double-stranded DNA molecule. At least one adaptor molecule comprises a truncated protelomerase target sequence, that when appended to a double-stranded DNA molecule comprising a truncated protelomerase target sequence, forms a functional protelomerase target sequence, such that protelomerase is able to cleave and religate the double stranded DNA to form covalently closed ends of the linear double stranded DNA molecule. One of the adaptor molecules may comprise a hairpin or a loop to form a closed linear DNA product with enhanced resistance to nuclease (e.g. exonuclease) digestion. In addition, the present inventors have discovered a method for efficient introduction of protected nucleotides in the form of a first adaptor molecule or a second adaptor molecule at both ends of a linear double-stranded region of the linear DNA product. The methods described herein may use a hairpin or a loop adaptor on one end of the DNA product and an adaptor comprising a truncated protelomerase target sequence that is subsequently closed by a protelomerase at the other end of the linear DNA product. The methods described herein may use a linear adaptor comprising protected nucleotides on one end of the DNA product and an adaptor comprising a truncated protelomerase target sequence at the other end of the DNA product.. That is to say that any type of adaptors described herein may be used at the other end of the linear DNA product to the adaptor comprising the truncated protelomerase sequence that is subsequently closed by a protelomerase, as long as the final DNA product is protected from nuclease (e.g. exonuclease) digestion. The methods of the invention provide protection from digestion by exonucleases that cleave the 3'-end nucleotides (e.g. exonuclease III) and exonucleases that cleave the 5'-end nucleotides (e.g. exonuclease VIII). Thus, the linear DNA product produced by the methods of the invention has prolonged in vivo expression when compared to a linear DNA product that does not comprise an adaptor molecule described herein.

[0046] As used herein the term "protected nucleotide" or "nuclease-resistant nucleotide" is intended to encompass any type of molecule that provides or enhances resistance to nuclease digestion (especially exonuclease digestion). Although the adaptor molecules are described herein as comprising phosphorothioated nucleotides, the skilled person would appreciate that the adaptor molecules may instead comprise any molecules that provide resistance to nuclease digestion (e.g. exonuclease III digestion). For example, the adaptor molecules may comprise nuclease resistant nucleotides i.e. modified nucleotides that provide or increase resistance to nucleases (e.g. exonucleases). The adaptor molecules may comprise a peptide, polypeptide or protein that provides or increases resistance to nucleases (e.g. exonucleases) digestion. The adaptor molecules may comprise 2'-O-methyl nucleotides or 2'-O-methoxyethyl (MOE) nucleotides.

[0047] As used herein, the term "phosphorothioated nucleotide" refers to a nucleotide that has an altered phosphate backbone, wherein, the sugar moieties are linked by a phosphorothioate bond. In the phosphate backbone of an oligonucleotide sequence, the phosphorothioate bond contains a sulphur atom as a substitute for a non-bridging oxygen atom. This modification renders the internucleotide linkage resistant to nuclease degradation.

[0048] As used herein, "truncated protelomerase target sequence" refers to a protelomerase target sequence that has had sufficient base pairs removed such that protelomerase no longer processes the DNA to produce closed ends, i.e., the protelomerase is unable to cleave and religate the double-stranded DNA to form covalently closed linear DNA. In other words, the truncated protelomerase sequence as used herein refers to a non-functional protelomerase sequence. With reference to the TelN sequence shown in FIG. 1, as an example, the base pairs may be absent from the telR sequence of a protelomerase target sequence or from the telL sequence of a protelomerase target sequence. The base pairs may be absent from the 5' end of the telR sequence, or from the 3' end of the telR sequence. The base pairs may be absent from the 5' end of the telL sequence, or from the 3' end of the telL sequence. Base pairs may also be removed from the 5' or 3' of the telO sequence of a protelomerase target sequence. By truncating the sequence by removing some of the base pairs from the telR or telL sequence, the protelomerase target sequence is rendered non-functional.

[0049] When the double stranded DNA molecule comprises two truncated protelomerase sequences, one at a first end and one at a second end (again with reference to the TelN sequence shown in FIG. 1, as an example), the truncated protelomerase sequences at the first end and the second end may have base pairs deleted from the telR sequence; the truncated protelomerase sequences at the first end and the second end may have base pairs deleted from the telL sequence; the truncated protelomerase sequence at the first end may have base pairs deleted form the telR sequence and the truncated protelomerase sequence at the second may have base pairs deleted from the telL sequence; the truncated protelomerase sequence at the first end may have base pairs deleted form the telL sequence and the truncated protelomerase sequence at the second may have base pairs deleted from the telR sequence. The number of base pairs deleted from each truncated sequence may be the same or may be different. For example, if different adaptor molecules are to be appended to the first end and the second end, different number of case pairs may be deleted in order to truncate the protelomerase sequence, provided that the number of base pairs deleted is sufficient to render the truncated protelomerase target sequence non-functional. One example method to test for whether a truncated sequence is non-functional is described in Example 2.

[0050] The linear DNA product (e.g. the closed linear DNA product) produced by the methods of the present invention has additional advantageous properties, such as a substantial lack of a bacterial backbone and / or antibiotic resistant genes. Lack of these features is particularly beneficial in the production of a cell delivery system, such as a viral vector or a nanoparticle, for example, for cell therapy. Lack of these features makes the linear DNA product produced by the methods of the present invention particularly suitable for use in a pharmaceutical composition.

[0051] Unexpectedly, the present inventors have discovered a method for production of a linear DNA product of enhanced resistance to nuclease digestion which allows for efficient production of large quantities of the linear DNA product with enhanced resistance to exonuclease digestion. The large-scale manufacture of the product may be in a cell-free system, which results in the production of a pure sample comprising the linear DNA product substantively free of bacterial contaminants (e.g. remaining after cell lysis).1. Methods for producing closed linear DNA product

[0052] The methods described herein may be used to produce a closed linear DNA product e.g. a covalently closed linear DNA product.

[0053] The invention provides a method for producing a closed linear DNA product, the method comprises the steps of: a. digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end, wherein the truncated protelomerase sequences are non-functional; b. appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and c. incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

[0054] The step of appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule is preferably performed in the presence of a ligase. Thus, the invention provides a method for producing a closed linear DNA product, wherein the method comprises: a. digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end, wherein the truncated protelomerase sequences are non-functional; b. ligating a first adaptor molecule to the first end of the digested double-stranded DNA molecule and ligating a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and c. incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

[0055] The step of appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule may be performed in the absence of a ligase, and may rely on hybridisation. Thus, the invention provides a method for producing a closed linear DNA product, wherein the method comprises: a. digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end, wherein the truncated protelomerase sequences are non-functional; b. hybridizing a first adaptor molecule to the first end of the digested double-stranded DNA molecule and hybridizing a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and c. incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

[0056] A closed linear DNA product has particular utility as a therapeutic agent (i.e. DNA therapeutic) which can be used to express a gene product in vivo. This is because its closed structure (e.g. covalently closed structure) prevents attack by enzymes such as exonucleases, leading to enhanced stability and longevity of gene expression as compared to "open" DNA molecules with exposed DNA ends. Linear double-stranded open-ended cassettes have been demonstrated to be inefficient with respect to gene expression when introduced into host tissue. This has been attributed to cassette instability due to the action of exonucleases in the extracellular space.

[0057] Sequestering DNA ends inside closed structures also has other advantages. The DNA ends are prevented from integrating with genomic DNA and so closed linear DNA products offer improved safety. In addition, the closed linear structure reduces concatamerisation of DNA products inside host cells and thus expression levels of the gene product can be regulated in a more sensitive manner.

[0058] The method of the invention may be used for production of DNA for in vitro expression in a host cell, for example, in DNA vaccines. DNA vaccines typically encode a modified form of an infectious organism's DNA. DNA vaccines are administered to a subject where they then express the selected protein of the infectious organism, initiating an immune response against that protein which is typically protective. DNA vaccines may also encode a tumour antigen in a cancer immunotherapy approach.

[0059] The method may produce other types of therapeutic DNA molecules e.g. those used in gene therapy. For example, such DNA molecules can be used to express a functional gene where a subject has a genetic disorder caused by a dysfunctional version of that gene. Examples of such diseases include sickle cell anaemia, cystic fibrosis, Huntington disease, Duchenne's Muscular Dystrophy, Haemophilia A, α1-antitrypsin deficiency, primary ciliary dyskinesia, or respiratory distress syndrome of prematurity. Other diseases where gene therapy may be useful include metabolic diseases, respiratory diseases, inflammatory diseases, autoimmune, chronic and infectious diseases, including such disorders as AIDS, cancer, neurological diseases, cardiovascular disease, hypercholesterolemia, various blood disorders including various anaemias, thalassemia and haemophilia, and emphysema. For the treatment of solid tumours, genes encoding toxic peptides (i.e., chemotherapeutic agents such as ricin, diptheria toxin and cobra venom factor), tumour suppressor genes such as p53, genes coding for mRNA sequences which are antisense to transforming oncogenes, antineoplastic peptides such as tumor necrosis factor (TNF) and other cytokines, or transdominant negative mutants of transforming oncogenes, may be expressed.

[0060] The double-stranded DNA molecule comprises a truncated protelomerase sequence; the truncated protelomerase target sequence may consist of SEQ ID NO:s 4, 5, 6 or 20. The truncated protelomerase sequence may consist of SEQ ID NO: 4. The truncated protelomerase sequence may consist of SEQ ID NO: 5. The truncated protelomerase sequence may consist of SEQ ID NO: 6. The truncated protelomerase sequence may consist of SEQ ID NO: 20.

[0061] The first adaptor molecule and / or the second adaptor molecule may comprise a sequence selected from SEQ ID NO:8, 9, 10, 11, 12 or 13. The first adaptor and / or the second adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 8 and 9, SEQ ID NO:s 10 and 11 or SEQ ID NO:s 12 or 13.

[0062] The truncated protelomerase sequence may consist of SEQ ID NO:23, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. The truncated protelomerase sequence may consist of SEQ ID NO: 23. The truncated protelomerase sequence may consist of SEQ ID NO: 30. The truncated protelomerase sequence may consist of SEQ ID NO: 31. The truncated protelomerase sequence may consist of SEQ ID NO: 32.

[0063] The first adaptor molecule and / or the second adaptor molecule may comprise a sequence selected from SEQ ID NO:24, 25, 26, 27, 28 or 29. The first adaptor and / or the second adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 24 and 25, SEQ ID NO:s 26 and 27 or SEQ ID NO:s 28 or 29.

[0064] The closing of the linear double-stranded region of the precursor double-stranded DNA molecule by the first adaptor molecule and / or the second adaptor molecule may be performed by hybridization or ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule may be hybridized to the first end of the linear double-stranded region. The second adaptor molecule may be hybridized to the second end of the linear double-stranded region. The first adaptor molecule may be ligated to the first end of the linear double-stranded region. The second adaptor molecule may be ligated to the second end of the linear double-stranded region. The closing of the linear double-stranded region by the first adaptor molecule and the second adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule may be hybridized and ligated to the first end of the linear double-stranded region. The second adaptor molecule may be hybridized and ligated to the second end of the linear double-stranded region.

[0065] When the method of the invention is performed in a single contiguous aqueous volume, it may be performed under conditions that promote appending (or linking) of the first and second adaptor molecules to the linear double-stranded region to produce the closed linear DNA product. The appending may be performed by creating a covalent link between the first and / or second adaptor molecule and the first and / or second end of the linear double-stranded region.

[0066] The single contiguous aqueous volume may be incubated under conditions that promote digestion of the double-stranded DNA molecule to produce the linear region of the double-stranded DNA molecule. The digestion of the double-stranded DNA molecule to produce the digested double-stranded DNA molecule may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0067] If the step of digesting the double-stranded DNA molecule with an endonuclease that that cleaves an endonuclease target sequence to generate a digested double stranded DNA molecule is carried out as a separate reaction, it may be performed under conditions that promote digestion of the double-stranded DNA molecule to produce the linear region of the double-stranded DNA molecule. The digestion of the double-stranded DNA molecule to produce the digested double-stranded DNA molecule may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0068] When the method if performed as a single contiguous aqueous volume, the single contiguous aqueous volume may be incubated under conditions that promote ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules. The ligation may be 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the digested double-stranded DNA molecules may be incorporated into closed linear DNA products. Preferably, the ligation is at least 15% efficient.

[0069] If the step of appending or ligating a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending or ligating a second adaptor molecule to the second end of the digested double stranded DNA molecule to generate a precursor double-stranded DNA molecule is carried out as a separate reaction, it may be performed under conditions that promote ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules. The ligation may be 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the digested double-stranded DNA molecules may be incorporated into closed linear DNA products. Preferably, the ligation is at least 15% efficient.

[0070] Ligation efficiency may be established based on DNA quantification values before and after the digestion / ligation reaction. Thus, ligation efficiency may be established based on the equation: (starting amplified DNA amount) / (final linear DNA amount) x 100%.

[0071] Ligation efficiency may also be established based on DNA quantification values before and after the digestion / ligation reaction and the subsequent exonuclease treatment to remove remaining open DNA constructs and adaptor molecules excess.

[0072] For example, the double-stranded DNA molecule generated by the rolling circle amplification is first quantified so that the amount of the double-stranded DNA molecule used as the starting material during the digestion / ligation / protelomerase reaction is known. After all the enzymatic reactions, the linear DNA product is quantified to calculate the ligation efficiency as per the equation above.

[0073] DNA quantification methods are known to a person skilled in the art. For example, DNA quantifications may be carried out using the Qubit dsDNA BR assay from ThermoFisher (https: / / www.thermofisher.com / order / catalog / product / Q32850# / Q32850).

[0074] The step of ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules may be performed at a second temperature of 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C.

[0075] The single contiguous aqueous volume may be incubated under conditions that promote the action of protelomerase to cleave and covalently close the protelomerase target sequence to form the closed linear DNA product. The incubation of the precursor double-stranded DNA molecule with protelomerase may be carried out at a third temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 26°C-34°C, 27°C-33°C 25°C-35°C, 28°C-32°C, 29°C-31°C or at about 30°C.

[0076] If the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product is performed as a separate reaction, it may be performed at a third temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 26°C-34°C, 27°C-33°C 25°C-35°C, 28°C-32°C, 29°C-31°C or at about 30°C.

[0077] The single contiguous aqueous volume may be performed in a step comprising incubating at a first temperature and then incubating at a second temperature and / or then incubating at a third temperature. The first temperature may be 1°C-100°C, 1°C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C-45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or about 37°C. The second temperature may be 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C. The third temperature may be 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 26°C-34°C, 27°C-33°C 25°C-35°C, 28°C-32°C, 29°C-31°C or at about 30°C.

[0078] Preferably, the first temperature is 35°C-39°C and the second temperature is 14°C-18°C and the third temperature is 28°C-32°C. Using these conditions, the endonuclease may be a Type IIS restriction endonuclease (e.g. Bsal) and the ligase may be T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase and the protelomerase may be TelN protelomerase from E. coli phage N15 TelN protelomerase along with the sequence defined herein. Alternatively, the protelomerase may be Klebsiella phage Phi K02 protelomerase, Yersinia phage PY54 protelomerase, Vibrio phage VP882 protelomerase, Borrelia burgdorferi protelomerase.

[0079] If a protelomerase other than TelN is used, the truncated protelomerase target sequence may be a truncated variant of a protelomerase target sequence specific for the protelomerase enzyme that is used.

[0080] The single contiguous aqueous volume may be incubated isothermally. The step of incubating the single contiguous aqueous volume may comprise incubating at a constant temperature. The constant temperature promotes simultaneous digestion of the double-stranded DNA molecule to produce the digested double-stranded DNA molecule and ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules and closing of the precursor double-stranded DNA molecule with protelomerase. For example, the constant temperature may be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0081] Preferably, the constant temperature is 30°C. The constant temperature is intended to mean that the temperature does not significantly change during the reaction. The constant temperature is intended to mean that the temperature variation during the step of incubating the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment the temperature during the step of incubating the single contiguous aqueous does not deviate by more than 5°C, preferably by not more than 3°C, even more preferably not more than 1°C. Thus, the constant temperature may be a temperature in a range of 20°C-30°C, 22°C-32°C, 24°C-34°C, 26°C-36°C, 28°C-38°C, 30°C-40°C, 22°C-28°C, 32°C-38°C, 25°C-35°C, 26°C-34°C, 27°C- 33°C, 27.5°C-32.5°C, 28°C-32°C, 28.5°C-31.5°C, 29°C-31°C, or 29.5°C-30.5°C. Preferably, the constant temperature is a temperature in a range of 27.5°C-32.5°C. Alternatively, the constant temperature may be a temperature in a range of 32°C-42°C, 33°C-41°C, 34°C-40°C, 35°C-39°C, 36°C-38°C. Preferably, the constant temperature is a temperature in a range of 34.5°C-39.5°C.

[0082] The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature 2-100, 5-80, 10-70, 20-60, or 30-60 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature 2-20, 5-29, 61-100, or 65-80 times.

[0083] The method may further comprise, before step (a) (i.e. the step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Thus, the invention provides a method for producing a closed linear DNA product, the method comprises: a. amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; b. digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end; c. appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and d. incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

[0084] Steps b) to d) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0085] The step of amplifying may be performed by in vitro or in vivo amplification. Preferably, the step of amplifying is performed by in vitro amplification. For example, the step of amplifying may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the step of amplifying is performed by rolling circle amplification. Thus, the invention provides a method for producing a closed linear DNA product, the method comprises: a. amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule by rolling circle amplification, wherein the truncated protelomerase sequence is non-functional; b. digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end; c. appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and d. incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

[0086] Steps b) to d) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0087] Rolling circle amplification may be performed without any primers, or in the presence of a primer or multiple primers. For example, the primer may be a synthetic primer. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be TthPrimPol. Preferably, if the rolling circle amplification is performed without any primers, it is performed in the presence of a primase, such as TthPrimPol. Similarly, if a primer is used during amplification reaction, a primase is not used. The double-stranded DNA product may be generated by the rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0088] In the methods described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Thus, the DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, the at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences would be known to the skilled person. The cleavable target sequence may be a Type IIS restriction endonuclease target sequence. For example, the restriction endonuclease target sequence may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF51, BstMAI, BstV11, BstV21, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104I, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva12691, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequence. The at least one cleavable sequence (e.g. endonuclease target sequence) may be a native cleavable sequence (i.e. a cleavable sequence present in the template molecule). Alternatively, the at least one cleavable sequence (e.g. endonuclease target sequence) may be introduced to the DNA template molecule prior to the production of the closed linear DNA product.

[0089] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF51, BstMAI, BstV11, BstV2I, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam11041, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269I, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0090] The ligase may be a DNA ligase, such as a T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0091] The protelomerase may be TelN protelomerase from E. coli phage N15 TelN protelomerase along with the sequence defined herein. Alternatively, the protelomerase may be Klebsiella phage Phi K02 protelomerase, Yersinia phage PY54 protelomerase, Vibrio phage VP882 protelomerase, Borrelia burgdorferi protelomerase.

[0092] If a protelomerase other than TelN is used, the truncated protelomerase target sequence may be a truncated variant of a protelomerase target sequence specific for the protelomerase enzyme that is used.

[0093] The DNA template molecule used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a natural circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from recombinase reaction, preferably Cre recombinase reaction, or (ii) a circular DNA molecule obtained from ligase reaction, preferably using the golden gate assembly. The DNA template molecule may be an enzymatically produced covalently-closed linear DNA molecule. For example, the DNA template molecule may be (i) a DNA molecule processed with TelN protelomerase; or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA template molecule may comprise an element that is double-stranded and an element that is single-stranded. For example, the template DNA molecule may comprise a double-stranded DNA and a single-stranded hairpin loop.

[0094] The DNA template molecule may be linear. If the DNA template molecule is linear, prior to amplification (e.g. rolling circle amplification), a DNA template molecule may be circularized to produce a DNA template molecule suitable for use in the methods described herein.

[0095] The template DNA molecule may comprise a cassette. The cassette may be a mammalian expression cassette. The cassette may further comprise a promoter. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence. The cassette may further comprise a LoxP sequence, preferably two LoxP sequences. If the two LoxP sequences are oriented in the same direction, the DNA sequence between the two LoxP sequences is excised as a circular loop of DNA. If the two LoxP sequences are oriented in the opposite direction, the DNA sequence between the two LoxP sequences is inverted. Thus, preferably, the two LoxP sequences are in the same orientation (i.e. the same direction) in the template DNA molecule.

[0096] The DNA template molecule may comprise a homopolymeric sequence at a 5'-end or a 3'-end or both a 5'-end and a 3'-end. The homopolymeric sequence may be added to the DNA template molecule before circularization. The homopolymeric sequence may be a polyA, a polyC, a polyG, or a polyT sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the linear DNA product, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0097] The method may further comprise (after the step of amplification and before the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule) a step of heat-deactivation. Thus, the invention provides a method for producing a closed linear DNA product, the method comprises: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat-deactivation of the reaction of step (a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and . (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target.

[0098] Steps c) to e) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0099] Preferably, amplification is rolling-circle amplification.

[0100] The step of heat-deactivation may be performed under conditions sufficient to inactive the reagents used during the amplification reaction. The step of heat-deactivation may be performed at a temperature of at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. The step of heat-deactivation may be performed for at least 1 min, at least 3 mins, at least 5 mins, at least 10 mins, at least 15 mins, or at least 20 mins.

[0101] In the method described herein, after the step of amplification, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase and first and second adaptor molecules to form a single contiguous aqueous volume may be performed without purifying the product of the amplification reaction. That is to say that the step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, may be performed directly after the step of amplification. The step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA moleculemay be performed directly after the step of heat-deactivation.

[0102] The method may further comprise, after the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, a step of purification of the closed linear DNA product.

[0103] The method may further comprise, after the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, a step of nuclease digestion. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The step of nuclease digestion may take place before or after the step of purification. This step allows for removal of any double-stranded DNA molecules and / or adaptor molecules which have not been used in the course of performing the method. Thus, the method may comprise the steps: a. amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; b. digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end; c. appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; d. incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences; and e. incubating the single contiguous aqueous volume with a nuclease (e.g. exonuclease).

[0104] Steps c) to e) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0105] In the method described herein, after the step of amplification, the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed without purifying the product of the amplification reaction. That is to say that the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed directly after the step of amplification. The step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed directly after the step of heat-deactivation.

[0106] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end;; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences; (e) purifying the closed linear DNA product; and (f) incubating the purified product of step (e) with a nuclease (e.g. exonuclease).

[0107] Steps b) to d) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0108] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat deactivation of the reaction of step (a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end;; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences; (f) purifying the closed linear DNA product; and (g) incubating the purified product of step (f) with a nuclease (e.g. exonuclease).

[0109] Steps c) to e) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0110] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end;; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences; (e) incubating the reaction of step (d) with a nuclease (e.g. exonuclease); and (f) purifying the closed linear DNA product.

[0111] Steps b) to d) may be carried out in a single contiguous aqueous volume, or they may be performed sequentially in separate reactions.

[0112] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat deactivation of the reaction of step (a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences; (f) incubating the reaction of step (e) with a nuclease (e.g. exonuclease); and (g) purifying the closed linear DNA product.

[0113] Steps c) to f) may be carried out in a single contiguous aqueous volume,or they may be performed sequentially in separate reactions. The single contiguous aqueous volume (or the purified product of step (g)) may be incubated with a nuclease and may be performed at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30-45°C or 35-40°C. The step of incubating the single contiguous aqueous volume (or the purified product of step (g)) with a nuclease may be performed for at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 min. The step of incubating the single contiguous aqueous volume (or the purified product of step (g)) may be performed at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product of step (g)) may be performed at 15-40°C for 10-60 minutes followed by a temperature of 60-90°C for 10-30 min. The higher temperature typically inactivates the nuclease (e.g. exonuclease). Thus, the method further provides a step of inactivating the nuclease (e.g. exonuclease). The step of incubating the single contiguous aqueous volume (or the purified product of step (g)) may be performed at 37°C for 30 min and 80°C for 20 min. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed at a temperature of 70-80°C. The step of inactivating the nuclease (e.g. exonuclease) may be performed for at least 1, at least 5, at least 10, at least 20 or at least 30 minutes. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed for at least 5 minutes.

[0114] The methods of the invention may comprise a (further) step of heat-deactivation after the step of incubating with the protelomerase and prior to treatment with an exonuclease. The heat-deactivation may be carried out after the steps of digesting a double-stranded DNA molecule to generate a digested double-stranded molecule; appending a first adaptor molecule to a first end of the digested double stranded DNA molecule and appending a second adaptor molecule to a second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule; and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed DNA product, whether the steps are performed in a single contiguous aqueous volume or whether the step are carried out sequentially in separate reactions.

[0115] Thus, the method of the invention may comprise: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences; and (d) heat-deactivation of the reaction of steps (a) to (c), or step (c).

[0116] Heat-deactivation may be carried out at a temperature of 50-90°C, preferably from 60-80°C, more preferably from 70-80°C, or 72-77°C or 75°C. The heat deactivation may be performed from 1 to 10 minutes, from 2 to 9 minutes, for 3 to 8 minutes, from 4 to 7 minutes, for 6 minutes or, most preferably for 5 minutes.

[0117] The method may be a cell-free method.

[0118] The closed linear DNA product may be partially double-stranded and / or partially single-stranded. The closed linear DNA product may comprise a portion that is double-stranded and a portion that is single-stranded.

[0119] The closed linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0120] The closed linear DNA product may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long.

[0121] The closed linear DNA product may comprise an inverted terminal repeat sequence.

[0122] The closed linear DNA product may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the closed linear DNA product is at least 50 base pairs long.

[0123] The double-stranded DNA molecule may be circular, or branched.

[0124] The double-stranded DNA molecule may not comprise an adaptor. The double-stranded DNA molecule may not comprise a hairpin, a loop or a stem-loop structure.

[0125] The double-stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0126] The double-stranded DNA molecule may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve an amplification yield of the double-stranded DNA molecule.

[0127] The double-stranded DNA molecule may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0128] The double-stranded DNA molecule may comprise one or more cleavable (e.g. endonuclease) target sequences. The double-stranded DNA molecule may comprise two cleavable (e.g. endonuclease) target sequences. The one or more cleavable (e.g. endonuclease) target sequences may be Type IIS endonuclease target sequences. The one or more cleavable (e.g. endonuclease) target sequences may be Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF5I, BstMAI, BstV1I, BstV21, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam11041, Earl, Ecil, Eco31I, Eco571, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269I, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0129] The double-stranded DNA molecule may be a product of amplification. Preferably, the amplification is rolling circle amplification.

[0130] The linear double-stranded region may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0131] The linear double-stranded region may comprise a sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequence of the double-stranded DNA molecule.

[0132] The first end and the second end of the linear double-stranded region (may be resistant to nuclease digestion. Preferably, the first end and the second end of the linear double-stranded region are resistant to the exonuclease digestion, such as exonuclease III digestion and / or exonuclease I digestion.

[0133] The linear double-stranded region may comprise a 3'-OH group at first and / or second ends. The 3'-OH group may facilitate ligation to the first and / or second adaptor molecule(s) (which may comprise a 5' phosphate). The linear double-stranded region may comprise a 5' phosphate at first and / or second ends. The 5' phosphate may facilitate ligation to the first and / or second adaptor molecule(s) (which may comprise a 3'-OH group).

[0134] The linear double-stranded region (e.g. of the double-stranded DNA molecule) may comprise an overhang. For example, the linear double-stranded region may comprise a 5' overhang or a 3' overhang. The linear double-stranded region may comprise a blunt end or blunt ends. The linear double-stranded region may comprise: a 5' overhang and a blunt end, two 5' overhangs, a 3' overhang and a blunt end, two 3' overhangs, or a 5' overhang and a 3' overhang. The overhang may have at least 3 nucleotides (preferably from 4 to 8 nucleotides). The overhang may be in the sense strand or the antisense strand of the linear double-stranded region.

[0135] The linear region of the double-stranded DNA molecule (e.g. of the double-stranded DNA molecule) may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, double-stranded DNA molecule is at least 50 base pairs long.

[0136] The double-stranded DNA molecule comprises a truncated protelomerase sequence, wherein the truncated protelomerase sequence is non-functional. The truncated protelomerase target sequence may be a truncated TelN protelomerase target sequence. The truncated protelomerase target sequence may consist of SEQ ID NO:s 4, 5, 6 or 20. The truncated protelomerase sequence may consist of SEQ ID NO: 4. The truncated protelomerase sequence may consist of SEQ ID NO: 5. The truncated protelomerase sequence may consist of SEQ ID NO: 6. The truncated protelomerase sequence may consist of SEQ ID NO: 20. The truncated protelomerase sequence may consist of SEQ ID NO:23, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. The truncated protelomerase sequence may consist of SEQ ID NO: 23. The truncated protelomerase sequence may consist of SEQ ID NO: 30. The truncated protelomerase sequence may consist of SEQ ID NO: 31. The truncated protelomerase sequence may consist of SEQ ID NO: 32.

[0137] The first adaptor molecule and / or the second adaptor molecule may be a synthetic adaptor molecule.

[0138] The first adaptor molecule may be a nucleic acid adaptor molecule. The second adaptor molecule may be a nucleic acid adaptor molecule. The second adaptor molecule may comprise a self-complementary element which creates a loop, such as a hairpin loop or a stem loop. The second adaptor molecule may comprise a hairpin or a stem-loop. The second adaptor molecule may comprise a double-stranded portion comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are linked together in a hairpin such that the sense strand is hybridized to the antisense strand. The double-stranded portion of an adaptor may comprise a 3' overhang or a 5' overhang of at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably the 3' overhang or the 5' overhang is 4-8 nucleotides. Each end of the linear double-stranded region (or of the digested double-stranded DNA molecule) may comprise a 3' or a 5' overhang. A portion of the first adaptor molecule (e.g. the overhang) may be complementary to the first end of the linear double-stranded region. A portion of the second adaptor molecule may be complementary to the second end of the linear double-stranded region.

[0139] The first adaptor molecule and / or the second adaptor molecule may comprise a truncated protelomerase target sequence. The first and / or second adaptor molecule may or may not comprise all base pairs that are deleted from the truncated protelomerase sequence present in the double-stranded DNA molecule, e.g., if the truncated protelomerase target sequence in the double stranded DNA molecule is missing eight base pairs, those eight base pairs may be present in the adaptor molecule sequence, or fewer than those eight base pairs may be present in the adaptor molecule. The portion of a protelomerase target sequence present in the first / and or second adaptor molecule are sufficient such that, wherein the first and / or second adaptor molecule is appended to a first and / or second end of the double-stranded DNA molecule comprising a truncated protelomerase target sequence the protelomerase target sequence is rendered functional and the protelomerase is able to cleave and ligate the sequence to covalently close the ends of the adaptor molecules and therefore the ends of the DNA molecule. The protelomerase target sequence may not be fully complete when the adaptor molecule is appended, provided that some activity is restored. The amount of activity restored may be 20%, 30%, 40%, 50%, 60% 70%, 80%, 90% or 100% of the full protelomerase activity with reference to the full protelomerase target sequence. The truncated protelomerase target sequences of the DNA molecule and / or of the adaptor molecule may comprise one or more point mutations, provided that activity is restored by the protelomerase target sequence formed by the adaptor molecule appending to the DNA molecule. The point mutations may be present in the truncated protelomerase target sequence of the double-stranded DNA molecule and / or in the truncated protelomerase target sequence of the adaptor molecule(s).

[0140] The first adaptor molecule and / or the second adaptor molecule may comprise a truncated TelN protelomerase target sequence. The first and / or second adaptor molecules may comprise a sequence selected from SEQ ID NO:8, 9, 10, 11, 12 or 13. The first adaptor and / or the second adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 8 and 9, SEQ ID NO:s 10 and 11 or SEQ ID NO:s 12 or 13. The first adaptor molecule and / or the second adaptor molecule may comprise a sequence selected from SEQ ID NO:24, 25, 26, 27, 28 or 29. The first adaptor and / or the second adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 24 and 25, SEQ ID NO:s 26 and 27 or SEQ ID NO:s 28 or 29.

[0141] The closed linear DNA product may be a covalently closed linear DNA product. Thus, in embodiments where each of the adaptor molecules comprise a truncated protelomerase target sequence, protelomerase covalently closes the ends of the adaptor molecules appended to the ends of linear double stranded region, which forms a first and / or second functional protelomerase target sequence, to form a covalently closed linear DNA product. In embodiments where one of the adaptor molecules comprises a loop (e.g. a hairpin), the adaptor molecule closes an end of the linear double-stranded region and protelomerase closes the other end of the linear double-stranded region (via the adaptor molecule comprising the truncated protelomerase sequence together with the truncated sequence of the digested double-stranded DNA molecule), forming a covalently closed linear DNA product.

[0142] The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end, wherein the truncated protelomerase sequences are non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the first end of the digested double-stranded DNA molecule and (ii) the second adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the second end of the digested double-stranded DNA molecule, and wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

[0143] The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end, wherein the truncated protelomerase sequences are non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the first end of the digested double-stranded DNA molecule and (ii) the second adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the second end of the digested double-stranded DNA molecule, and wherein the first adaptor molecule is ligated to the first end of the digested double-stranded DNA molecule and the second adaptor molecule is ligated to the second end of the digested double-stranded DNA molecule, and wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

[0144] The first adaptor molecule and / or the second adaptor molecule may not be a plasmid or a vector DNA.

[0145] The first adaptor and / or the second adaptor molecule comprise a truncated protelomerase target sequence. The truncated protelomerase target sequence may comprise a partial telR sequence. The truncated protelomerase target sequence may comprise a partial telR sequence and a partial telO sequence. The first and / or second adaptor molecule may comprise 10, 11, 12, 13, 14, 15, 16 17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 base pairs of a telR sequence. Preferably, the adaptor comprises at least 21 base pairs of a telR sequence. The truncated protelomerase target sequence may comprise a partial telL sequence. The truncated protelomerase target sequence may comprise a partial telL sequence and a partial telO sequence. The first and / or second adaptor molecule may comprise 10, 11, 12, 13, 14, 15, 16 17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 base pairs of a telL sequence. Preferably, the adaptor comprises at least 21 base pairs of a telL sequence. The first and / or second adaptor molecules may comprise the same sequence or different sequences, i.e., the first adaptor molecule may comprise a different sequence in the overhang portion such that it only appends (by hybridization and / or ligation) to a first end of the digested double-stranded DNA molecule, and not to the second end. Different adaptor molecules may hybridize or ligate to the first end and the second end of the digested double-stranded DNA molecule.

[0146] The first adaptor molecule and / or the second adaptor molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The double-stranded portion may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 base pairs.

[0147] The first adaptor molecule and / or the second adaptor molecule may comprise a 5' phosphate. The 5' phosphate may facilitate ligation to the linear double-stranded region (which may comprise a 3'-OH group at first and / or second ends). The first adaptor molecule and / or the second adaptor molecule may comprise a 3'-OH. The 3'-OH may facilitate ligation to the linear double-stranded region (which may comprise a 5' phosphate at first and / or second ends).

[0148] The first and / or second adaptor molecule may comprise the sequence of SEQ ID NO: 14 or a portion thereof. The first and / or second adaptor molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 14. The double-stranded portion of the first and / or second adaptor molecule may comprise the sequence of SEQ ID NO: 15 or a portion thereof. The double-stranded portion of the first and / or second adaptor molecule may comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 or at least 15 contiguous nucleotides of SEQ ID NO: 15.

[0149] The first and second adaptor molecules may comprise an identical nucleic acid sequence. The first and second adaptor molecules may comprise a different nucleic acid sequence.

[0150] The first adaptor molecule may comprise a portion that is complementary to the first end of the linear double-stranded region (of the digested double-stranded DNA molecule). The second adaptor molecule may comprise a portion that is complementary to the second end of the linear double-stranded region (of the digested double-stranded DNA molecule). The first adaptor molecule may comprise a portion that anneals to the first end of the linear double-stranded region (of the digested double-stranded DNA molecule). The second adaptor molecule may comprise a portion that anneals to the second end of the linear double-stranded region (of the digested double-stranded DNA molecule). The first adaptor molecule may comprise a portion that is complementary and anneals to the first end of the linear double-stranded region (of the digested double-stranded DNA molecule). The second adaptor molecule may comprise a portion that is complementary and anneals to the second end of the linear double-stranded region (of the digested double-stranded DNA molecule).

[0151] The portion that is complementary or anneals to the first or second end of the linear double-stranded region (of the digested double-stranded DNA molecule) may be a 5' overhang or a 3' overhang of the first and / or second adaptor molecule. The overhang of the first adaptor molecule may be complementary to the first end of the linear double-stranded region and / or the overhang of the second adaptor molecule may be complementary to the second end of the double-stranded region. The overhang of the first adaptor molecule may anneal to the first end of the linear double-stranded region and / or the overhang of the second adaptor molecule may anneal to the second end of the linear double-stranded region. The overhang of the first adaptor molecule may be complementary to and anneal to the first end of the linear double-stranded region and / or the overhang of the second adaptor molecule may be complementary to and anneal to the second end of the linear double-stranded region.

[0152] The first adaptor molecule and / or the second adaptor molecule may not comprise a Type IIS endonuclease target sequence. The first adaptor molecule and / or the second adaptor molecule may not comprise Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BslFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF5I, BstMAI, BstV11, BstV2I, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam11041, Earl, Ecil, Eco31I, Eco571, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva12691, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0153] The first adaptor molecule and / or the second adaptor molecule may comprise a Type IIS endonuclease target sequence, provided it is different to a type IIS endonuclease target sequence that is present in the double-stranded DNA molecule.

[0154] The first adaptor molecule and / or the second adaptor molecule may comprise one or more locked nucleic acids (LNAs).

[0155] The first adaptor molecule and / or the second adaptor molecule may comprise one or more protected nucleotides (i.e. nuclease-resistant nucleotides), such as phosphorothioated nucleotides. The protected nucleotides may be located in the single-stranded portion (e.g. hairpin portion) or the double-stranded portion. The protected nucleotides may be located in the overhang portion of the adaptor molecules.

[0156] The closed linear DNA product may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the closed linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the closed linear DNA product comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand.

[0157] The internal positions may not be located between the second and penultimate nucleotide of the closed linear DNA product.

[0158] The linear double-stranded region (of the double-stranded DNA molecule) may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the linear double-stranded region (of the double-stranded DNA molecule) may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least or 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the linear double-stranded region (of the double-stranded DNA molecule) comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. The internal positions may not be located between the second and penultimate nucleotide of the linear double-stranded region (of the double-stranded DNA molecule).

[0159] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) suitable for use in the methods described herein may be phosphorothioated nucleotides. For example, phosphorothioated nucleotides may be α-S-dATP (i.e. 2'-deoxyadenosine-5'-(α-thio)-triphosphate), α-S-dCTP (i.e. 2'-deoxycytidine-5'-(α-thio)-triphosphate), α-S-dGTP (i.e. 2'-deoxyguanosine-5'-(α-thio)-triphosphate), α-S-dTTP (i.e. 2'-deoxythymidine-5'-(α-thio)-triphosphate), α-S-dUTP (i.e. 2'-deoxyuridine-5'-(α-thio)-triphosphate), and / or uridine 2', 3'-cyclophosphorothioate.

[0160] The phosphorothioated nucleotides may be Sp-isomers, Rp-isomers or a mixture of both Sp- and Rp-isomers.

[0161] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be 2'-O-methyl nucleotides or 2'-O-methoxyethyl (MOE) nucleotides. For example, the MOE nucleotides may be 2'-O-methoxy-ethyl guanosine, 2'-O-methoxy-ethyl cytidine, 2'-O-methoxy-ethyl adenosine, and / or 2'-O-methoxy-ethyl thymidine.

[0162] The first end of the digested double-stranded DNA molecule may be complementary to a portion of the first adaptor molecule. The second end of the digested double-stranded DNA molecule may be complementary to a portion of the second adaptor molecule. The first end and / or the second end of the digested double-stranded DNA molecule may be generated by endonuclease digestion.

[0163] The first adaptor molecule and / or the second adaptor molecule may comprise a functional portion. The functional portion may be a binding molecule, a targeting sequence, or a probe.

[0164] The functional portion may be a probe. As used herein, the term "probe" refers to a fragment of DNA, RNA or DNA / RNA chimera of variable length (e.g. 3-1000 bases long), which is used to detect the presence of target nucleotide sequences that are complementary to the sequence in the probe. Typically, the probe hybridizes to single-stranded nucleic acid whose base sequence allows probe-target base pairing due to complementarity between the probe and target. Thus, the functional portion may be a DNA sequence, a RNA sequence or a DNA / RNA chimera sequence. As used herein, the term "complementary" refers to the pairing of nucleotide sequences according to Watson / Crick pairing rules. For example, a sequence 5'-GCGGTCCCA-3' has the complementary sequence of 5'-TGGGACCGC-3'. A complement sequence can also be a sequence of RNA complementary to the DNA sequence.

[0165] The functional portion may be a binding molecule. The term "binding molecule" refers to any molecule capable of binding to the linear DNA product described herein and / or that is capable of binding to a further molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal antibody or a polyclonal antibody. The binding molecule may be an antibody fragment.

[0166] The functional portion may facilitate detection of the DNA product by binding to capture molecules (e.g. capture antibodies bound by protein-protein interactions). The functional portion may bind to a cell target, for example, a cell receptor.

[0167] The functional portion may be a label. The 'label' can be any chemical entity which enable the detection of the double-stranded nucleic acid molecule via, physical, chemical and / or biological means. The label may be a chromophore, a fluorophore and / or a radioactive molecule.

[0168] The functional portion may be a targeting sequence. The targeting sequence may be a fragment of DNA or RNA of variable length, which is used to target the DNA product to a specific location in a cell. The targeting sequence may be used to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the closed linear DNA product. For example, the targeting sequence may be a DNA nuclear targeting sequences (i.e. a recognition sequence for endogenous DNA-binding proteins), such as SV40 enhancer sequence (preferably downstream from the cassette).

[0169] To facilitate detection and / or quantification of the DNA product, the functional portion may comprise a fluorophore, a radioactive compound or a barcode.

[0170] A signal corresponding to the presence, absence and / or level of the closed linear DNA product may be measured using a barcode. The barcode may comprise at least one binding moiety linked to a barcoded portion, wherein the barcoded portion comprises at least one nucleotide (i.e. wherein the barcoded portion comprises a nucleotide sequence at least one nucleotide in length), and wherein the binding moiety is capable of binding to the 3' overhang, the 5' overhang or the blunt end of the closed linear DNA product. The binding moiety is capable of binding to 3' and / or 5' end of the closed linear DNA product. The signal may be measured by determining the presence, absence and / or level of the barcoded portion of the barcode (e.g. by sequencing or PCR). The barcoded portion may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. The barcode may comprise at least 2 binding moieties (e.g. a first binding moiety and a second binding moiety). For example, the first binding moiety linked to the first barcoded portion may bind to the 3' end of the closed linear DNA product and the second binding moiety linked to the second barcoded portion may bind to the 5' end of the closed linear DNA product. The 3' and 5' ends may comprise a 3' overhang, a 5' overhang or a blunt end.

[0171] A signal corresponding to the presence, absence and / or level of the closed linear DNA product may be measured using a fluorophore (i.e. a fluorescently-labelled molecule), which is attached or bound to the 3' overhang, the 5' overhang or the blunt end of the closed linear DNA product. The signal may be measured by flow cytometry and / or fluorescence-activated cell sorting.

[0172] The functional portion may also facilitate DNA sequencing. For example, the functional portion may be a sequencing adaptor. The term "sequencing adaptor" is intended to encompass one or more nucleic acid domains that include at least a portion of a nucleic acid sequence (or complement thereof) utilized by a sequencing platform of interest, such as a sequencing platform provided by Illumina ®< (e.g. the HiSeg ™< , MiSeq ™< and / or Genome Analyzer ™< sequencing systems), Oxford Nanopore ™< Technologies (e.g. the MinION sequencing system), Ion Torrent ™< (e.g. the Ion PGM ™< and / or Ion Proton ™< sequencing systems), Pacific Biosciences (e.g. the PACBIO RS II sequencing system); Life Technologies ™< (e.g. a SOLiD sequencing system), Roche (e.g. the 454 GS FLX+ and / or GS Junior sequencing systems), or any other sequencing platform of interest.

[0173] The first adaptor molecule and / or the second adaptor molecule may comprise an inverted terminal repeat sequence. The inverted terminal repeat sequences of the first adaptor molecule and the second adaptor molecule may be symmetrical (i.e. have the same symmetrical three-dimensional organization with respect to each other) or asymmetrical (i.e. have different three-dimensional organization with respect to each other). The inverted terminal repeat sequences of the first adaptor molecule and the second adaptor molecule may be from the same or different serotypes. An inverted terminal repeat sequence may comprise a terminal resolution site and a Rep binding site.

[0174] The first adaptor molecule and / or the second adaptor molecule may comprise an aptamer.

[0175] The first adaptor molecule and / or the second adaptor molecule may confer resistance to the nuclease digestion, such as exonuclease digestion (e.g. exonuclease I and / or exonuclease III digestion).

[0176] The closing of the precursor double-stranded DNA at the first end by protelomerase may generate a first closed end of the closed linear DNA product. The closing of the precursor double-stranded DNA molecule at the second end may generate a second closed end of the closed linear DNA product. The first closed end and the second closed end of the closed linear DNA product may be resistant to nuclease digestion. The nuclease digestion may be exonuclease digestion. Preferably, the nuclease digestion is exonuclease III digestion and / or exonuclease I digestion.2. Methods for producing a closed linear DNA product

[0177] The methods described herein may be used to produce a closed linear DNA product e.g. a covalently closed linear DNA product.

[0178] The invention provides a method for producing a closed linear DNA product, the method comprises the steps of: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences, and the second end of the precursor double-stranded product is closed by the second adaptor molecule.

[0179] The step of appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule is preferably performed in the presence of a ligase. Thus, the invention provides a method for producing a closed linear DNA product, wherein the method comprises: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; (b) ligating a first adaptor molecule to the first end of the digested double-stranded DNA molecule and ligating a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor.

[0180] The step of appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule may be performed in the absence of a ligase, and may rely on hybridisation. Thus, the invention provides a method for producing a closed linear DNA product, wherein the method comprises: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; (b) hybridizing a first adaptor molecule to the first end of the digested double-stranded DNA molecule and hybridizing a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the second end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor.

[0181] A closed linear DNA product has particular utility as a therapeutic agent (i.e. DNA therapeutic) which can be used to express a gene product in vivo. This is because its closed structure (e.g. covalently closed structure) prevents attack by enzymes such as exonucleases, leading to enhanced stability and longevity of gene expression as compared to "open" DNA molecules with exposed DNA ends. Linear double-stranded open-ended cassettes have been demonstrated to be inefficient with respect to gene expression when introduced into host tissue. This has been attributed to cassette instability due to the action of exonucleases in the extracellular space.

[0182] Sequestering DNA ends inside closed structures also has other advantages. The DNA ends are prevented from integrating with genomic DNA and so closed linear DNA products offer improved safety. In addition, the closed linear structure reduces concatamerisation of DNA products inside host cells and thus expression levels of the gene product can be regulated in a more sensitive manner.

[0183] The method of the invention may be used for production of DNA for in vitro expression in a host cell, for example, in DNA vaccines. DNA vaccines typically encode a modified form of an infectious organism's DNA. DNA vaccines are administered to a subject where they then express the selected protein of the infectious organism, initiating an immune response against that protein which is typically protective. DNA vaccines may also encode a tumour antigen in a cancer immunotherapy approach.

[0184] The method may produce other types of therapeutic DNA molecules e.g. those used in gene therapy. For example, such DNA molecules can be used to express a functional gene where a subject has a genetic disorder caused by a dysfunctional version of that gene. Examples of such diseases include sickle cell anaemia, cystic fibrosis, Huntington disease, Duchenne's Muscular Dystrophy, Haemophilia A, α1-antitrypsin deficiency, primary ciliary dyskinesia, or respiratory distress syndrome of prematurity. Other diseases where gene therapy may be useful include metabolic diseases, respiratory diseases, inflammatory diseases, autoimmune, chronic and infectious diseases, including such disorders as AIDS, cancer, neurological diseases, cardiovascular disease, hypercholesterolemia, various blood disorders including various anaemias, thalassemia and haemophilia, and emphysema. For the treatment of solid tumours, genes encoding toxic peptides (i.e., chemotherapeutic agents such as ricin, diptheria toxin and cobra venom factor), tumour suppressor genes such as p53, genes coding for mRNA sequences which are antisense to transforming oncogenes, antineoplastic peptides such as tumor necrosis factor (TNF) and other cytokines, or transdominant negative mutants of transforming oncogenes, may be expressed.

[0185] The closing of the linear double-stranded region of the precursor double-stranded DNA molecule by the first adaptor molecule and / or the second adaptor molecule may be performed by hybridization or ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule may be hybridized to the first end of the linear double-stranded region. The second adaptor molecule may be hybridized to the second end of the linear double-stranded region. The first adaptor molecule may be ligated to the first end of the linear double-stranded region. The second adaptor molecule may be ligated to the second end of the linear double-stranded region. The closing of the linear double-stranded region by the first adaptor molecule and the second adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule may be hybridized and ligated to the first end of the linear double-stranded region. The second adaptor molecule may be hybridized and ligated to the second end of the linear double-stranded region.

[0186] The steps of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule; appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule; and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, may be performed in a single contiguous aqueous volume. When the method of the invention is performed in a single contiguous aqueous volume, it may be performed under conditions that promote appending (or linking) of the first and second adaptor molecules to the linear double-stranded region to produce the closed linear DNA product. The appending may be performed by creating a covalent link between the first and / or second adaptor molecule and the first and / or second end of the linear double-stranded region.

[0187] When the method of the invention is performed in a single contiguous aqueous volume, it may be performed under conditions that promote appending (or linking) of the first and second adaptor molecules to the linear double-stranded region to produce the closed linear DNA product. The appending may be performed by creating a covalent link between the first and / or second adaptor molecule and the first and / or second end of the linear double-stranded region.

[0188] The single contiguous aqueous volume may be incubated under conditions that promote digestion of the double-stranded DNA molecule to produce the linear region of the double-stranded DNA molecule. The digestion of the double-stranded DNA molecule to produce the digested double-stranded DNA molecule may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0189] If the step of digesting the double-stranded DNA molecule with an endonuclease that that cleaves an endonuclease target sequence to generate a digested double stranded DNA molecule is carried out as a separate reaction, it may be performed under conditions that promote digestion of the double-stranded DNA molecule to produce the linear region of the double-stranded DNA molecule. The digestion of the double-stranded DNA molecule to produce the digested double-stranded DNA molecule may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0190] When the method if performed as a single contiguous aqueous volume, the single contiguous aqueous volume may be incubated under conditions that promote ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules. The ligation may be 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the digested double-stranded DNA molecules may be incorporated into closed linear DNA products. Preferably, the ligation is at least 15% efficient.

[0191] If the step of appending or ligating a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending or ligating a second adaptor molecule to the second end of the digested double stranded DNA molecule to generate a precursor double-stranded DNA molecule is carried out as a separate reaction, it may be performed under conditions that promote ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules. The ligation may be 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the digested double-stranded DNA molecules may be incorporated into closed linear DNA products. Preferably, the ligation is at least 15% efficient.

[0192] Ligation efficiency may be established based on DNA quantification values before and after the digestion / ligation reaction. Thus, ligation efficiency may be established based on the equation: (starting amplified DNA amount) / (final linear DNA amount) x 100%.

[0193] Ligation efficiency may also be established based on DNA quantification values before and after the digestion / ligation reaction and the subsequent exonuclease treatment to remove remaining open DNA constructs and adaptor molecules excess.

[0194] For example, the double-stranded DNA molecule generated by the rolling circle amplification is first quantified so that the amount of the double-stranded DNA molecule used as the starting material during the digestion / ligation / protelomerase reaction is known. After all the enzymatic reactions, the linear DNA product is quantified to calculate the ligation efficiency as per the equation above.

[0195] DNA quantification methods are known to a person skilled in the art. For example, DNA quantifications may be carried out using the Qubit dsDNA BR assay from ThermoFisher (https: / / www.thermofisher.com / order / catalog / product / Q32850# / Q32850).

[0196] The step of ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules may be performed at a second temperature of 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C.

[0197] The single contiguous aqueous volume may be incubated under conditions that promote the action of protelomerase to cleave and covalently close the protelomerase target sequence to form the closed linear DNA product. The incubation of the precursor double-stranded DNA molecule with protelomerase may be carried out at a third temperature of 1°C-100°C, 1°C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 26°C-34°C, 27°C-33°C 25°C-35°C, 28°C-32°C, 29°C-31°C or at about 30°C.

[0198] If the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product is performed as a separate reaction, it may be performed at a third temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 26°C-34°C, 27°C-33°C 25°C-35°C, 28°C-32°C, 29°C-31°C or at about 30°C.

[0199] The single contiguous aqueous volume may be performed in a step comprising incubating at a first temperature and then incubating at a second temperature and / or then incubating at a third temperature. The first temperature may be 1°C-100°C, 1°C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C-45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or about 37°C. The second temperature may be 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C. The third temperature may be 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 26°C-34°C, 27°C-33°C 25°C-35°C, 28°C-32°C, 29°C-31°C or at about 30°C.

[0200] Preferably, the first temperature is 35°C-39°C and the second temperature is 14°C-18°C and the third temperature is 28°C-32°C. Using these conditions the endonuclease may be a Type IIS restriction endonuclease (e.g. Bsal) and the ligase may be T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase and the protelomerase may be TelN protelomerase from E. coli phage N15 TelN protelomerase along with the sequence defined herein. Alternatively, the protelomerase may be Klebsiella phage Phi K02 protelomerase, Yersinia phage PY54 protelomerase, Vibrio phage VP882 protelomerase, Borrelia burgdorferi protelomerase.

[0201] If a protelomerase other than TelN is used, the truncated protelomerase target sequence may be a truncated variant of a protelomerase target sequence specific for the protelomerase enzyme that is used.

[0202] The single contiguous aqueous volume may be incubated isothermally. The step of incubating the single contiguous aqueous volume may comprise incubating at a constant temperature. The constant temperature promotes simultaneous digestion of the double-stranded DNA molecule to produce the digested double-stranded DNA molecule and ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules and closing of the precursor double-stranded DNA molecule with protelomerase. For example, the constant temperature may be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. The constant temperature is intended to mean that the temperature does not significantly change during the reaction. The constant temperature is intended to mean that the temperature variation during the step of incubating the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment the temperature during the step of incubating the single contiguous aqueous does not deviate by more than 5°C, preferably by not more than 3°C, even more preferably not more than 1°C. Thus, the constant temperature may be a temperature in a range of 20°C-30°C, 22°C-32°C, 24°C-34°C, 26°C-36°C, 28°C-38°C, 30°C-40°C, 22°C-28°C, 32°C-38°C, 25°C-35°C, 26°C-34°C, 27°C- 33°C, 27.5°C-32.5°C, 28°C-32°C, 28.5°C-31.5°C, 29°C-31°C, or 29.5°C-30.5°C. Preferably, the constant temperature is a temperature in a range of 27.5°C-32.5°C. Alternatively, the constant temperature may be a temperature in a range of 32°C-42°C, 33°C-41°C, 34°C-40°C, 35°C-39°C, 36°C-38°C. Preferably, the constant temperature is a temperature in a range of 34.5°C-39.5°C.

[0203] The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature 2-100, 5-80, 10-70, 20-60, or 30-60 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature 2-20, 5-29, 61-100, or 65-80 times.

[0204] The method may further comprise, before step (a) (i.e. the step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Thus, the invention provides a method for producing a closed linear DNA product, the method comprises: a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor.

[0205] The step of amplifying may be performed by in vitro or in vivo amplification. Preferably, the step of amplifying is performed by in vitro amplification. For example, the step of amplifying may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the step of amplifying is performed by rolling circle amplification. Thus, the invention provides a method for producing a closed linear DNA product, the method comprises: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule by rolling circle amplification, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor.

[0206] Rolling circle amplification may be performed without any primers, or in the presence of a primer or multiple primers. For example, the primer may be a synthetic primer. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be TthPrimPol. Preferably, if the rolling circle amplification is performed without any primers, it is performed in the presence of a primase, such as TthPrimPol. Similarly, if a primer is used during amplification reaction, a primase is not used. The double-stranded DNA product may be generated by the rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0207] In the methods described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Thus, the DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, the at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences would be known to the skilled person. The cleavable target sequence may be a Type IIS restriction endonuclease target sequence. For example, the restriction endonuclease target sequence may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso311, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF51, BstMAI, BstV11, BstV21, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104I, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva12691, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequence. The at least one cleavable sequence (e.g. endonuclease target sequence) may be a native cleavable sequence (i.e. a cleavable sequence present in the template molecule). Alternatively, the at least one cleavable sequence (e.g. endonuclease target sequence) may be introduced to the DNA template molecule prior to the production of the closed linear DNA product.

[0208] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF51, BstMAI, BstV11, BstV2I, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104I, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269I, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0209] The ligase may be a DNA ligase, such as a T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0210] The protelomerase may be TelN protelomerase from E. coli phage N15 TelN protelomerase along with the sequence defined herein. Alternatively, the protelomerase may be Klebsiella phage Phi K02 protelomerase, Yersinia phage PY54 protelomerase, Vibrio phage VP882 protelomerase, Borrelia burgdorferi protelomerase.

[0211] If a protelomerase other than TelN is used, the truncated protelomerase target sequence may be a truncated variant of a protelomerase target sequence specific for the protelomerase enzyme that is used.

[0212] The DNA template molecule used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a natural circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from recombinase reaction, preferably Cre recombinase reaction, or (ii) a circular DNA molecule obtained from ligase reaction, preferably using the golden gate assembly. The DNA template molecule may be an enzymatically produced covalently-closed linear DNA molecule. For example, the DNA template molecule may be (i) a DNA molecule processed with TelN protelomerase; or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA template molecule may comprise an element that is double-stranded and an element that is single-stranded. For example, the template DNA molecule may comprise a double-stranded DNA and a single-stranded hairpin loop.

[0213] The DNA template molecule may be linear. If the DNA template molecule is linear, prior to amplification (e.g. rolling circle amplification), a DNA template molecule may be circularized to produce a DNA template molecule suitable for use in the methods described herein.

[0214] The template DNA molecule may comprise a cassette. The cassette may be a mammalian expression cassette. The cassette may further comprise a promoter. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence. The cassette may further comprise a LoxP sequence, preferably two LoxP sequences. If the two LoxP sequences are oriented in the same direction, the DNA sequence between the two LoxP sequences is excised as a circular loop of DNA. If the two LoxP sequences are oriented in the opposite direction, the DNA sequence between the two LoxP sequences is inverted. Thus, preferably, the two LoxP sequences are in the same orientation (i.e. the same direction) in the template DNA molecule.

[0215] The DNA template molecule may comprise a homopolymeric sequence at a 5'-end or a 3'-end or both a 5'-end and a 3'-end. The homopolymeric sequence may be added to the DNA template molecule before circularization. The homopolymeric sequence may be a polyA, a polyC, a polyG, or a polyT sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the linear DNA product, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0216] The method may further comprise (after the step of amplification and before the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule) a step of heat-deactivation. Thus, the invention provides a method for producing a closed linear DNA product, the method comprises: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat-deactivation of the reaction of step (a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and . (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor.

[0217] Preferably, amplification is rolling-circle amplification.

[0218] The step of heat-deactivation may be performed under conditions sufficient to inactive the reagents used during the amplification reaction. The step of heat-deactivation may be performed at a temperature of at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. The step of heat-deactivation may be performed for at least 1 min, at least 3 mins, at least 5 mins, at least 10 mins, at least 15 mins, or at least 20 mins.

[0219] In the method described herein, after the step of amplification, the step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed without purifying the product of the amplification reaction. That is to say that the step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, may be performed directly after the step of amplification. The step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed directly after the step of heat-deactivation.

[0220] The method may further comprise, after the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, a step of purification of the closed linear DNA product.

[0221] The method may further comprise, after the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, a step of nuclease digestion. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The step of nuclease digestion may take place before or after the step of purification. This step allows for removal of any double-stranded DNA molecules and / or adaptor molecules which have not been used in the course of performing the method. Thus, the method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor; and (e) incubating the reaction of step (d) with a nuclease (e.g. exonuclease).

[0222] In the method described herein, after the step of amplification, the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed without purifying the product of the amplification reaction. That is to say that the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed directly after the step of amplification. The step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed directly after the step of heat-deactivation.

[0223] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor; (e) purifying the closed linear DNA product; and (f) incubating the purified product of step (e) with a nuclease (e.g. exonuclease).

[0224] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat deactivation of the reaction of step (a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor; (f) purifying the closed linear DNA product; and (g) incubating the purified product of step (f) with a nuclease (e.g. exonuclease).

[0225] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first ens of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor; (e) incubating the reaction of step (d) with a nuclease (e.g. exonuclease); and (f) purifying the closed linear DNA product.

[0226] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat deactivation of the reaction of step (a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA; (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor; (f) incubating the reaction of step (e) with a nuclease (e.g. exonuclease); and (g) purifying the closed linear DNA product.

[0227] Steps c) to f) may be carried out in a single contiguous aqueous volume. The single contiguous aqueous volume (or the purified product of step (g)) may be incubated with a nuclease and may be performed at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30-45°C or 35-40°C. The step of incubating the single contiguous aqueous volume (or the purified product of step (g)) with a nuclease may be performed for at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 min. The step of incubating the single contiguous aqueous volume (or the purified product of step (g)) may be performed at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product of step (g)) may be performed at 15-40°C for 10-60 minutes followed by a temperature of 60-90°C for 10-30 min. The higher temperature typically inactivates the nuclease (e.g. exonuclease). Thus, the method further provides a step of inactivating the nuclease (e.g. exonuclease). The step of incubating the single contiguous aqueous volume (or the purified product of step (g)) may be performed at 37°C for 30 min and 80°C for 20 min. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed at a temperature of 70-80°C. The step of inactivating the nuclease (e.g. exonuclease) may be performed for at least 1, at least 5, at least 10, at least 20 or at least 30 minutes. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed for at least 5 minutes.

[0228] The methods of the invention may comprise a (further) step of heat-deactivation after the step of incubating with the protelomerase and prior to treatment with an exonuclease. The heat-deactivation may be carried out after the steps of digesting a double-stranded DNA molecule to generate a digested double-stranded molecule; appending a first adaptor molecule to a first end of the digested double stranded DNA molecule and appending a second adaptor molecule to a second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule; and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed DNA product, whether the steps are performed in a single contiguous aqueous volume or whether the step are carried out sequentially in separate reactions.

[0229] Thus, the method of the invention may comprise: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence and the second end of the precursor double-stranded DNA molecule is closed by the second adaptor; and (d) heat-deactivation of the reaction of steps (a) to (c), or step (c).

[0230] Heat-deactivation may be carried out at a temperature of 50-90°C, preferably from 60-80°C, more preferably from 70 to 80°C, 72-77 or 75°C. The heat deactivation may be performed from 1 to 10 minutes, from 2 to 9 minutes, for 3 to 8 minutes, from 4 to 7 minutes, for 6 minutes or, most preferably for 5 minutes.

[0231] The method may be a cell-free method.

[0232] The closed linear DNA product may be partially double-stranded and / or partially single-stranded. The closed linear DNA product may comprise a portion that is double-stranded and a portion that is single-stranded.

[0233] The closed linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0234] The closed linear DNA product may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long.

[0235] The closed linear DNA product may comprise an inverted terminal repeat sequence.

[0236] The closed linear DNA product may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the closed linear DNA product is at least 50 base pairs long.

[0237] The double-stranded DNA molecule may be circular, or branched.

[0238] The double-stranded DNA molecule may not comprise an adaptor. The double-stranded DNA molecule may not comprise a hairpin, a loop or a stem-loop structure.

[0239] The double-stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0240] The double-stranded DNA molecule may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve an amplification yield of the double-stranded DNA molecule.

[0241] The double-stranded DNA molecule may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0242] The double-stranded DNA molecule may comprise one or more cleavable (e.g. endonuclease) target sequences. The double-stranded DNA molecule may comprise two cleavable (e.g. endonuclease) target sequences. The one or more cleavable (e.g. endonuclease) target sequences may be Type IIS endonuclease target sequences. The one or more cleavable (e.g. endonuclease) target sequences may be Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AcIWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF5I, BstMAI, BstV1I, BstV21, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104I, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269I, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0243] The double-stranded DNA molecule may be a product of amplification. Preferably, the amplification is rolling circle amplification.

[0244] The linear double-stranded region of the double-stranded DNA molecule) may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0245] The double-stranded DNA molecule comprises a truncated protelomerase sequence, wherein the truncated protelomerase sequence is non-functional. The truncated protelomerase target sequence may be a truncated TelN protelomerase target sequence. The truncated protelomerase target sequence may consist of SEQ ID NO:s 4, 5, 6 or 20. The truncated protelomerase sequence may consist of SEQ ID NO: 4. The truncated protelomerase sequence may consist of SEQ ID NO: 5. The truncated protelomerase sequence may consist of SEQ ID NO: 6. The truncated protelomerase sequence may consist of SEQ ID NO: 20. The truncated protelomerase sequence may consist of SEQ ID NO:23, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. The truncated protelomerase sequence may consist of SEQ ID NO: 23. The truncated protelomerase sequence may consist of SEQ ID NO: 30. The truncated protelomerase sequence may consist of SEQ ID NO: 31. The truncated protelomerase sequence may consist of SEQ ID NO: 32.

[0246] The linear double-stranded region may comprise a sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequence of the double-stranded DNA molecule.

[0247] The first end and the second end of the linear double-stranded region (e.g. of the double-stranded DNA molecule) may be resistant to nuclease digestion. Preferably, the first end and the second end of the linear double-stranded region are resistant to the exonuclease digestion, such as exonuclease III digestion and / or exonuclease I digestion.

[0248] The linear double-stranded region of the digested double-stranded DNA molecule may comprise a 3'-OH group at first and / or second ends. The 3'-OH group may facilitate ligation to the first and / or second adaptor molecule(s) (which may comprise a 5' phosphate). The linear double-stranded region may comprise a 5' phosphate at first and / or second ends. The 5' phosphate may facilitate ligation to the first and / or second adaptor molecule(s) (which may comprise a 3'-OH group).

[0249] The linear double-stranded region (e.g. of the digested double-stranded DNA molecule) may comprise an overhang. For example, the linear double-stranded region may comprise a 5' overhang or a 3' overhang. The linear double-stranded region may comprise a blunt end or blunt ends. The linear double-stranded region may comprise: a 5' overhang and a blunt end, two 5' overhangs, a 3' overhang and a blunt end, two 3' overhangs, or a 5' overhang and a 3' overhang. The overhang may have at least 3 nucleotides (preferably from 4 to 8 nucleotides). The overhang may be in the sense strand or the antisense strand of the linear double-stranded region.

[0250] The linear region of the double-stranded DNA molecule (e.g. of the double-stranded DNA molecule) may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, double-stranded DNA molecule is at least 50 base pairs long.

[0251] The first adaptor molecule and / or the second adaptor molecule may be a synthetic adaptor molecule.

[0252] The first adaptor molecule may be a nucleic acid adaptor molecule. The second adaptor molecule may be a nucleic acid adaptor molecule. The second adaptor molecule may comprise a self-complementary element which creates a loop, such as a hairpin loop or a stem loop. The second adaptor molecule may comprise a hairpin or a stem-loop. The second adaptor molecule may comprise a double-stranded portion comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are linked together in a hairpin such that the sense strand is hybridized to the antisense strand. The double-stranded portion of an adaptor may comprise a 3' overhang or a 5' overhang of at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably the 3' overhang or the 5' overhang is 4-8 nucleotides. Each end of the linear double-stranded region (of the double-stranded DNA molecule) may comprise a 3' or a 5' overhang.

[0253] A portion of the first adaptor molecule (e.g. the overhang) may be complementary to the first end of the linear double-stranded region. A portion of the second adaptor molecule may be complementary to the second end of the linear double-stranded region.

[0254] The first adaptor molecule and / or the second adaptor molecule may comprise a truncated protelomerase target sequence. The first and / or second adaptor molecule may or may not comprise all base pairs that are deleted from the truncated protelomerase sequence present in the double-stranded DNA molecule, e.g., if the truncated protelomerase target sequence in the double stranded DNA molecule is missing eight base pairs, those eight base pairs may be present in the adaptor molecule sequence, or fewer than those eight base pairs may be present in the adaptor molecule. The portion of a protelomerase target sequence present in the first / and or second adaptor molecule are sufficient such that, wherein the first and / or second adaptor molecule is appended to a first and / or second end of the double-stranded DNA molecule comprising a truncated protelomerase target sequence the protelomerase target sequence is rendered functional and the protelomerase is able to cleave and ligate the sequence to covalently close the ends of the adaptor molecules and therefore the ends of the DNA molecule. The protelomerase target sequence may not be fully complete when the adaptor molecule is appended, provided that some activity is restored. The amount of activity restored may be 20%, 30%, 40%, 50%, 60% 70%, 80%, 90% or 100% of the full protelomerase activity with reference to the full TelN target sequence. The truncated protelomerase target sequences of the DNA molecule and / or of the adaptor molecule may comprise one or more point mutations, provided that activity is restored by the protelomerase target sequence formed by the adaptor molecule appending to the DNA molecule. The point mutations may be present in the truncated protelomerase target sequence of the double-stranded DNA molecule and / or in the truncated protelomerase target sequence of the adaptor molecule(s).

[0255] The first adaptor molecule and / or the second adaptor molecule may comprise a truncated TelN protelomerase target sequence. The first and / or second adaptor molecules may comprise a sequence selected from SEQ ID NO:8, 9, 10, 11, 12 or 13. The first adaptor and / or the second adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 8 and 9, SEQ ID NO:s 10 and 11 or SEQ ID NO:s 12 or 13. The first adaptor molecule and / or the second adaptor molecule may comprise a sequence selected from SEQ ID NO:24, 25, 26, 27, 28 or 29. The first adaptor and / or the second adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 24 and 25, SEQ ID NO:s 26 and 27 or SEQ ID NO:s 28 or 29.

[0256] The closed linear DNA product may be a covalently closed linear DNA product. Thus, in embodiments where an adaptor molecules comprises a truncated protelomerase target sequence, protelomerase covalently closes the end of the adaptor molecule appended to the end of linear double stranded region which forms a first functional protelomerase target sequence, to form a covalently closed linear DNA product. In embodiments where one of the adaptor molecules comprises a loop (e.g. a hairpin), the adaptor molecule closes an end (e.g., the first end) of the linear double-stranded region and protelomerase closes the other end (e.g., the second end) of the linear double-stranded region, to form a covalently closed linear DNA product.

[0257] The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule, wherein (i) the first adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the first end of the linear double-stranded region and (ii) the second adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the second end of the linear double-stranded region; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences and the second end of the precursor double stranded DNA molecule is closed by the second adaptor molecule.

[0258] The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule, wherein (i) the first adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the first end of the linear double-stranded region and (ii) the second adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the second end of the linear double-stranded region, and wherein the first adaptor molecule is ligated to the first end of the linear double-stranded region and the second adaptor molecule is ligated to the second end of the linear double-stranded region; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences and the second end of the precursor double stranded DNA molecule is closed by the second adaptor molecule.

[0259] The first adaptor molecule and / or the second adaptor molecule may not be a plasmid or a vector DNA.

[0260] The first adaptor and / or the second adaptor molecule comprise a truncated protelomerase target sequence. The first adaptor and / or the second adaptor molecule may comprise a truncated TelN protelomerase target sequence. The truncated protelomerase target sequence may comprise a partial telR sequence. The truncated protelomerase target sequence may comprise a partial telR sequence and a partial telO sequence. The first and / or second adaptor molecule may comprise 10, 11, 12, 13, 14, 15, 16 17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 base pairs of a telR sequence. Preferably, the adaptor comprises at least 21 base pairs of a telR sequence. The truncated protelomerase target sequence may comprise a partial telL sequence. The truncated protelomerase target sequence may comprise a partial telL sequence and a partial telO sequence. The first and / or second adaptor molecule may comprise 10, 11, 12, 13, 14, 15, 16 17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 base pairs of a telL sequence. Preferably, the adaptor comprises at least 21 base pairs of a telL sequence. The first and / or second adaptor molecules may comprise the same sequence or different sequences, i.e., the first adaptor molecule may comprise a different sequence in the overhang portion such that it only appends (by hybridization and / or ligation) to a first end of the digested double-stranded DNA molecule, and not to the second end.

[0261] The second adaptor molecule may comprise a single-stranded portion. The single-stranded portion may form a hairpin or a stem-loop. Thus, the second adaptor molecule may comprise a loop portion. The single-stranded portion may comprise less than 10, 9, 8, 7, 6, 5, 4, 3, 2 nucleotides. Preferably, the single-stranded portion comprises 5 nucleotides.

[0262] The first adaptor molecule and / or the second adaptor molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The double-stranded portion may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 base pairs.

[0263] The first adaptor molecule and / or the second adaptor molecule may comprise a 5' phosphate. The 5' phosphate may facilitate ligation to the linear double-stranded region (which may comprise a 3'-OH group at first and / or second ends). The first adaptor molecule and / or the second adaptor molecule may comprise a 3'-OH. The 3'-OH may facilitate ligation to the linear double-stranded region (which may comprise a 5' phosphate at first and / or second ends).

[0264] The first and / or second adaptor molecule may comprise the sequence of SEQ ID NO: 14 or a portion thereof. The first and / or second adaptor molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 14. The double-stranded portion of the first and / or second adaptor molecule may comprise the sequence of SEQ ID NO: 15 or a portion thereof. The double-stranded portion of the first and / or second adaptor molecule may comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 or at least 15 contiguous nucleotides of SEQ ID NO: 15. The single-stranded portion of the first and / or second adaptor molecule may comprise a sequence of ACTCA. The single-stranded portion of the first adaptor molecule and / or the second adaptor molecule may comprise at least 1, at least 2, at least 3, at least 4 or at least 5 contiguous nucleotides of the sequence ACTCA. The second adaptor molecule may comprise or consist of the sequence of SEQ ID NO:17.

[0265] The first and second adaptor molecules may comprise an identical nucleic acid sequence. The first and second adaptor molecules may comprise a different nucleic acid sequence.

[0266] The first adaptor molecule may comprise a portion that is complementary to the first end of the digested double-stranded DNA molecule. The second adaptor molecule may comprise a portion that is complementary to the second end of the digested double-stranded DNA molecule. The first adaptor molecule may comprise a portion that anneals to the first end of the digested double-stranded DNA molecule. The second adaptor molecule may comprise a portion that anneals to the second end of the digested double-stranded DNA molecule. The first adaptor molecule may comprise a portion that is complementary and anneals to the first end of the digested double-stranded DNA molecule. The second adaptor molecule may comprise a portion that is complementary and anneals to the second end of the digested double-stranded DNA molecule.

[0267] The portion that is complementary or anneals to the first or second end of digested double-stranded DNA molecule may be a 5' overhang or a 3' overhang of the first and / or second adaptor molecule. The overhang of the first adaptor molecule may be complementary to the first end of the digested double-stranded DNA molecule and / or the overhang of the second adaptor molecule may be complementary to the second end of the digested double-stranded DNA molecule. The overhang of the first adaptor molecule may anneal to the first end of the digested double-stranded DNA molecule and / or the overhang of the second adaptor molecule may anneal to the second end of the digested double-stranded DNA molecule. The overhang of the first adaptor molecule may be complementary to and anneal to the first end of the digested double-stranded DNA molecule and / or the overhang of the second adaptor molecule may be complementary to and anneal to the second end of the digested double-stranded DNA molecule.

[0268] The first adaptor molecule and / or the second adaptor molecule may not comprise a Type IIS endonuclease target sequence. The first adaptor molecule and / or the second adaptor molecule may not comprise Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BslFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF5I, BstMAI, BstV11, BstV2I, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104I, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva12691, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0269] The first adaptor molecule and / or the second adaptor molecule may comprise a Type IIS endonuclease target sequence, provided it is different to a type IIS endonuclease target sequence that is present in the double-stranded DNA molecule.

[0270] The first adaptor molecule and / or the second adaptor molecule may comprise one or more locked nucleic acids (LNAs).

[0271] The first adaptor molecule and / or the second adaptor molecule may comprise one or more protected nucleotides (i.e. nuclease-resistant nucleotides), such as phosphorothioated nucleotides. The protected nucleotides may be located in the single-stranded portion (e.g. hairpin portion) or the double-stranded portion. The protected nucleotides may be located in the overhang portion of the adaptor molecules.

[0272] The closed linear DNA product may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the closed linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the closed linear DNA product comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand.

[0273] The internal positions may not be located between the second and penultimate nucleotide of the closed linear DNA product.

[0274] The linear double-stranded region (of the double-stranded DNA molecule) may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the linear double-stranded region (of the double-stranded DNA molecule) may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least or 500 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. Preferably, the linear double-stranded region (of the double-stranded DNA molecule) comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions in each strand. The internal positions may not be located between the second and penultimate nucleotide of the linear double-stranded region (of the double-stranded DNA molecule).

[0275] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) suitable for use in the methods described herein may be phosphorothioated nucleotides. For example, phosphorothioated nucleotides may be α-S-dATP (i.e. 2'-deoxyadenosine-5'-(a-thio)-triphosphate), α-S-dCTP (i.e. 2'-deoxycytidine-5'-(α-thio)-triphosphate), α-S-dGTP (i.e. 2'-deoxyguanosine-5'-(α-thio)-triphosphate), α-S-dTTP (i.e. 2'-deoxythymidine-5'-(α-thio)-triphosphate), α-S-dUTP (i.e. 2'-deoxyuridine-5'-(α-thio)-triphosphate), and / or uridine 2', 3'-cyclophosphorothioate.

[0276] The phosphorothioated nucleotides may be Sp-isomers, Rp-isomers or a mixture of both Sp- and Rp-isomers.

[0277] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be 2'-O-methyl nucleotides or 2'-O-methoxyethyl (MOE) nucleotides. For example, the MOE nucleotides may be 2'-O-methoxy-ethyl guanosine, 2'-O-methoxy-ethyl cytidine, 2'-O-methoxy-ethyl adenosine, and / or 2'-O-methoxy-ethyl thymidine.

[0278] The first end of the linear double-stranded region may be complementary to a portion of the first adaptor molecule. The second end of the linear double-stranded region may be complementary to a portion of the second adaptor molecule. The first end and / or the second end of the linear double-stranded region may be generated by endonuclease digestion.

[0279] The first adaptor molecule and / or the second adaptor molecule may comprise a functional portion. The functional portion may be a binding molecule, a targeting sequence, or a probe.

[0280] The functional portion may be a probe. As used herein, the term "probe" refers to a fragment of DNA, RNA or DNA / RNA chimera of variable length (e.g. 3-1000 bases long), which is used to detect the presence of target nucleotide sequences that are complementary to the sequence in the probe. Typically, the probe hybridizes to single-stranded nucleic acid whose base sequence allows probetarget base pairing due to complementarity between the probe and target. Thus, the functional portion may be a DNA sequence, a RNA sequence or a DNA / RNA chimera sequence. As used herein, the term "complementary" refers to the pairing of nucleotide sequences according to Watson / Crick pairing rules. For example, a sequence 5'-GCGGTCCCA-3' has the complementary sequence of 5'-TGGGACCGC-3'. A complement sequence can also be a sequence of RNA complementary to the DNA sequence.

[0281] The functional portion may be a binding molecule. The term "binding molecule" refers to any molecule capable of binding to the linear DNA product described herein and / or that is capable of binding to a further molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal antibody or a polyclonal antibody. The binding molecule may be an antibody fragment.

[0282] The functional portion may facilitate detection of the DNA product by binding to capture molecules (e.g. capture antibodies bound by protein-protein interactions). The functional portion may bind to a cell target, for example, a cell receptor.

[0283] The functional portion may be a label. The 'label' can be any chemical entity which enable the detection of the double-stranded nucleic acid molecule via, physical, chemical and / or biological means. The label may be a chromophore, a fluorophore and / or a radioactive molecule.

[0284] The functional portion may be a targeting sequence. The targeting sequence may be a fragment of DNA or RNA of variable length, which is used to target the DNA product to a specific location in a cell. The targeting sequence may be used to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the closed linear DNA product. For example, the targeting sequence may be a DNA nuclear targeting sequences (i.e. a recognition sequence for endogenous DNA-binding proteins), such as SV40 enhancer sequence (preferably downstream from the cassette).

[0285] To facilitate detection and / or quantification of the DNA product, the functional portion may comprise a fluorophore, a radioactive compound or a barcode.

[0286] A signal corresponding to the presence, absence and / or level of the closed linear DNA product may be measured using a barcode. The barcode may comprise at least one binding moiety linked to a barcoded portion, wherein the barcoded portion comprises at least one nucleotide (i.e. wherein the barcoded portion comprises a nucleotide sequence at least one nucleotide in length), and wherein the binding moiety is capable of binding to the 3' overhang, the 5' overhang or the blunt end of the closed linear DNA product. The binding moiety is capable of binding to 3' and / or 5' end of the closed linear DNA product. The signal may be measured by determining the presence, absence and / or level of the barcoded portion of the barcode (e.g. by sequencing or PCR). The barcoded portion may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. The barcode may comprise at least 2 binding moieties (e.g. a first binding moiety and a second binding moiety). For example, the first binding moiety linked to the first barcoded portion may bind to the 3' end of the closed linear DNA product and the second binding moiety linked to the second barcoded portion may bind to the 5' end of the closed linear DNA product. The 3' and 5' ends may comprise a 3' overhang, a 5' overhang or a blunt end.

[0287] A signal corresponding to the presence, absence and / or level of the closed linear DNA product may be measured using a fluorophore (i.e. a fluorescently-labelled molecule), which is attached or bound to the 3' overhang, the 5' overhang or the blunt end of the closed linear DNA product. The signal may be measured by flow cytometry and / or fluorescence-activated cell sorting.

[0288] The functional portion may also facilitate DNA sequencing. For example, the functional portion may be a sequencing adaptor. The term "sequencing adaptor" is intended to encompass one or more nucleic acid domains that include at least a portion of a nucleic acid sequence (or complement thereof) utilized by a sequencing platform of interest, such as a sequencing platform provided by Illumina ®< (e.g. the HiSeq ™< , MiSeq ™< and / or Genome Analyzer ™< sequencing systems), Oxford Nanopore ™< Technologies (e.g. the MinION sequencing system), Ion Torrent ™< (e.g. the Ion PGM ™< and / or Ion Proton ™< sequencing systems), Pacific Biosciences (e.g. the PACBIO RS II sequencing system); Life Technologies ™< (e.g. a SOLiD sequencing system), Roche (e.g. the 454 GS FLX+ and / or GS Junior sequencing systems), or any other sequencing platform of interest.

[0289] The first adaptor molecule and / or the second adaptor molecule may comprise an inverted terminal repeat sequence. The inverted terminal repeat sequences of the first adaptor molecule and the second adaptor molecule may be symmetrical (i.e. have the same symmetrical three-dimensional organization with respect to each other) or asymmetrical (i.e. have different three-dimensional organization with respect to each other). The inverted terminal repeat sequences of the first adaptor molecule and the second adaptor molecule may be from the same or different serotypes. An inverted terminal repeat sequence may comprise a terminal resolution site and a Rep binding site.

[0290] The first adaptor molecule and / or the second adaptor molecule may comprise an aptamer.

[0291] The first adaptor molecule and / or the second adaptor molecule may confer resistance to the nuclease digestion, such as exonuclease digestion (e.g. exonuclease I and / or exonuclease III digestion).

[0292] The closing of the precursor double-stranded DNA molecule at the first end by protelomerase may generate a first closed end of the closed linear DNA product. The closing of the precursor double-stranded DNA molecule at the second end by the appending, or ligation, of the second adaptor may generate a second closed end of the closed linear DNA product. The first closed end and the second closed end of the closed linear DNA product may be resistant to nuclease digestion. The nuclease digestion may be exonuclease digestion. Preferably, the nuclease digestion is exonuclease III digestion and / or exonuclease I digestion.3. Methods for producing a partially closed linear DNA product comprising nuclease-resistant nucleotides

[0293] The methods described herein may be used to produce a partially closed linear DNA product comprising nuclease-resistant (i.e. protected nucleotides).

[0294] The methods described herein may be used to produce a partially closed DNA product e.g. a covalently partially closed DNA product.

[0295] The invention provides a method for producing a partially closed DNA product, the method comprises the steps of: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences.

[0296] The step of appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule is preferably performed in the presence of a ligase. Thus, the invention provides a method for producing a partially closed DNA product, wherein the method comprises: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; (b) ligating a first adaptor molecule to the first end of the digested double-stranded DNA molecule and ligating a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

[0297] The step of appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule may be performed in the absence of a ligase, and may rely on hybridisation. Thus, the invention provides a method for producing a partially closed DNA product, wherein the method comprises: a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; b) hybridizing a first adaptor molecule to the first end of the digested double-stranded DNA molecule and hybridizing a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; and c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

[0298] A partially closed DNA product has particular utility as a therapeutic agent (i.e. DNA therapeutic) which can be used to express a gene product in vivo. This is because its partially closed structure prevents attack by enzymes such as exonucleases, leading to enhanced stability and longevity of gene expression as compared to "open" DNA molecules with exposed unprotected DNA ends. Linear double-stranded open-ended cassettes have been demonstrated to be inefficient with respect to gene expression when introduced into host tissue. This has been attributed to cassette instability due to the action of exonucleases in the extracellular space. Having one end of the partially closed DNA product closed gives it increased stability and efficiency, particularly when used in IVT

[0299] Sequestering DNA ends inside closed structures also has other advantages. The DNA ends are prevented from integrating with genomic DNA and so partially closed DNA products offer improved safety. In addition, the closed linear structure reduces concatamerisation of DNA products inside host cells and thus expression levels of the gene product can be regulated in a more sensitive manner.

[0300] The method of the invention may be used for production of DNA for in vitro expression in a host cell, for example, in DNA vaccines. DNA vaccines typically encode a modified form of an infectious organism's DNA. DNA vaccines are administered to a subject where they then express the selected protein of the infectious organism, initiating an immune response against that protein which is typically protective. DNA vaccines may also encode a tumour antigen in a cancer immunotherapy approach.

[0301] The method may produce other types of therapeutic DNA molecules e.g. those used in gene therapy. For example, such DNA molecules can be used to express a functional gene where a subject has a genetic disorder caused by a dysfunctional version of that gene. Examples of such diseases include sickle cell anaemia, cystic fibrosis, Huntington disease, Duchenne's Muscular Dystrophy, Haemophilia A, α1-antitrypsin deficiency, primary ciliary dyskinesia, or respiratory distress syndrome of prematurity. Other diseases where gene therapy may be useful include metabolic diseases, respiratory diseases, inflammatory diseases, autoimmune, chronic and infectious diseases, including such disorders as AIDS, cancer, neurological diseases, cardiovascular disease, hypercholesterolemia, various blood disorders including various anaemias, thalassemia and haemophilia, and emphysema. For the treatment of solid tumours, genes encoding toxic peptides (i.e., chemotherapeutic agents such as ricin, diptheria toxin and cobra venom factor), tumour suppressor genes such as p53, genes coding for mRNA sequences which are antisense to transforming oncogenes, antineoplastic peptides such as tumor necrosis factor (TNF) and other cytokines, or transdominant negative mutants of transforming oncogenes, may be expressed.

[0302] The closing of the linear double-stranded region of the precursor double-stranded DNA molecule by the first adaptor molecule and the protecting of the second end by the second adaptor molecule may be performed by hybridization or ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule may be hybridized to the first end of the linear double-stranded region. The second adaptor molecule may be hybridized to the second end of the linear double-stranded region. The first adaptor molecule may be ligated to the first end of the linear double-stranded region. The second adaptor molecule may be ligated to the second end of the linear double-stranded region. The closing of the linear double-stranded region by the first adaptor molecule and the second adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule may be hybridized and ligated to the first end of the linear double-stranded region. The second adaptor molecule may be hybridized and ligated to the second end of the linear double-stranded region.

[0303] The steps of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule; appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule; and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, may be performed in a single contiguous aqueous volume. When the method of the invention is performed in a single contiguous aqueous volume, it may be performed under conditions that promote appending (or linking) of the first and second adaptor molecules to the linear double-stranded region to produce the partially closed DNA product. The appending may be performed by creating a covalent link between the first and / or second adaptor molecule and the first and / or second end of the linear double-stranded region.

[0304] The single contiguous aqueous volume may be incubated under conditions that promote digestion of the double-stranded DNA molecule to produce the linear region of the double-stranded DNA molecule. The digestion of the double-stranded DNA molecule to produce the digested double-stranded DNA molecule may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0305] If the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double stranded DNA molecule is carried out as a separate reaction, it may be performed under conditions that promote digestion of the double-stranded DNA molecule to produce the linear region of the double-stranded DNA molecule. The digestion of the double-stranded DNA molecule to produce the digested double-stranded DNA molecule may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0306] When the method if performed as a single contiguous aqueous volume, the single contiguous aqueous volume may be incubated under conditions that promote ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules. The ligation may be 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the digested double-stranded DNA molecules may be incorporated into closed linear DNA products. Preferably, the ligation is at least 15% efficient.

[0307] If the step of appending or ligating a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending or ligating a second adaptor molecule to the second end of the digested double stranded DNA molecule to generate a precursor double-stranded DNA molecule is carried out as a separate reaction, it may be performed under conditions that promote ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules. The ligation may be 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, 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 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the digested double-stranded DNA molecules may be incorporated into closed linear DNA products. Preferably, the ligation is at least 15% efficient.

[0308] Ligation efficiency may be established based on DNA quantification values before and after the digestion / ligation reaction. Thus, ligation efficiency may be established based on the equation: (starting amplified DNA amount) / (final linear DNA amount) x 100%.

[0309] Ligation efficiency may also be established based on DNA quantification values before and after the digestion / ligation reaction and the subsequent exonuclease treatment to remove remaining open DNA constructs and adaptor molecules excess.

[0310] For example, the double-stranded DNA molecule generated by the rolling circle amplification is first quantified so that the amount of the double-stranded DNA molecule used as the starting material during the digestion / ligation / protelomerase reaction is known. After all the enzymatic reactions, the linear DNA product is quantified to calculate the ligation efficiency as per the equation above.

[0311] DNA quantification methods are known to a person skilled in the art. For example, DNA quantifications may be carried out using the Qubit dsDNA BR assay from ThermoFisher (https: / / www.thermofisher.com / order / catalog / product / Q32850# / Q32850).

[0312] The step of ligation of the digested double-stranded DNA molecule to the first and second adaptor molecules may be performed at a second temperature of 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C.

[0313] The single contiguous aqueous volume may be incubated under conditions that promote the action of protelomerase to cleave and covalently close the protelomerase target sequence to form the closed linear DNA product. The incubation of the precursor double-stranded DNA molecule with protelomerase may be carried out at a third temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 26°C-34°C, 27°C-33°C 25°C-35°C, 28°C-32°C, 29°C-31°C or at about 30°C.

[0314] If the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product is performed as a separate reaction, it may be performed at a third temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 26°C-34°C, 27°C-33°C 25°C-35°C, 28°C-32°C, 29°C-31°C or at about 30°C.

[0315] The single contiguous aqueous volume may be performed in a step comprising incubating at a first temperature and then incubating at a second temperature and / or then incubating at a third temperature. The first temperature may be 1°C-100°C, 1°C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C-45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or about 37°C. The second temperature may be 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C. The third temperature may be 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 26°C-34°C, 27°C-33°C 25°C-35°C, 28°C-32°C, 29°C-31°C or at about 30°C.

[0316] Preferably, the first temperature is 35°C-39°C and the second temperature is 14°C-18°C and the third temperature is 28°C-32°C. Using these conditions the endonuclease may be a Type IIS restriction endonuclease (e.g. Bsal) and the ligase may be T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase and the protelomerase may be TelN protelomerase from E. coli phage N15 TelN protelomerase along with the sequence defined herein. Alternatively, the protelomerase may be Klebsiella phage Phi K02 protelomerase, Yersinia phage PY54 protelomerase, Vibrio phage VP882 protelomerase, Borrelia burgdorferi protelomerase.

[0317] If a protelomerase other than TelN is used, the truncated protelomerase target sequence may be a truncated variant of a protelomerase target sequence specific for the protelomerase enzyme that is used.

[0318] The single contiguous aqueous volume may be incubated isothermally. The step of incubating the single contiguous aqueous volume may comprise incubating at a constant temperature. The constant temperature promotes simultaneous digestion of the double-stranded DNA molecule to produce the digested double-stranded DNA molecule and ligation of the linear double-stranded region to the first and second adaptor molecules and closing of the precursor double-stranded DNA molecule with protelomerase. For example, the constant temperature may be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. The constant temperature is intended to mean that the temperature does not significantly change during the reaction. The constant temperature is intended to mean that the temperature variation during the step of incubating the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment the temperature during the step of incubating the single contiguous aqueous does not deviate by more than 5°C, preferably by not more than 3°C, even more preferably not more than 1°C. Thus, the constant temperature may be a temperature in a range of 20°C-30°C, 22°C-32°C, 24°C-34°C, 26°C-36°C, 28°C-38°C, 30°C-40°C, 22°C-28°C, 32°C-38°C, 25°C-35°C, 26°C-34°C, 27°C- 33°C, 27.5°C-32.5°C, 28°C-32°C, 28.5°C-31.5°C, 29°C-31°C, or 29.5°C-30.5°C. Preferably, the constant temperature is a temperature in a range of 27.5°C-32.5°C. Alternatively, the constant temperature may be a temperature in a range of 32°C-42°C, 33°C-41°C, 34°C-40°C, 35°C-39°C, 36°C-38°C. Preferably, the constant temperature is a temperature in a range of 34.5°C-39.5°C.

[0319] The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature 2-100, 5-80, 10-70, 20-60, or 30-60 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature and / or the third temperature 2-20, 5-29, 61-100, or 65-80 times.

[0320] The method may further comprise, before step (a) (i.e. the step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Thus, the invention provides a method for producing a partially closed DNA product, the method comprises: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; and (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

[0321] The step of amplifying may be performed by in vitro or in vivo amplification. Preferably, the step of amplifying is performed by in vitro amplification. For example, the step of amplifying may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the step of amplifying is performed by rolling circle amplification. Thus, the invention provides a method for producing a partially closed DNA product, the method comprises: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule by rolling circle amplification, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; and (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

[0322] Rolling circle amplification may be performed without any primers, or in the presence of a primer or multiple primers. For example, the primer may be a synthetic primer. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be TthPrimPol. Preferably, if the rolling circle amplification is performed without any primers, it is performed in the presence of a primase, such as TthPrimPol. Similarly, if a primer is used during amplification reaction, a primase is not used. The double-stranded DNA product may be generated by the rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0323] In the methods described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Thus, the DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, the at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences would be known to the skilled person. The cleavable target sequence may be a Type IIS restriction endonuclease target sequence. For example, the restriction endonuclease target sequence may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso311, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF51, BstMAI, BstV11, BstV21, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104I, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva12691, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequence. The at least one cleavable sequence (e.g. endonuclease target sequence) may be a native cleavable sequence (i.e. a cleavable sequence present in the template molecule). Alternatively, the at least one cleavable sequence (e.g. endonuclease target sequence) may be introduced to the DNA template molecule prior to the production of the partially closed DNA product.

[0324] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF51, BstMAI, BstV11, BstV21, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104I, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269I, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0325] The ligase may be a DNA ligase, such as a T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0326] The protelomerase may be TelN protelomerase from E. coli phage N15 TelN protelomerase along with the sequence defined herein. Alternatively, the protelomerase may be Klebsiella phage Phi K02 protelomerase, Yersinia phage PY54 protelomerase, Vibrio phage VP882 protelomerase, Borrelia burgdorferi protelomerase.

[0327] If a protelomerase other than TelN is used, the truncated protelomerase target sequence may be a truncated variant of a protelomerase target sequence specific for the protelomerase enzyme that is used.

[0328] The DNA template molecule used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a natural circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from recombinase reaction, preferably Cre recombinase reaction, or (ii) a circular DNA molecule obtained from ligase reaction, preferably using the golden gate assembly. The DNA template molecule may be an enzymatically produced covalently-closed linear DNA molecule. For example, the DNA template molecule may be (i) a DNA molecule processed with TelN protelomerase; or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA template molecule may comprise an element that is double-stranded and an element that is single-stranded. For example, the template DNA molecule may comprise a double-stranded DNA and a single-stranded hairpin loop.

[0329] The DNA template molecule may be linear. If the DNA template molecule is linear, prior to amplification (e.g. rolling circle amplification), a DNA template molecule may be circularized to produce a DNA template molecule suitable for use in the methods described herein.

[0330] The template DNA molecule may comprise a cassette. The cassette may be a mammalian expression cassette. The cassette may further comprise a promoter. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence. The cassette may further comprise a LoxP sequence, preferably two LoxP sequences. If the two LoxP sequences are oriented in the same direction, the DNA sequence between the two LoxP sequences is excised as a circular loop of DNA. If the two LoxP sequences are oriented in the opposite direction, the DNA sequence between the two LoxP sequences is inverted. Thus, preferably, the two LoxP sequences are in the same orientation (i.e. the same direction) in the template DNA molecule.

[0331] The DNA template molecule may comprise a homopolymeric sequence at a 5'-end or a 3'-end or both a 5'-end and a 3'-end. The homopolymeric sequence may be added to the DNA template molecule before circularization. The homopolymeric sequence may be a polyA, a polyC, a polyG, or a polyT sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the linear DNA product, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0332] The method may further comprise (after the step of amplification and before the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule) a step of heat-deactivation. Thus, the invention provides a method for producing a partially closed DNA product, the method comprises: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat-deactivation of the reaction of step (a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; and . (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

[0333] Preferably, amplification is rolling-circle amplification.

[0334] The step of heat-deactivation may be performed under conditions sufficient to inactive the reagents used during the amplification reaction. The step of heat-deactivation may be performed at a temperature of at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. The step of heat-deactivation may be performed for at least 1 min, at least 3 mins, at least 5 mins, at least 10 mins, at least 15 mins, or at least 20 mins.

[0335] In the method described herein, after the step of amplification, the step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed without purifying the product of the amplification reaction. That is to say that the step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, may be performed directly after the step of amplification. The step of digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed directly after the step of heat-deactivation.

[0336] The method may further comprise, after the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, a step of purification of the partially closed DNA product.

[0337] The method may further comprise, after the step of incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, a step of nuclease digestion. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The step of nuclease digestion may take place before or after the step of purification. This step allows for removal of any double-stranded DNA molecules and / or adaptor molecules which have not been used in the course of performing the method. Thus, the method may comprise the steps: a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence; and e) incubating the reaction of step (d) with a nuclease (e.g. exonuclease).

[0338] In the method described herein, after the step of amplification, the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed without purifying the product of the amplification reaction. That is to say that the step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed directly after the step of amplification. The step of digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule may be performed directly after the step of heat-deactivation.

[0339] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence; (e) purifying the partially closed DNA product; and (f) incubating the purified product of step (e) with a nuclease (e.g. exonuclease).

[0340] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat deactivation of the reaction of step (a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence; (f) purifying the partially closed DNA product; and (g) incubating the purified product of step (f) with a nuclease (e.g. exonuclease).

[0341] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (c) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; (d) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first ens of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence; (e) incubating the reaction of step (d) with a nuclease (e.g. exonuclease); and (f) purifying the partially closed DNA product.

[0342] The method may comprise the steps: (a) amplifying a template DNA molecule comprising a truncated protelomerase target sequence and an endonuclease target sequence to generate a double-stranded DNA molecule, wherein the truncated protelomerase sequence is non-functional; (b) heat deactivation of the reaction of step (a); (c) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (d) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; (e) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence; (f) incubating the reaction of step (e) with a nuclease (e.g. exonuclease); and (g) purifying the partially closed DNA product.

[0343] Steps c) to f) may be carried out in a single contiguous aqueous volume. The single contiguous aqueous volume (or the purified product of step (g)) may be incubated with a nuclease and may be performed at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30-45°C or 35-40°C. The step of incubating the single contiguous aqueous volume (or the purified product of step (g)) with a nuclease may be performed for at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 min. The step of incubating the single contiguous aqueous volume (or the purified product of step (g)) may be performed at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product of step (g)) may be performed at 15-40°C for 10-60 minutes followed by a temperature of 60-90°C for 10-30 min. The higher temperature typically inactivates the nuclease (e.g. exonuclease). Thus, the method further provides a step of inactivating the nuclease (e.g. exonuclease). The step of incubating the single contiguous aqueous volume (or the purified product of step (g)) may be performed at 37°C for 30 min and 80°C for 20 min. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed at a temperature of 70-80°C. The step of inactivating the nuclease (e.g. exonuclease) may be performed for at least 1, at least 5, at least 10, at least 20 or at least 30 minutes. Preferably, the step of inactivating the nuclease (e.g. exonuclease) is performed for at least 5 minutes.

[0344] The methods of the invention may comprise a (further) step of heat-deactivation after the step of incubating with the protelomerase and prior to treatment with an exonuclease. The heat-deactivation may be carried out after the steps of digesting a double-stranded DNA molecule to generate a digested double-stranded molecule; appending a first adaptor molecule to a first end of the digested double stranded DNA molecule and appending a second adaptor molecule to a second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule; and incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, whether the steps are performed in a single contiguous aqueous volume or whether the step are carried out sequentially in separate reactions.

[0345] Thus, the method of the invention may comprise: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides; (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence; and (d) heat-deactivation of the reaction of steps (a) to (c), or step (c).

[0346] Heat-deactivation may be carried out at a temperature of 50-90°C, preferably from 60-80°C, more preferably from 70 to 80°C, 72-77 or 75°C. The heat deactivation may be performed from 1 to 10 minutes, from 2 to 9 minutes, for 3 to 8 minutes, from 4 to 7 minutes, for 6 minutes or, most preferably for 5 minutes.

[0347] The method may be a cell-free method.

[0348] The partially closed DNA product may be partially double-stranded and / or partially single-stranded. The partially closed DNA product may comprise a portion that is double-stranded and a portion that is single-stranded.

[0349] The partially closed DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0350] The partially closed DNA product may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long.

[0351] The partially closed DNA product may comprise an inverted terminal repeat sequence.

[0352] The partially closed DNA product may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the partially closed DNA product is at least 50 base pairs long.

[0353] The double-stranded DNA molecule may be circular, or branched.

[0354] The double-stranded DNA molecule may not comprise an adaptor. The double-stranded DNA molecule may not comprise a hairpin, a loop or a stem-loop structure.

[0355] The double-stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example a gene encoding a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosomal binding site and a translational termination sequence. The cassette may additionally comprise sequences aiding protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas mediated homology directed repair (HDR). The cassette may encode CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, poly C, polyT or polyG sequence. The homopolymeric sequence may be between 3-200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the cassette, in which case, the homopolymeric sequence may be between 4-12 nucleotides in length, or between 5-10 nucleotides in length. The homopolymeric sequence may be used to improve mRNA expression, in which case, the homopolymeric sequence may be between 10-200 nucleotides in length, preferably between 80-150 nucleotides in length. The homopolymeric sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides in length. Preferably, the homopolymeric sequence is at least 100 nucleotides in length. More preferably still, the homopolymeric sequence is at least 120 nucleotides in length. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides.

[0356] The double-stranded DNA molecule may comprise a spacer. The spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve an amplification yield of the double-stranded DNA molecule.

[0357] The double-stranded DNA molecule may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0358] The double-stranded DNA molecule may comprise one or more cleavable (e.g. endonuclease) target sequences. The double-stranded DNA molecule may comprise two cleavable (e.g. endonuclease) target sequences. The one or more cleavable (e.g. endonuclease) target sequences may be Type IIS endonuclease target sequences. The one or more cleavable (e.g. endonuclease) target sequences may be Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, Alol, Alw261, Alwl, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEI, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF5I, BstMAI, BstV1I, BstV21, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104I, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269I, NmeAIII, PaqCI, PciSI, Pctl, Plel, PpsI, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0359] The double-stranded DNA molecule may be a product of amplification. Preferably, the amplification is rolling circle amplification.

[0360] The linear double-stranded region (e.g. of the double-stranded DNA molecule) may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long.

[0361] The linear double-stranded region (e.g. of the double-stranded DNA molecule) may comprise a sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequence of the double-stranded DNA molecule.

[0362] The first end and the second end of the linear double-stranded region (e.g. of the double-stranded DNA molecule) may be resistant to nuclease digestion. Preferably, the first end and the second end of the linear double-stranded region are resistant to the exonuclease digestion, such as exonuclease III digestion and / or exonuclease I digestion.

[0363] The linear double-stranded region may comprise a 3'-OH group at first and / or second ends. The 3'-OH group may facilitate ligation to the first and / or second adaptor molecule(s) (which may comprise a 5' phosphate). The linear double-stranded region may comprise a 5' phosphate at first and / or second ends. The 5' phosphate may facilitate ligation to the first and / or second adaptor molecule(s) (which may comprise a 3'-OH group).

[0364] The linear double-stranded region (e.g. of the double-stranded DNA molecule) may comprise an overhang. For example, the linear double-stranded region may comprise a 5' overhang or a 3' overhang. The linear double-stranded region may comprise a blunt end or blunt ends. The linear double-stranded region may comprise: a 5' overhang and a blunt end, two 5' overhangs, a 3' overhang and a blunt end, two 3' overhangs, or a 5' overhang and a 3' overhang. The overhang may have at least 3 nucleotides (preferably from 4 to 8 nucleotides). The overhang may be in the sense strand or the antisense strand of the linear double-stranded region.

[0365] The linear region of the double-stranded DNA molecule may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, double-stranded DNA molecule is at least 50 base pairs long.

[0366] The double-stranded DNA molecule comprises a truncated protelomerase sequence, wherein the truncated protelomerase sequence is non-functional. The truncated protelomerase target sequence may be a truncated TelN protelomerase target sequence. The truncated protelomerase target sequence may consist of SEQ ID NO:s 4, 5, 6 or 20. The truncated protelomerase sequence may consist of SEQ ID NO: 4. The truncated protelomerase sequence may consist of SEQ ID NO: 5. The truncated protelomerase sequence may consist of SEQ ID NO: 6. The truncated protelomerase sequence may consist of SEQ ID NO: 20. The truncated protelomerase sequence may consist of SEQ ID NO:23, SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. The truncated protelomerase sequence may consist of SEQ ID NO: 23. The truncated protelomerase sequence may consist of SEQ ID NO: 30. The truncated protelomerase sequence may consist of SEQ ID NO: 31. The truncated protelomerase sequence may consist of SEQ ID NO: 32.

[0367] The first adaptor molecule and / or the second adaptor molecule may be a synthetic adaptor molecule.

[0368] The first adaptor molecule may be a nucleic acid adaptor molecule. The second adaptor molecule may be a nucleic acid adaptor molecule. The double-stranded portion of an adaptor may comprise a 3' overhang or a 5' overhang of at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably the 3' overhang or the 5' overhang is 4-8 nucleotides. Each end of the linear double-stranded region ( of the double-stranded DNA molecule) may comprise a 3' or a 5' overhang.

[0369] A portion of the first adaptor molecule (e.g. the overhang) may be complementary to the first end of the linear double-stranded region. A portion of the second adaptor molecule may be complementary to the second end of the linear double-stranded region.

[0370] The first adaptor molecule may comprise a truncated protelomerase target sequence. The first and / or second adaptor molecule may or may not comprise all base pairs that are deleted from the truncated protelomerase sequence present in the double-stranded DNA molecule, e.g., if the truncated protelomerase target sequence in the double stranded DNA molecule is missing eight base pairs, those eight base pairs may be present in the adaptor molecule sequence, or fewer than those eight base pairs may be present in the adaptor molecule. The portion of a protelomerase target sequence present in the first adaptor molecule are sufficient such that, wherein the first adaptor molecule is appended to a first end of the double-stranded DNA molecule comprising a truncated protelomerase target sequence, the protelomerase target sequence is rendered functional and the protelomerase is able to cleave and ligate the sequence to covalently close the ends of the adaptor molecules and therefore the ends of the DNA molecule. The protelomerase target sequence may not be fully complete when the adaptor molecule is appended, provided that some activity is restored. The amount of activity restored may be 20%, 30%, 40%, 50%, 60% 70%, 80%, 90% or 100% of the full protelomerase activity with reference to the full protelomerase target sequence. The truncated protelomerase target sequences of the DNA molecule and / or of the adaptor molecule may comprise one or more point mutations, provided that activity is restored by the protelomerase target sequence formed by the adaptor molecule appending to the DNA molecule. The point mutations may be present in the truncated protelomerase target sequence of the double-stranded DNA molecule and / or in the truncated protelomerase target sequence of the adaptor molecule(s).

[0371] The first adaptor molecule comprises a truncated protelomerase target sequence, which may be a truncated TelN protelomerase target sequence. The first adaptor molecule may comprise a sequence selected from SEQ ID NO:8, 9, 10, 11, 12 or 13. The first adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 8 and 9, SEQ ID NO:s 10 and 11 or SEQ ID NO:s 12 or 13. The first adaptor molecule may comprise a sequence selected from SEQ ID NO:24, 25, 26, 27, 28 or 29. The first adaptor molecule may consist of a pair of complementary sequences selected from SEQ ID NO:s 24 and 25, SEQ ID NO:s 26 and 27 or SEQ ID NO:s 28 or 29.

[0372] The partially closed DNA product may be a partially covalently closed DNA product. Thus, in embodiments where an adaptor molecules comprises a truncated protelomerase target sequence, protelomerase covalently closes the end of the adaptor molecule appended to the end of linear double stranded region which forms a first functional protelomerase target sequence, to form a covalently partially closed DNA product. In embodiments where protelomerase closes one end (e.g., the first end) of the linear double-stranded region, the second adaptor does not close the other end of the precursor double stranded DNA molecule, (i.e., it remains open-ended and protected from nuclease digestion by protected nucleotides), to form a covalently partially closed DNA product.

[0373] The invention provides a method for producing a covalently partially closed DNA product, wherein the method comprises: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides, wherein (i) the first adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the first end of the linear double-stranded region and (ii) the second adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the second end of the linear double-stranded region; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

[0374] The invention provides a method for producing a covalently partially closed DNA product, wherein the method comprises: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease resistant nucleotides, wherein (i) the first adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the first end of the linear double-stranded region and (ii) the second adaptor molecule comprises an overhang that is complementary to and anneals to an overhang at the second end of the linear double-stranded region, and wherein the first adaptor molecule is ligated to the first end of the linear double-stranded region and the second adaptor molecule is ligated to the second end of the linear double-stranded region; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences.

[0375] The first adaptor molecule and / or the second adaptor molecule may not be a plasmid or a vector DNA.

[0376] The first adaptor molecule comprises a truncated protelomerase target sequence, which may be a truncated TelN protelomerase target sequence. The truncated protelomerase target sequence may comprise a partial telR sequence. The truncated protelomerase target sequence may comprise a partial telR sequence and a partial telO sequence. The first adaptor molecule may comprise 10, 11, 12, 13, 14, 15, 16 17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 base pairs of a telR sequence. Preferably, the adaptor comprises at least 21 base pairs of a telR sequence. The truncated protelomerase target sequence may comprise a partial telL sequence. The truncated protelomerase target sequence may comprise a partial tell sequence and a partial telO sequence. The first adaptor molecule may comprise 10, 11, 12, 13, 14, 15, 16 17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 base pairs of a telL sequence. Preferably, the adaptor comprises at least 21 base pairs of a telL sequence.

[0377] The first and / or second adaptor molecules may comprise the same sequence or different sequences, i.e., the first adaptor molecule may comprise a different sequence in the overhang portion such that it only appends (by hybridization and / or ligation) to a first end of the digested double-stranded DNA molecule, and not to the second end.

[0378] The first and second adaptor molecules may comprise one or more phosphorothioated nucleotides, such that, once the adaptor molecules are appended (e.g. ligated) to the linear double-stranded region, the partially closed linear DNA product is resistant to nuclease digestion or has improved or enhanced resistance to nuclease digestion. The partially closed linear DNA product may be resistant to 3'-end exonuclease digestion (e.g. by exonuclease III) and / or 5'-end exonuclease digestion (e.g. by exonuclease VIII).

[0379] The adaptor molecule may comprise a plurality of phosphorothioated nucleotides. For example, the adaptor molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides in each strand.

[0380] The adaptor molecule may be a nucleic acid adaptor molecule. The adaptor molecule may be double-stranded. The adaptor molecule may comprise a portion that is double-stranded.

[0381] The first and / or second adaptor molecules may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 base pairs.

[0382] The first and / or second adaptor molecule may comprise a plurality of phosphorothioated nucleotides in each strand. For example, the adaptor molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides in each strand.

[0383] The adaptor molecule may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the adaptor molecules may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides at internal positions in each strand. Preferably, the adaptor molecule comprises at least 2 phosphorothioated nucleotides at internal positions in each strand.

[0384] The internal positions may not be located between the second and penultimate nucleotide of the adaptor molecule. The internal positions may be any position in the adaptor molecules other than the last nucleotide at the end of each strand.

[0385] The adaptor molecule may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of protected nucleotides.

[0386] Once the adaptor molecules are appended to the linear double-stranded region, the partially closed linear DNA product may comprise a protected nucleotide (e.g. phosphorothioated nucleotide) at the 5'-end (or at the 5'-end region) of one or both strands. Preferably, the partially closed linear DNA product comprises a phosphorothioated nucleotide at the 5'-end (or at the 5'-end region) of one strand. The partially closed linear DNA product may comprise a phosphorothioated nucleotide at the 5'-end (or at the 5'-end region) of one strand. As most exonucleases, for example exonuclease III, remove nucleotides from the 3'-end of the polynucleotide chain, the linear DNA product may comprise a protected nucleotide at the 3'-end (or at the 3'-end region) of one strand. Preferably, the partially closed linear DNA product comprises a phosphorothioated nucleotide at the 3'-end (or the 3'-end region) of one strand. The partially closed linear DNA product may comprise at least one phosphorothioated nucleotide at the 3'-end (or the 3'-end region) and at least one phosphorothioated nucleotide at the 5'-end (or the 5'-end region) of one strand. The partially closed linear DNA product may comprise at least one phosphorothioated nucleotide at the 3'-end (or the 3'-end region) of the sense strand and the 5'-end (or the 5'-end region) if the antisense strand. The partially closed linear DNA product may comprise at least one phosphorothioated nucleotide at the 5'-end (or the 5'-end region) of the sense strand and the 3'-end (or the 3'-end region) if the antisense strand. Thus, both the sense and antisense strands in the double stranded partially closed linear DNA product may be protected from nuclease digestion by using nuclease-resistant nucleotides at one end of the partially closed linear DNA product.

[0387] The first adaptor molecule and / or the second adaptor molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The double-stranded portion may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 base pairs.

[0388] The first adaptor molecule and / or the second adaptor molecule may comprise a 5' phosphate. The 5' phosphate may facilitate ligation to the linear double-stranded region (which may...

Claims

1. A method of producing a linear DNA product, wherein the method comprises the steps of: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, wherein the truncated protelomerase target sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequences.

2. A method of producing a closed linear DNA product, wherein the method comprises the steps of: (a) digesting the double-stranded DNA molecule with an endonuclease that cleaves the endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end, wherein the truncated protelomerase target sequences are non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

3. A method of producing a closed linear DNA product, wherein the method comprises the steps of: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, a truncated protelomerase sequence at a first end and a truncated protelomerase sequence at a second end, wherein the truncated protelomerase target sequences are non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule and wherein the second adaptor molecule comprises a truncated protelomerase target sequence that forms a second functional protelomerase target sequence with the truncated protelomerase sequence at the second end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first and second ends of the precursor double-stranded DNA molecule at the first and second functional protelomerase target sequences.

4. The method of any one of claims 1 to 3, wherein the first and / or the second adaptor molecules are nucleic acid adaptor molecules.

5. The method of claims 1 to 4, wherein the first adaptor molecule and / or the second adaptor molecule comprises a double stranded region with an overhang.

6. The method of claims 1 to 5, wherein the first adaptor molecule hybridizes to the first end of the digested double-stranded DNA molecule and the second adaptor hybridizes to the second end of the digested double-stranded DNA molecule.

7. The method of any one of claims claim 1 to 6, wherein steps (a)-(c) are performed in a single contiguous aqueous volume.

8. The method of anyone of claims 1 to 6, wherein steps (a)-(c) are performed sequentially in separate reactions.

9. The method of anyone of claims 1 to 8, wherein the first adaptor is ligated to the first end of the linear double-stranded region and the second adaptor is ligated to the second end of the linear double-stranded region.

10. The method of claims 1 to 9, wherein the endonuclease is a type II endonuclease, a blunt end endonuclease or a type IIS endonuclease.

11. The method of claim 10, wherein the endonuclease is a Type IIS restriction endonuclease, optionally wherein the endonuclease is Bbsl, Bsal, BsmBI, BspOI, BtgZI, Esp3I,SapI, Aarl, Acc361, AclWI, Acul, Ajul, AloI, Alw261, AlwI, Arsl, AsuHPI, Bael, Barl, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAl, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bse11, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAl, BsmFI, Bsml, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF51, BstMAI, BstV11, BstV21, Bsul, BtgZI, BtsCl, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam11041, Earl, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109I, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269I, NmeAIII, PaqCI, PciSI, Pctl, Plel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction endonuclease.

12. A method of producing a partially closed linear DNA product, wherein the method comprises the steps of: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, and wherein the truncated protelomerase sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule, wherein the truncated protelomerase target sequence of the first adaptor is non-functional and wherein the second adaptor molecule is a nucleic acid molecule that comprises one or more nuclease-resistant nucleotides; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the partially closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence.

13. The method of claim 12 wherein the one or more nuclease-resistant nucleotides are one or more phosphorothioated nucleotides.

14. A method of producing a closed linear DNA product, wherein the method comprises the steps of: (a) digesting a double-stranded DNA molecule with an endonuclease that cleaves an endonuclease target sequence to generate a digested double-stranded DNA molecule, wherein the digested double-stranded DNA molecule comprises a linear double-stranded region, and a truncated protelomerase sequence at a first end, and wherein the truncated protelomerase sequence is non-functional; (b) appending a first adaptor molecule to the first end of the digested double-stranded DNA molecule and appending a second adaptor molecule to the second end of the digested double-stranded DNA molecule to generate a precursor double-stranded DNA molecule, wherein the first adaptor molecule comprises a truncated protelomerase target sequence that forms a first functional protelomerase target sequence with the truncated protelomerase sequence at the first end of the digested double-stranded DNA molecule; and (c) incubating the precursor double-stranded DNA molecule with a protelomerase to generate the closed linear DNA product, wherein the protelomerase closes the first end of the precursor double-stranded DNA molecule at the first functional protelomerase target sequence, and the second end is closed by the second adaptor molecule.

15. The method of claim 14, wherein the second adaptor molecule comprises a hairpin.