Double-stranded circular DNA vector

Abstract

The present invention addresses the problem of providing a novel method for removing an unnecessary functional sequence contained in unnecessary linear covalently-closed DNA that is generated as a by-product during the production of target linear covalently-closed DNA.　Provided is a double-stranded circular DNA vector which includes a pair of protelomerase recognition sequences, wherein a first region that constitutes one of two regions constituting the double-stranded circular DNA vector between the pair of protelomerase recognition sequences includes two or more endonuclease recognition sequences and a functional sequence disposed between the two or more endonuclease recognition sequences, and a second region that constitutes the other of the two regions includes a target nucleic acid sequence and does not include the endonuclease recognition sequence.

Description

double-stranded circular DNA vector

[0001] The present invention relates to a double-stranded circular DNA vector, a transformed cell, a method for producing linear covalently closed DNA, a method for removing a functional sequence, and the like.

[0002] Double-stranded circular DNA vectors are widely used as DNA vectors that can be easily manipulated. In addition to a target gene sequence, double-stranded circular DNA vectors contain sequences necessary for maintaining and replicating a plasmid, such as a replication origin sequence and an antibiotic resistance gene. When a double-stranded circular DNA vector is introduced into animal tissues or cells, these sequences are also delivered together with the target gene sequence, which is undesirable from the viewpoint of avoiding immunogenicity.

[0003] Linear covalently closed DNA (LCC DNA) has been used as a means for introducing only a desired gene sequence into animal tissues or cells while avoiding the delivery of unnecessary sequences contained in double-stranded circular DNA vectors (Patent Document 1).

[0004] Linear covalently closed DNA is a linear double-stranded DNA whose termini are closed with hairpin structures. It can be produced by utilizing the activity of protelomerase, which forms hairpin structures at the cleaved termini when cleaving double-stranded DNA. For example, a double-stranded circular DNA vector containing a target gene sequence between a pair of protelomerase recognition sequences is prepared. Next, protelomerase is applied to this double-stranded circular DNA vector to produce a linear covalently closed DNA containing only the target gene sequence and whose termini are closed with hairpin structures. The linear covalently closed DNA produced in this manner has the advantages of being stable due to its resistance to exonucleases and having a small size that allows for high transfection efficiency into cells. Furthermore, the placement of protelomerase recognition sequences allows the removal of replication origins, antibiotic resistance genes, etc. from the linear covalently closed DNA containing the target gene sequence, thereby reducing the risk of immunogenicity, etc.

[0005] Patent Document 1 discloses a method for producing linear covalently closed DNA in a cell-free system. The method described in Patent Document 1 involves synthesizing double-stranded DNA molecules in a cell-free system using a DNA polymerase, followed by treatment with protelomerase to cleave the double-stranded DNA molecules, thereby producing linear covalently closed DNA. However, this method requires a complicated procedure for reacting various enzymes in a cell-free system. Furthermore, in the reaction that produces the desired linear covalently closed DNA containing the desired gene sequence, unwanted linear covalently closed DNA is simultaneously produced as a by-product. Various enzymes are required to degrade and remove this unwanted linear covalently closed DNA in a cell-free system, resulting in a high cost problem.

[0006] Therefore, a new method for removing unwanted linear covalently closed DNA generated as a by-product during the preparation of the desired linear covalently closed DNA is needed. In particular, sequences with specific functions, such as replication origins and antibiotic resistance genes, are thought to have a significant impact on product performance and safety, so reliable removal is required even in large-scale preparation.

[0007] U.S. Patent Publication No. 9,109,250

[0008] An object of the present invention is to provide a new method for removing unnecessary sequences contained in unnecessary linear covalently closed DNA that is generated as a by-product when preparing a desired linear covalently closed DNA.

[0009] In the reaction in which a double-stranded circular DNA vector is cleaved by protelomerase activity to generate linear covalently closed DNA, in addition to the desired linear covalently closed DNA containing the desired gene sequence, unnecessary linear covalently closed DNA containing unnecessary sequences (referred to as "functional sequences" in this specification) such as replication origins and antibiotic resistance genes are generated as by-products.

[0010] To solve the above-mentioned problems, the present inventors placed a pair of endonuclease recognition sequences on both sides of a functional sequence in a region that would result in unwanted linear covalently closed DNA produced as a by-product. This double-stranded circular DNA vector was cleaved with protelomerase and endonuclease, then dissociated into single strands, which were then rapidly hybridized and separated into two fractions: a soluble fraction and an insoluble fraction. As a result, they found that the desired linear covalently closed DNA was recovered as the soluble fraction, while the DNA strand containing the functional sequence was removed as the insoluble fraction. The present invention is based on the above findings and provides the following:

[0011] (1) A method for producing a linear covalently closed DNA, comprising: (a) a double-stranded circular DNA vector comprising a pair of protelomerase recognition sequences; a first region constituting one of two regions constituting a double-stranded circular DNA vector between the pair of protelomerase recognition sequences and comprising two or more endonuclease recognition sequences and a functional sequence disposed between the two or more endonuclease recognition sequences; and a second region constituting the other of the two regions and comprising a nucleic acid sequence of interest but not comprising the endonuclease recognition sequence; or (b) a linear double-stranded DNA comprising two or more protelomerase recognition sequences comprising a nucleic acid sequence of interest therebetween and two or more endonuclease recognition sequences comprising a functional sequence therebetween, the double-stranded circular DNA vector comprising a pair of protelomerase recognition sequences; a first region constituting one of two regions constituting a double-stranded circular DNA vector between the pair of protelomerase recognition sequences and comprising two or more endonuclease recognition sequences and a functional sequence disposed between the two or more endonuclease recognition sequences; The method includes a hybridization step of hybridizing single-stranded circular DNA derived from the linear covalently closed DNA obtained after the dissociation step, and a separation step of separating the linear covalently closed DNA after the hybridization step. (2) The method according to (1), in which the hybridization step is carried out within 30 seconds. (3) The method according to (1) or (2), in which the dissociation step and the hybridization step use alkaline denaturation and neutralization, respectively. (4) The method according to (3), in which the alkaline denaturation is carried out at a pH of 11 or higher by adding an alkaline aqueous solution. (5) The method according to (3) or (4), in which the alkaline denaturation is carried out in the presence of a surfactant. (6) The method according to any one of (3) to (5), in which the neutralization is carried out at a pH of 9 or lower by adding an acidic aqueous solution. (7) The method according to (1) or (2), in which the dissociation step and the hybridization step use heating to 95°C or higher and cooling to 4°C or lower, respectively. (8) The method according to any one of (1) to (7), wherein the separation step removes single-stranded DNA and / or looped fragments derived from the DNA fragments.(9) The method according to (8), wherein the removal involves centrifugation, filtration, precipitation, and / or sedimentation of an insoluble fraction containing single-stranded DNA derived from the DNA fragment, and / or ultrafiltration of the looped fragment. (10) The method according to any one of (1) to (9), wherein the production step involves using a transformed cell containing the double-stranded circular DNA vector and expressing the protelomerase or an active fragment thereof. (11) The method according to (10), wherein the transformed cell further expresses the endonuclease or an active fragment thereof. (12) The method according to (10) or (11), wherein the production step involves performing cleavage with the protelomerase or an active fragment thereof in the transformed cell, and then performing cleavage with the endonuclease or an active fragment thereof in a cell-free system. (13) The method according to any one of (1) to (9), wherein the production step is performed in a cell-free system. (14) The method according to any one of (1) to (13), wherein the functional sequence is 100 bases or more in length, and / or each of the two or more endonuclease recognition sequences is located within 100 bases from each of the pair of protelomerase recognition sequences. (15) A method for removing a functional sequence contained in linear covalently closed DNA, the linear covalently closed DNA containing two or more endonuclease recognition sequences and a functional sequence located between the two or more endonuclease recognition sequences, the method comprising: a cleaving step of cleaving the linear covalently closed DNA with the endonuclease or an active fragment thereof, a dissociation step of dissociating the DNA fragments containing the functional sequence obtained after the cleaving step into single strands by alkali denaturation or heating to 95°C or higher, an insoluble fraction formation step of forming an insoluble fraction by neutralizing or cooling to 4°C or lower the single-stranded DNA derived from the DNA fragments obtained by alkali denaturation or heating, respectively, and a removal step of removing the insoluble fraction. (16) The method according to (15), wherein the cleaving step is performed in the presence of yet another linear covalently closed DNA that does not contain the endonuclease recognition sequence, and the yet another linear covalently closed DNA is separated by the removal step.(17) The method according to (15) or (16), wherein the functional sequence is 100 bases or more in length and / or each of the two or more endonuclease recognition sequences is located within 100 bases from each of the covalently closed ends of the linear covalently closed DNA. (18) A double-stranded circular DNA vector comprising a pair of protelomerase recognition sequences, wherein a first region constituting one of two regions constituting the double-stranded circular DNA vector between the pair of protelomerase recognition sequences comprises two or more endonuclease recognition sequences and a functional sequence located between the two or more endonuclease recognition sequences, and a second region constituting the other of the two regions comprises a nucleic acid sequence of interest but does not comprise the endonuclease recognition sequence. (19) The double-stranded circular DNA vector according to (18), wherein the functional sequence comprises a gene sequence and / or an untranslated sequence. (20) The double-stranded circular DNA vector according to (19), wherein the gene is a selection marker gene and / or an enzyme gene. (21) The double-stranded circular DNA vector according to (20), wherein the selection marker gene is an antibiotic resistance gene and / or an auxotrophy complementation gene. (22) The double-stranded circular DNA vector according to (20) or (21), wherein the enzyme gene is a gene sequence encoding the protelomerase or an active fragment thereof and / or a gene sequence encoding the endonuclease or an active fragment thereof, both of which are placed under the control of an expression-controllable promoter. (23) The double-stranded circular DNA vector according to any of (19) to (22), wherein the non-translated sequence is a nucleic acid sequence encoding a replication origin and / or a non-translated RNA. (24) The double-stranded circular DNA vector according to any of (18) to (23), wherein the functional sequence is 100 bases or more in length. (25) The double-stranded circular DNA vector according to any one of (18) to (24), wherein the two or more endonuclease recognition sequences are the same or different from each other. (26) The double-stranded circular DNA vector according to any one of (18) to (25), wherein the target nucleic acid sequence comprises a target gene sequence encoding a target protein or a fragment thereof.(27) The double-stranded circular DNA vector according to any one of (18) to (26), wherein each of the two or more endonuclease recognition sequences is located within 100 base lengths from each of the pair of protelomerase recognition sequences. (28) The double-stranded circular DNA vector according to any one of (18) to (27), wherein the protelomerase is TelN protelomerase, TelA protelomerase, or TelK protelomerase. (29) The double-stranded circular DNA vector according to any one of (18) to (28), wherein the endonuclease is a homing endonuclease. (30) A single-stranded DNA selected from the two DNA strands constituting the double-stranded circular DNA vector according to any one of (18) to (29). (31) A transformed cell comprising the double-stranded circular DNA vector according to any one of (18) to (29). This specification includes the disclosure of Japanese Patent Application No. 2024-139220, from which the present application claims priority.

[0012] According to the present invention, a new method is provided for removing unnecessary sequences contained in unnecessary linear covalently closed DNA that is generated as a by-product when preparing a desired linear covalently closed DNA.

[0013] This diagram shows a schematic diagram of a method for removing unnecessary sequences (functional sequences) contained in linear covalently closed DNA. Figure 1A shows that when an I-SceI recognition sequence located on only one side of a functional sequence is cleaved, double-stranded DNA containing the functional sequence is recovered by dissociating the cleavage product into single strands and then hybridizing. Figure 1B shows that when I-SceI recognition sequences located on both sides of a functional sequence are cleaved, the cleaved functional sequence is removed during the dissociation into single strands and hybridization steps. The structures of the S1 and S2 vectors are shown, along with the results of alkaline denaturation and neutralization treatments after cleavage of the S1 and S2 vectors in E. coli. Figure 2A shows the structure of the S1 vector and the results after alkaline denaturation and neutralization treatments. Figure 2B shows the structure of the S2 vector and the results after alkaline denaturation and neutralization treatments. The results of HindIII cleavage, alkaline denaturation, and neutralization treatments were performed in a cell-free system on linear covalently closed DNA containing two HindIII recognition sequences. Figure 3A shows the results of electrophoresis after various treatments. Figure 3B shows that uncleaved linear covalently closed DNA is not removed by alkaline denaturation and neutralization treatment (upper panel), and that target sequences cleaved with HindIII and lacking covalently closed ends at both ends are removed by alkaline denaturation and neutralization treatment (lower panel). The electrophoresis results after various treatments are shown for cases where protelomerase cleavage, I-SceI endonuclease cleavage, alkaline denaturation, and neutralization treatment were all performed in a cell-free system.

[0014] 1. Double-stranded circular DNA vector 1-1. Overview A first aspect of the present invention is a double-stranded circular DNA vector. The double-stranded circular DNA vector of this aspect includes a pair of protelomerase recognition sequences, and between the pair of protelomerase recognition sequences, includes a first region including a functional sequence and a second region including a nucleic acid sequence of interest. The double-stranded circular DNA vector of this aspect allows for the simple and efficient removal of the functional sequence, making it possible to simply and efficiently prepare linear covalently closed DNA including a nucleic acid sequence of interest.

[0015] 1-2. Definition of Terms The following terms frequently used in this specification are defined. As used herein, "linear covalently closed DNA (LCC DNA)" refers to linear double-stranded DNA whose ends are closed with a hairpin structure. At each end of linear covalently closed DNA, the 5' and 3' ends of two DNA strands are linked to form a hairpin structure. In linear covalently closed DNA, the end portions protected by the hairpin structure are resistant to degradation by exonucleases. Note that linear covalently closed DNA becomes single-stranded circular DNA when the double strands are dissociated by alkali denaturation, heat denaturation, or the like. This single-stranded circular DNA can return to the original linear covalently closed DNA when it self-hybridizes to form a double strand.

[0016] As used herein, "protelomerase" refers to a polypeptide that has the activity of recognizing a specific sequence in a double-stranded DNA molecule, cleaving the double-stranded DNA molecule at a position within or near the recognition sequence, and linking the 5' and 3' ends of the cleaved ends to form a covalently closed end (hereinafter referred to as "protelomerase activity"). As used herein, the type of protelomerase is not particularly limited as long as it has the above activity. For example, protelomerase derived from Agrobacterium fabrum (TelA protelomerase, ACCESSION No. AAK88254), protelomerase derived from Halomonas virus HAP1 (ACCESSION No. ABY90402), protelomerase derived from Vibrio virus VP882 (ACCESSION No. ABM73418), protelomerase derived from Klebsiella phage phiKO2 (TelK protelomerase, ACCESSION No. AAR83042), protelomerase derived from Rhizobium pusense (ACCESSION No. AAR83043), and protelomerase derived from Rhizobium pusense (ACCESSION No. AAR83044). Examples of the protelomerase include protelomerase derived from a Feldmannia species virus (ACCESSION No. ACH46812), protelomerase derived from Vibrio phage vB_VpaM_MAR (ACCESSION No. AFV81380), protelomerase derived from Yersinia phage PY54 (Tel protelomerase, ACCESSION No. CAD91792), protelomerase derived from Escherichia virus N15 (TelN protelomerase, ACCESSION No. AAB81106), or a mutant of any of the above protelomerases. In this specification, protelomerase is not included in the endonucleases described below.

[0017] As used herein, an "active fragment" of protelomerase refers to a polypeptide fragment that contains a partial region of protelomerase and retains protelomerase activity, e.g., 1% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or equivalent thereof, of the activity of the full-length protein. The length of the amino acids constituting the polypeptide is not particularly limited. For example, the active fragment may be a region of at least 100, 150, 200, 250, 300, 350, or 400 consecutive amino acids that includes the catalytic domain of protelomerase.

[0018] As used herein, a "protelomerase gene" refers to a gene that encodes a protelomerase or an active fragment thereof. Examples of protelomerase genes include genes that encode any of the above-mentioned protelomerases or active fragments thereof. Examples include a protelomerase gene that encodes a TelN protelomerase consisting of the amino acid sequence shown in SEQ ID NO: 2, and a TelN protelomerase gene consisting of the nucleotide sequence shown in SEQ ID NO: 1.

[0019] As used herein, the term "protelomerase recognition sequence" refers to a sequence recognized by protelomerase. Protelomerase recognizes the protelomerase recognition sequence, cleaves the double-stranded DNA molecule at a position within or near the protelomerase recognition sequence, and rejoins the cleaved ends. Protelomerase recognition sequences are known to be palindromic or palindrome-like sequences. A pair of sequences constituting a palindromic or palindrome-like sequence is sometimes referred to as "telR" and "telL," and a palindrome or palindrome-like sequence composed of telR and telL is sometimes referred to as "telRL." In this case, "protelomerase recognition sequence" refers to a palindrome or palindrome-like sequence composed of telR and telL, and "a pair of protelomerase recognition sequences" refers to a pair (i.e., two sets) of telRL.

[0020] As used herein, the term "endonuclease" refers to an enzyme that has the activity of cleaving phosphodiester bonds within a nucleotide chain. Among endonucleases, endonucleases that recognize a specific sequence and cleave double-stranded DNA molecules within or near that sequence are specifically called restriction enzymes. Restriction enzymes include naturally occurring restriction enzymes, as well as artificial restriction enzymes such as TALEN and zinc finger nucleases (ZFNs), genome editing enzymes such as Cas9 proteins, or mutants of any of the above endonucleases. The endonuclease may be either an enzyme that has the activity of cleaving two DNA strands, or an enzyme (nickases) that cleaves only one DNA strand.

[0021] As used herein, any type of "restriction enzyme" known in the art can be used. Specific examples of restriction enzymes include, but are not limited to, AccI, AgeI, AfaI, ApaI, ApaLI, AseI, BamHI, BglI, BglIII, BmtI, DraI, DpnI, EcoRI, EcoRV, HindIII, KpnI, NaeI, NcoI, NdeI, NheI, NotI, NruI, NsiI, PvuI, PvuII, PsI, SacI, SacII, SalI, SbfI, ScaI, SmaI, SmiI, SnaBI, SpeI, SphI, XbaI, XhoI, and XmaI. The restriction enzyme may be a modified enzyme such as a high-fidelity (HF) restriction enzyme that has improved specificity for the restriction enzyme recognition sequence.

[0022] Among restriction enzymes, those found in inteins, introns, etc. are particularly called "homing endonucleases." Homing endonucleases are generally known to recognize long recognition sequences of 12 to 40 bp, and it is also known that the recognition sequence is not a palindromic sequence. In this specification, the type of homing endonucleases is not particularly limited. For example, I-SceI (ACCESSION No. NP009324), I-CeuI (ACCESSION No. CAA78934), I-CreI (ACCESSION No. No. CAA26008), I-AniI, I-ChuI, I-DmoI, I-CsmI, PI-SceI, PI-TIiI, PI-MtuI, I-SceII, I-SceIII, HO, PI-CivI, PI-Ct rI, PI-AaeI, PI-BsuI, PI-DhaI, PI-DraI, PI-MavI, PI-MchI, PI-MfuI, PI-MflI, PI-MgaI, PI-MgoI, PI-MinI, PI-M Examples of homing endonucleases include kaI, PI-MmaI, PI-MshI, PI-MsmI, PI-MsoI, PI-MthI, PI-MtuI, PI-MxeI, PI-NpuI, PI-PfuI, PI-RmaI, PI-SpbI, PI-SspI, PI-FacI, PI-MjaI, PI-PhoI, PI-TagI, PI-ThyI, PI-TkoI, PI-TspI, or a mutant of any of the above homing endonucleases. Among these, I-SceI, I-ChuI, I-DmoI, I-CreI, I-CsmI, PI-PfuI, PI-SceI, PI-TliI, I-MsoI, PI-MtuI, I-CeuI, I-SceII, I-SceIII, HO, or a mutant of any of the above homing endonucleases is preferred, and I-SceI, I-ChuI, I- DmoI, I-CreI, I-CsmI, PI-SceI, PI-PfuI, PI-TliI, I-MsoI, PI-MtuI, I-CeuI, or a mutant of any of the above homing endonucleases is more preferred, and I-SceI, I-CeuI, I-CreI, or a mutant of any of the above homing endonucleases is even more preferred.

[0023] As used herein, an "active fragment" of an endonuclease refers to a polypeptide fragment that contains a partial region of the endonuclease and retains endonuclease activity, for example, 1% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or equivalent thereof, of the activity of the full-length protein. The length of the amino acids constituting the polypeptide is not particularly limited. For example, the length may be at least 100, 150, 200, 250, 300, 350, or 400 consecutive amino acids that comprise the catalytic domain of the endonuclease.

[0024] As used herein, an "endonuclease gene" refers to a gene that encodes an endonuclease or an active fragment thereof. Examples of endonuclease genes include genes that encode any of the above-mentioned endonucleases or active fragments thereof. Examples include a gene that encodes an I-SceI homing endonuclease consisting of the amino acid sequence shown in SEQ ID NO: 4, and an I-SceI homing endonuclease gene consisting of the nucleotide sequence shown in SEQ ID NO: 3.

[0025] As used herein, the term "endonuclease recognition sequence" refers to a sequence recognized by an endonuclease, which recognizes the endonuclease recognition sequence and cleaves a double-stranded DNA molecule at a position within or near the endonuclease recognition sequence.

[0026] In the present invention, a "double-stranded circular DNA vector" refers to a circular double-stranded DNA molecule that can be maintained and / or replicated in a cell. The double-stranded circular DNA vector may contain sequences necessary for intracellular maintenance and replication, such as a replication origin and / or a gene encoding an antibiotic resistance protein. The double-stranded circular DNA vector may be, for example, a plasmid vector or a bacmid vector. The vector may also be a shuttle vector that can replicate between bacteria such as E. coli and mammalian cells.

[0027] 1-3. Configuration The double-stranded circular DNA vector of this embodiment contains a pair of protelomerase recognition sequences as essential components.

[0028] The type of the pair of protelomerase recognition sequences contained in the double-stranded circular DNA vector of this embodiment is not particularly limited, and may be any sequence other than the pair of protelomerase recognition sequences in the double-stranded circular DNA vector, particularly a sequence not contained in the nucleic acid sequence of interest. The length of the protelomerase recognition sequence is not limited, and may be 5 bp or more, 6 bp or more, 7 bp or more, 8 bp or more, 9 bp or more, 10 bp or more, 15 bp or more, 20 bp or more, 25 bp or more, 30 bp or more, 35 bp or more, 40 bp or more, 45 bp or more, 50 bp or more, 55 bp or more, 60 bp or more, 65 bp or more, 70 bp or more, 80 bp or more, or 90 bp or more. Particularly preferred examples of protelomerase recognition sequences include sequences recognized by TelN protelomerase, TelA protelomerase, or TelK protelomerase, or sequences recognized by mutants thereof. More specifically, examples of such sequences include a base sequence recognized by TelA protelomerase (SEQ ID NO: 5), a base sequence recognized by protelomerase derived from Halomonas virus HAP1 (SEQ ID NO: 6), a base sequence recognized by protelomerase derived from Vibrio virus VP882 (SEQ ID NO: 7), a base sequence recognized by TelK protelomerase (SEQ ID NO: 8), a base sequence recognized by protelomerase derived from Rhizobium pusense, a base sequence recognized by protelomerase derived from Feldmannia species virus, a base sequence recognized by protelomerase derived from Vibrio phage vB_VpaM_MAR, a base sequence recognized by Tel protelomerase (SEQ ID NO: 9), and a base sequence recognized by TelN protelomerase (telRL sequence) (SEQ ID NO: 10).

[0029] Herein, the two regions that constitute the entire double-stranded circular DNA vector of this embodiment between a pair of protelomerase recognition sequences are referred to as the "first region" and the "second region," respectively. In the double-stranded circular DNA vector of this embodiment, the first region contains two or more endonuclease recognition sequences, and a functional sequence is contained between these two or more endonuclease recognition sequences. The second region is characterized by containing a nucleic acid sequence of interest but not containing the same recognition sequence as the endonuclease recognition sequences.

[0030] In the double-stranded circular DNA vector of this embodiment, the two or more endonuclease recognition sequences contained in the first region may be any sequences that are not contained in the sequence of the second region, particularly the nucleic acid sequence of interest. Examples of endonuclease recognition sequences include restriction enzyme recognition sequences, TALEN recognition sequences, ZFN recognition sequences, and CRISPR / Cas9 recognition sequences.

[0031] In one embodiment, the two or more endonuclease recognition sequences may be homing endonuclease recognition sequences. Examples of homing endonucleases include, but are not limited to, I-SceI, I-CeuI, I-CreI, I-AniI, I-ChuI, I-DmoI, I-CsmI, PI-SceI, PI-TIiI, PI-MtuI, I-CeuI, I-SceII, I-SceIII, HO, PI-CivI, PI-CtrI, PI-AaeI, PI-BsuI, PI-DhaI, PI-DraI, PI-MavI, PI-MchI, PI-MfuI, PI-M Examples of PIs include flI, PI-MgaI, PI-MgoI, PI-MinI, PI-MkaI, PI-MkaI, PI-MmaI, PI-MshI, PI-MsmI, PI-MsoI, PI-MthI, PI-MtuI, PI-MxeI, PI-NpuI, PI-PfuI, PI-RmaI, PI-SpbI, PI-SspI, PI-FacI, PI-MjaI, PI-PhoI, PI-TagI, PI-ThyI, PI-TkoI, PI-TspI, or mutants thereof. The mutant is not particularly limited and can be appropriately selected depending on the purpose. For example, the homology to the original amino acid sequence may be 80% or more, 90% or more, 95% or more, or 99% or more. Specific examples of homing endonuclease recognition sequences include the I-SceI recognition sequence (SEQ ID NO: 11), the I-CeuI recognition sequence (SEQ ID NO: 12), and the I-CreI recognition sequence (SEQ ID NO: 13). Homing endonucleases have long, asymmetric recognition sequences of, for example, 12 to 40 bp, and are unlikely to be contained in the genome of a host such as E. coli. Therefore, they have the advantage of not cleaving the genome of a host such as E. coli and not adversely affecting the growth or cultivation of E. coli.

[0032] In the double-stranded circular DNA vector of this embodiment, the number of endonuclease recognition sequences contained in the first region may be two or more, for example, two, three, four, five or more.

[0033] The two or more endonuclease recognition sequences contained in the first region may be sequences recognized by the same endonuclease (e.g., endonuclease recognition sequences consisting of the same base sequence), or may be sequences recognized by different endonucleases (e.g., endonuclease recognition sequences consisting of different base sequences).

[0034] As used herein, the term "functional sequence" refers to any sequence other than the nucleic acid sequence of interest. More specifically, it refers to a sequence having any function, such as a sequence that functions in the maintenance or replication of a vector, a sequence involved in the generation of linear covalently closed DNA, and / or a sequence necessary for the removal of the functional sequence itself, and which is desirably removed when generating linear covalently closed DNA containing the nucleic acid sequence of interest, as described below. In this specification, it may also be referred to as an "unnecessary sequence," etc.

[0035] The length of the functional sequence is not limited, as long as it can be cleaved by the endonuclease recognition sequences located on both ends thereof and then removed by the dissociation and separation steps of the method of the present invention, as described below. For example, it may be 100 or more bases long, 200 or more bases long, 300 or more bases long, 400 or more bases long, 500 or more bases long, 1,000 or more bases long, or 2,000 or more bases long, and / or 20,000 or less bases long, 15,000 or less bases long, 12,000 or less bases long, 10,000 or less bases long, or 5,000 or less bases long. Exemplary ranges include 100 to 20,000 bases long, 500 to 10,000 bases long, 1,000 to 5,000 bases long, or 2,000 to 4,000 bases long.

[0036] In one embodiment, the functional sequence comprises a gene sequence and / or an untranslated sequence.

[0037] The gene sequence contained in the functional sequence is not particularly limited and may have any function, such as a selection marker gene and / or an enzyme gene.

[0038] For example, the selectable marker gene may be an antibiotic resistance gene and / or an auxotrophy-complementing gene. Specific examples of antibiotic resistance genes include the β-lactamase gene (sometimes referred to as the "ampR gene") that confers ampicillin resistance, the aminoglycoside 3' phosphotransferase gene (sometimes referred to as the "kanR gene") that confers kanamycin resistance, the tetracycline efflux transporter gene that confers tetracycline resistance, and the CAT (chloramphenicol acetyltransferase) gene that confers chloramphenicol resistance.

[0039] As used herein, the term "auxotrophic complementary gene" refers to a gene capable of complementing the growth of an auxotrophic strain. The auxotrophic strain herein is not limited to any particular type, as long as it does not grow or grows at a low rate in a medium that does not sufficiently contain a particular medium component. Specific examples of auxotrophy include amino acid auxotrophy, vitamin auxotrophy, and nucleic acid auxotrophy. Examples of amino acid auxotrophy include methionine auxotrophy, leucine auxotrophy, cysteine ​​auxotrophy, histidine auxotrophy, and arginine auxotrophy. Examples of vitamin auxotrophy include biotin auxotrophy and thiamine auxotrophy. Examples of nucleic acid auxotrophy include uracil auxotrophy, adenine auxotrophy, and thymine auxotrophy. The specific type of auxotrophic complementary gene can be appropriately selected based on these auxotrophic requirements. Specific examples include a methionine synthase gene, a leucine synthase gene, a thiamine synthase gene, a uracil synthase gene, and an adenine synthase gene.

[0040] The type of enzyme gene is not limited herein, and examples include the antibiotic resistance genes and auxotrophy complementation genes described above, as well as genes encoding protelomerase and / or genes encoding endonuclease. The protelomerase or active fragment thereof encoded by the protelomerase gene preferably recognizes and cleaves a pair of protelomerase recognition sequences contained in the double-stranded circular DNA vector of this embodiment. Furthermore, the endonuclease or active fragment thereof encoded by the endonuclease gene preferably recognizes and cleaves two or more endonuclease recognition sequences contained in the double-stranded circular DNA vector of this embodiment. The protelomerase gene and endonuclease gene are preferably placed under the control of an expression-controllable promoter.

[0041] As used herein, the term "expression-controllable promoter" refers to a gene expression regulatory region that can control the expression of a gene or the like located downstream (on the 3' end) in a cell into which a double-stranded circular DNA vector has been introduced, and is a promoter that can induce the expression of a target gene or the like in the cell at any time. An expression-controllable promoter can be induced by conditions such as the presence or absence of a specific factor or temperature. There are no limitations on the type of expression-controllable promoter, and it can be selected appropriately depending on the purpose. Examples of such promoters include heat-inducible promoters (lambda PR, lambda PL, etc.), arabinose-inducible promoters (araBAD promoter, etc.), IPTG-inducible promoters (LAC promoter, LACUV5 promoter, TAC promoter, Trc promoter, LPP promoter, etc.), T7 promoter, cold-inducible promoters (cspA promoter, etc.), Trp promoter, rhamnose-inducible promoter (rhaT promoter, etc.), proU promoter, prpB promoter, phoA promoter, recA promoter, tetA promoter, cadA promoter, or variants thereof, with IPTG-inducible promoters and arabinose-inducible promoters being preferred.

[0042] As used herein, the phrase "placed under the control of a promoter whose expression can be controlled" means that a gene to be expressed is placed downstream of the promoter whose expression can be controlled and is under the control of the promoter.

[0043] In one embodiment, when the functional sequence includes both a protelomerase gene sequence and an endonuclease gene sequence, the protelomerase gene sequence and the endonuclease gene sequence may be placed under the control of the same promoter. For example, the protelomerase gene sequence and the endonuclease gene sequence may be a fusion gene sequence encoding a fusion polypeptide containing a protelomerase and an endonuclease. Alternatively, a gene sequence encoding a self-cleaving peptide such as a 2A peptide may be placed between the protelomerase gene sequence and the endonuclease gene sequence.

[0044] Examples of gene sequences that do not fall under the category of selection marker genes or enzyme genes include sequences that encode activator or repressor proteins such as the LacI gene and the AraC gene.

[0045] As used herein, the term "non-translated sequence" refers to any sequence that does not encode an amino acid sequence such as a peptide or protein. Non-translated sequences are sequences that function as, for example, DNA or RNA sequences, and specific examples thereof include nucleic acid sequences that encode replication origin regions and / or non-translated RNA.

[0046] As used herein, the term "replication origin" refers to a sequence region capable of initiating DNA replication initiation. For example, a sequence region that enables DNA replication in a double-stranded circular DNA vector such as a plasmid DNA can be mentioned. Various base sequences are known as replication origin regions for double-stranded circular DNA vectors that can replicate in E. coli. Based on the base sequence of the replication origin region and / or the replication mechanism, they are classified into ColE1-type plasmid DNA (e.g., pUC-type plasmid DNA and pET-type plasmid DNA), p15A-type plasmid DNA (e.g., pACYC-type plasmid DNA), pSC101-type plasmid DNA, R6K-type plasmid DNA, etc.; however, the replication origin in this embodiment may be any of these.

[0047] The non-translated RNA may be, for example, an RNA selection marker that enables the selection of transformed cells. With the RNA selection marker, the non-translated RNA or non-coding RNA transcribed from the nucleic acid sequence regulates the expression of a target gene from the chromosome, thereby enabling the selection of transformed cells. Specific examples of RNA selection markers include antisense RNA, siRNA, suppressor tRNA, RNA-OUT (WO 2008 / 153733), RNA II precursor, mature RNA II, RNA I (antisense RNA of RNA II), and the like. Non-translated RNA is also called non-coding RNA (ncRNA).

[0048] Further examples of non-coding sequences include (multiple) cloning sites, overlapping regions for use in Clontech's In-Fusion cloning system, New England Biolabs' Gibson Assembly system, and the like.

[0049] In the double-stranded circular DNA vector of this embodiment, the second region contains the nucleic acid sequence of interest as described above, but does not contain an endonuclease recognition sequence contained in the first region. As used herein, the phrase "does not contain an endonuclease recognition sequence contained in the first region" or "does not contain the endonuclease recognition sequence" means that the second region does not contain any recognition sequences identical to the two or more endonuclease recognition sequences located on both sides of the functional sequence described above in the first region. In other words, the double-stranded circular DNA vector of this embodiment contains two or more endonuclease recognition sequences in the first region that are not contained in the second region.

[0050] As used herein, the term "nucleic acid sequence of interest" is not particularly limited. The nucleic acid sequence of interest is not limited to a sequence encoding a protein or a fragment thereof, or RNA, and may be, for example, a sequence that can function as a nucleic acid drug, or a sequence that can be used to introduce a nucleic acid sequence of interest. Specific examples of nucleic acid sequences include, but are not limited to, gene sequences encoding proteins or fragments thereof; nucleic acid sequences encoding non-coding RNA such as siRNA, shRNA, miRNA, lncRNA, or ribozymes; or nucleic acid drugs such as antisense nucleic acids, aptamers, or bait nucleic acids. Examples of sequences that can be used to introduce a nucleic acid sequence of interest include sequences that can be recognized / cleaved by nucleases such as restriction enzymes and genome editing enzymes, and multicloning sites containing sequences that can be recognized / cleaved by two or more nucleases.

[0051] In one embodiment, the nucleic acid sequence of interest includes a gene sequence of interest encoding a protein of interest or a fragment thereof. As used herein, "gene sequence of interest" refers to a gene sequence encoding a protein of interest or a fragment thereof, or an RNA molecule of interest. The protein of interest or a fragment thereof is not particularly limited and can be selected appropriately depending on the purpose. Examples include polypeptides constituting viruses, polypeptides produced by animals, plants, fungi, algae, bacteria, viruses, etc., and fragments thereof. These can be used as cell / gene therapy drugs, vaccines, disease therapy drugs, etc. The polypeptides produced by animals, plants, fungi, algae, bacteria, viruses, etc. are not particularly limited and can be selected appropriately depending on the purpose. For example, enzymes such as phytase, amylase, glucosidase, cellulase, lipase, protease, glutaminase, peptidase, oxidase, lactase, xylanase, trypsin, pectinase, and isomerase; antibody-binding proteins such as protein A, protein G, and protein L; human antibodies, humanized antibodies, chimeric antibodies, llama antibodies, alpaca antibodies, single-chain antibodies, heavy-chain antibodies, multivalent antibodies; Fab, F(ab'), F(ab') ２ , Fc, Fc fusion protein, bispecific antibody, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc(Fv) ２ disulfide-linked Fv (sdFv), diabodies, antibodies or antibody fragments such as antibody-like molecule targeting peptides (microantibodies), and other functional proteins, conjugates of compounds other than antibodies or antibody fragments with antibody fragments (antibody-like molecules), serum albumins such as human serum albumin, epidermal growth factors such as human epidermal growth factor, insulin, growth hormone, erythropoietin, interferon, blood coagulation factor VIII, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin, IL-1, IL-6, tissue plasminogen activator (TPA), urokinase, leptin, stem cell growth factor (SCF), fibroin, fluorescent proteins, hepatitis B virus surface antigen, hirudin, and the like.

[0052] A nucleic acid sequence of interest, such as a gene sequence of interest, can include a promoter in addition to the nucleic acid sequence of interest (e.g., a sequence encoding a protein of interest or a fragment thereof), and may also include further components such as introns, enhancers, terminators, and / or polyA signals, as needed.

[0053] The base length of the target nucleic acid sequence is not particularly limited as long as it can be accommodated in a double-stranded circular DNA vector. For example, it may be 6 bases or more, 10 bases or more, 20 bases or more, 50 bases or more, 100 bases or more, 200 bases or more, 500 bases or more, 1,000 bases or more, 2,000 bases or more, 5,000 bases or more, 10,000 bases or more, 12,000 bases or more, 15,000 bases or more, or 20,000 bases or more, and / or 30,000 bases or less, 25,000 bases or less, 20,000 bases or less, 15,000 bases or less, 12,000 bases or less, 10,000 bases or less, or 5,000 bases or less. Exemplary ranges include 5,000 to 30,000 bases in length, 10,000 to 25,000 bases in length, 12,000 to 20,000 bases in length, or 15,000 to 18,000 bases in length.

[0054] In the double-stranded circular DNA vector of this embodiment, the two or more endonuclease recognition sequences contained in the first region may be arranged adjacent to one or both of the protelomerase recognition sequences, or a spacer sequence may be arranged between one or both of the protelomerase recognition sequences.

[0055] As used herein, the term "spacer sequence" refers to a sequence inserted between each of two or more endonuclease recognition sequences contained in the first region of the double-stranded circular DNA vector of this embodiment and each of a pair of protelomerase recognition sequences. When the double-stranded circular DNA vector of this embodiment is cleaved with a protelomerase and an endonuclease, a nucleic acid fragment consisting of this spacer sequence (i.e., a loop-shaped fragment, as described below) can also be generated. Therefore, the spacer sequence is preferably a short sequence. The spacer sequence is, for example, 1,000 bases or less, 500 bases or less, 400 bases or less, 300 bases or less, or 200 bases or less in length, and preferably 100 bases or less, 90 bases or less, 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less, 40 bases or less, 30 bases or less, 20 bases or less, or 10 bases or less in length. Exemplary ranges include 1 to 300 bases in length, 1 to 250 bases in length, 1 to 200 bases in length, 1 to 150 bases in length, 1 to 100 bases in length, 1 to 70 bases in length, 1 to 50 bases in length, 1 to 40 bases in length, 1 to 30 bases in length, 1 to 20 bases in length, 1 to 10 bases in length, 2 to 10 bases in length, 3 to 10 bases in length, 4 to 9 bases in length, 5 to 9 bases in length, or 6 to 8 bases in length.

[0056] The method for producing the double-stranded circular DNA vector of this embodiment is not particularly limited and can be appropriately selected depending on the purpose. Examples include total synthesis, PCR, and methods using the In-Fusion cloning system from Clontech or the Gibson Assembly system from New England Biolabs. For example, the vector can be produced based on commercially available or known vectors, such as pUC vectors, pET vectors, and pGEM vectors. In particular, design based on a plasmid with a pUC-based ori, such as the pUC19 vector, is preferred in terms of high DNA replication efficiency. The double-stranded circular DNA vector of this embodiment can also be produced by adding a pair of protelomerase recognition sequences, two or more endonuclease recognition sequences, and the nucleic acid sequence of interest to these commercially available vectors.

[0057] 1-4. Effect: When the double-stranded circular DNA vector of this embodiment is cleaved with protelomerase or an active fragment thereof and an endonuclease or an active fragment thereof, linear covalently closed DNA containing the nucleic acid sequence of interest is generated from the second region, and DNA fragments containing the functional sequence contained in the first region and lacking hairpin structures at either end are generated. The linear covalently closed DNA and DNA fragments thus obtained are dissociated into single strands, and then only the single-stranded circular DNA derived from the linear covalently closed DNA is (self-)hybridized to return it to a double-stranded state. This allows the linear covalently closed DNA to be recovered as a soluble fraction, while the single-stranded DNA derived from the DNA fragments can be efficiently removed as an insoluble fraction.

[0058] According to the double-stranded circular DNA vector of this embodiment, a transforming composition containing the double-stranded circular DNA vector is also provided. The transforming composition of the present invention may contain a solvent and / or an additive as optional components. By transforming a host cell with the transforming composition of the present invention, a transformed cell of the third embodiment described below can be produced.

[0059] Furthermore, the double-stranded circular DNA vector of this embodiment also provides a transformation kit containing the double-stranded circular DNA vector. The transformation kit of the present invention may optionally contain other reagents required for transformation, such as a medium, a buffer, a transformation reagent, an antibiotic used to select transformed cells, and / or an instruction manual describing the transformation method.

[0060] 2. Single-stranded DNA The second aspect of the present invention is a single-stranded DNA. The single-stranded DNA of this aspect is selected from the two DNA strands that constitute the double-stranded circular DNA vector described in the first aspect.

[0061] The single-stranded DNA of this embodiment may be either circular or linear, and may be, for example, a single-stranded circular DNA obtained by single-stranding the double-stranded circular DNA vector described in the first embodiment, a single-stranded linear DNA obtained by cleaving a single-stranded circular DNA, or a DNA strand synthesized using a single-stranded circular DNA or a single-stranded linear DNA as a template.

[0062] In one embodiment, the single-stranded DNA of this aspect is a sense strand. In this aspect, the "sense strand" refers to the strand that encodes a protein, non-translated RNA, or the like when the nucleic acid sequence of interest contained in the second region of the double-stranded circular DNA vector includes a gene encoding a protein, non-translated RNA, or the like.

[0063] In another embodiment, the single-stranded DNA of this aspect is an antisense strand. In this aspect, the "antisense strand" refers to a strand having a base sequence complementary to a strand encoding a protein, non-translated RNA, or the like, when the nucleic acid sequence of interest contained in the second region of the double-stranded circular DNA vector includes a gene encoding a protein, non-translated RNA, or the like.

[0064] The single-stranded DNA of this embodiment is used as a template for synthesis with a DNA polymerase, and the complementary strand of the resulting DNA strand is further synthesized to obtain the linear double-stranded DNA described below in the fourth embodiment. For example, by using phi29 DNA polymerase in a DNA polymerase reaction, continuous DNA synthesis can be performed using the single-stranded DNA of this embodiment as a template. This type of continuous synthesis is called "rolling circle amplification" in U.S. Patent Publication No. 9,109,250, and is considered advantageous for mass production in a cell-free system because it allows unlimited extension.

[0065] 3. Transformed Cells The third aspect of the present invention is a transformed cell, which contains the double-stranded circular DNA vector described in the first aspect.

[0066] In this embodiment, the type of transformed cell is not limited. The transformed cell may be any cell into which a double-stranded circular DNA vector can be introduced and which can maintain and / or replicate the double-stranded circular DNA vector. Examples of transformed cells include archaea, bacteria, and eukaryotic cells. Bacteria may be Escherichia, lactic acid bacteria, or Bacillus (e.g., Bacillus subtilis). Examples of eukaryotic cells include fungal cells (e.g., yeast cells), algae cells, plant cells, protozoan cells, insect cells, nematode cells, fish cells, avian cells (e.g., chicken cells), and mammalian cells (e.g., mouse cells, chimpanzee cells, and human cells). Among these, cells capable of replicating the double-stranded circular DNA vector are preferred.

[0067] Examples of Escherichia include Escherichia coli, and more specifically, Escherichia coli (e.g., Escherichia coli K-12 strain, Escherichia coli B strain), etc.

[0068] Commercially available E. coli strains and E. coli strains from the Biological Resource Center can also be used, such as JM109 strain, DH5 strain, DH5α strain, DH10B strain, NEB10β strain, HST08 strain, HST16CR strain, HB101 strain, W3110 strain, MG1655 strain, BL21 strain, BL21(DE3) strain, etc. These strains can be obtained from New England Biolabs, Takara Bio Inc., Thermo Fisher Scientific, Toyobo Co., Ltd., ATCC (American Type Culture Collection), NBRP (National Bio Resource Project), etc.

[0069] In the present invention, strains derived from the above-mentioned E. coli strains can also be used, such as the methionine-requiring JW3973 strain (available from NBRP), the leucine-requiring JW2806 strain (available from NBRP), the cysteine-requiring JW3582 strain (available from NBRP), and the thiamine- and histidine-requiring ME5305 strain (available from NBRP).

[0070] 4. Method for Producing Linear Covalently Closed DNA 4-1. Overview A fourth aspect of the present invention is a method for producing linear covalently closed DNA. The production method of this aspect includes the essential steps of a generation step, a dissociation step, a hybridization step, and a separation step, and the optional steps of an introduction step and a culture step. The production method of this aspect can produce linear covalently closed DNA containing a target nucleic acid sequence from a double-stranded circular DNA vector or linear double-stranded DNA. The production method of this aspect can efficiently remove DNA fragments containing functional sequences that are generated as by-products.

[0071] 4-2. Method Hereinafter, each step in the method for producing linear covalently closed DNA of this embodiment will be specifically explained.

[0072] (Introduction step) In this embodiment, the "introduction step" refers to a step of introducing a double-stranded circular DNA vector into a host cell to obtain a transformed cell containing the double-stranded circular DNA vector. This step is a selective step. Note that the configuration of the double-stranded circular DNA vector in this embodiment is similar to that described in the first embodiment, and therefore a detailed description thereof will be omitted here.

[0073] The method for introducing the double-stranded circular DNA vector into the host cell in this step is not particularly limited. For example, a gene transfer method (transformation method) known in the art, such as that described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, may be used. Specific examples include the heat shock method, lipofection, electroporation, microinjection, calcium phosphate method, DEAE-dextran method, introduction using cationic lipids, introduction using cationic polymers (e.g., polyethyleneimine (PEI)), introduction using nanoparticles, introduction using viruses, and particle bombardment.

[0074] In this step, the host cells into which the double-stranded circular DNA vector has been introduced can be appropriately selected in a medium containing an antibiotic based on the gene encoding the antibiotic resistance protein in the double-stranded circular DNA vector.

[0075] (Culturing step) In this embodiment, the "culturing step" refers to a step of culturing transformed cells into which a double-stranded circular DNA vector has been introduced. The purpose of this step is to increase the amount of the double-stranded circular DNA vector by growing the transformed cells. This step is a selective step.

[0076] The method for culturing the transformed cells in this step is not particularly limited and can be appropriately selected depending on the type of transformed cells. For example, a culture method known in the art, such as that described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc., may be used.

[0077] The medium for culturing the transformed cells in this step may be either a liquid medium or a solid medium. The medium may contain one or more components selected from the group consisting of enzymatic protein hydrolysates such as peptone and tryptone, biological extracts such as potato dextrose and yeast extract, amino acids such as glutamic acid or salts thereof, sugars such as glucose, glycerol, and sucrose, and inorganic salts such as sodium chloride, magnesium chloride, and potassium dihydrogen phosphate. Specific media and compositions include LB medium (tryptone, yeast extract, sodium chloride), YPG medium (yeast extract, peptone, glucose), PD medium (potato dextrose), and TB medium (tryptone, yeast extract, dipotassium hydrogen phosphate, potassium dihydrogen phosphate).

[0078] When the double-stranded circular DNA vector contains a gene encoding an antibiotic resistance protein, this step can be carried out in the presence of an antibiotic corresponding to the gene, such as ampicillin, carbenicillin, kanamycin, tetracycline, or chloramphenicol.

[0079] The culture conditions for this step can be appropriately selected depending on the type of transformed cells. For example, the culture can be performed at 20 to 42°C, 25 to 40°C, 30 to 38°C, or 35 to 37°C.

[0080] The culture time in this step is not limited as long as a sufficient amount of double-stranded circular DNA vector can be obtained, and may be, for example, 1 hour or more, 2 hours or more, 4 hours or more, 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, or 1 week or more.

[0081] The pH of the medium used in this step is not particularly limited as long as it allows the transformed cells to grow. For example, the medium may have a pH of 3 or higher, 4 or higher, 5 or higher, 6 or higher, 7 or higher, 7.5 or higher, 8 or higher, 8.5 or higher, 9 or higher, or 9.5 or higher, and / or a pH of 11 or lower, 10.5 or lower, 10 or lower, 9.5 or lower, 9 or lower, 8.5 or lower, 8 or lower, or 7.5 or lower, e.g., a pH of 3 to 11, 4 to 10, 5 to 9, or 7 to 9, or a pH of 7.5 to 10.5, 8 to 10, or 8.5 to 9.5.

[0082] (Production step) In this embodiment, the "production step" refers to a step of cleaving the above-mentioned double-stranded circular DNA vector or linear double-stranded DNA with a protelomerase or an active fragment thereof that recognizes and cleaves a protelomerase recognition sequence, and an endonuclease or an active fragment thereof that recognizes and cleaves an endonuclease recognition sequence, to produce a linear covalently closed DNA containing a nucleic acid sequence of interest, and a DNA fragment containing a functional sequence.

[0083] In this embodiment, the "linear double-stranded DNA" comprises two or more protelomerase recognition sequences containing a nucleic acid sequence of interest therebetween, and two or more endonuclease recognition sequences containing a functional sequence therebetween. The number of protelomerase recognition sequences is not limited as long as it is two or more, and may be two, three, four, five, or more. For example, when the number of protelomerase recognition sequences is two, the linear double-stranded DNA comprises two protelomerase recognition sequences containing a nucleic acid sequence of interest therebetween, and two or more endonuclease recognition sequences containing a functional sequence therebetween, other than the region between the two protelomerase recognition sequences. When the number of protelomerase recognition sequences is three or more, the linear double-stranded DNA comprises three or more protelomerase recognition sequences, the nucleic acid sequence of interest, and two or more endonuclease recognition sequences containing a functional sequence therebetween, and the nucleic acid sequence of interest or the two or more endonuclease recognition sequences are contained between adjacent pairs of protelomerase recognition sequences. An example of a linear double-stranded DNA is one that contains a pair of protelomerase recognition sequences with a nucleic acid sequence of interest between them, and two or more endonuclease recognition sequences with a functional sequence between them, repeated alternately. U.S. Patent Publication No. 9,109,250 discloses that linear double-stranded DNA having such a structure can be obtained by synthesizing a DNA strand using a polymerase such as phi29 DNA polymerase using the single-stranded DNA of the third embodiment as a template, and then synthesizing the complementary strand of the resulting DNA strand. The method for producing the single-stranded DNA used as a template here is not limited, and it can also be obtained, for example, by dissociating the double-stranded circular DNA vector described in the first embodiment into single strands.

[0084] (1) When cleavage by protelomerase is performed intracellularly: In one embodiment, cleavage by protelomerase or an active fragment thereof in this step can be performed intracellularly. In this case, cleavage by protelomerase or an active fragment thereof can be performed using a transformed cell that expresses protelomerase or an active fragment thereof. In this case, the protelomerase or an active fragment thereof can be expressed from a double-stranded circular DNA vector or the genome of the transformed cell. When expressing protelomerase or an active fragment thereof from a double-stranded circular DNA vector, a gene encoding the protelomerase or an active fragment thereof may be included in the functional sequence of the double-stranded circular DNA vector. It is preferable that the gene encoding the protelomerase or an active fragment thereof is placed under the control of a promoter whose expression can be controlled.

[0085] In a further embodiment, the transformed cells used in this step further express an endonuclease or an active fragment thereof. The endonuclease or an active fragment thereof can be expressed from a double-stranded circular DNA vector or the genome of the transformed cells. When the endonuclease or an active fragment thereof is expressed from a double-stranded circular DNA vector, a gene encoding the endonuclease or an active fragment thereof can be included in the functional sequence of the double-stranded circular DNA vector. It is preferable that the gene encoding the endonuclease or an active fragment thereof is also placed under the control of a promoter whose expression can be controlled. The gene encoding the endonuclease or an active fragment thereof can also be placed under the control of a common promoter together with the gene encoding the above-mentioned protelomerase or an active fragment thereof.

[0086] When an expression-controllable promoter is used, the method for inducing the expression of the protelomerase or an active fragment thereof and / or the endonuclease or an active fragment thereof is not particularly limited and can be appropriately selected depending on the type of expression-controllable promoter. For example, an expression inducer can be added to the medium. Specifically, arabinose can be added as an expression inducer for an arabinose-inducible promoter, IPTG can be added as an expression inducer for an IPTG-inducible promoter, or rhamnose can be added as an expression inducer for a rhamnose-inducible promoter to the medium containing the transformed cells after the culturing step.

[0087] The time for culturing the transformed cells in the presence of the expression inducer is not particularly limited, as long as the expression of the protelomerase or an active fragment thereof and / or the endonuclease or an active fragment thereof is sufficiently induced, and may be, for example, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 6 hours or more, 12 hours or more, or 24 hours or more.

[0088] The pH of the medium used for inducing expression may be within a range in which the protelomerase or an active fragment thereof and / or the endonuclease or an active fragment thereof can function in the cells, and may be, for example, pH 3 to 11, pH 4 to 10, pH 5 to 9, or pH 7 to 9. For example, a pH range between the optimal pH for the protelomerase and the optimal pH for the endonuclease can be used.

[0089] The temperature at which transformed cells are cultured in the presence of an expression inducer may be, for example, 20° C. or higher, 25° C. or higher, 28° C. or higher, 30° C. or higher, or 31° C. or higher, and / or 42° C. or lower, 40° C. or lower, 39° C. or lower, 38° C. or lower, 37° C. or lower, 36° C. or lower, 35° C. or lower, 34° C. or lower, 33° C. or lower, 32° C. or lower, or 31° C. or lower, for example, 26 to 38° C., 27 to 37° C., 28 to 36° C., 29 to 35° C., 30 to 34° C., 30 to 33° C., 30 to 32° C., or 30 to 31° C. For example, a temperature range between the optimal temperature of protelomerase and the optimal temperature of endonuclease can also be used.

[0090] If the expression-controllable promoter is a heat-inducible promoter or a cold-inducible promoter, a heat shock or cold shock may be applied. The conditions for the heat shock or cold shock are not particularly limited, as long as the temperature and time are such that the expression of the protelomerase or an active fragment thereof and / or the endonuclease or an active fragment thereof is sufficiently induced. For example, heat shock can be performed at 36°C to 45°C, 37°C to 44°C, 38°C to 43°C, 39°C to 42°C, or 40°C to 41°C for 10 seconds or more, 20 seconds or more, 30 seconds or more, 40 seconds or more, 50 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, or 1 hour or more, and / or 24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, or 2 hours or less, for example, 10 seconds to 24 hours, 20 seconds to 12 hours, 25 seconds to 6 hours, 30 seconds to 3 hours, 40 seconds to 2 hours, 50 seconds to 1 hour, 1 minute to 30 minutes, or 2 minutes to 5 minutes. Furthermore, for example, in the case of cold shock, temperature conditions of 4°C to 35°C, 10°C to 30°C, 11°C to 25°C, 12°C to 20°C, or 13°C to 17°C and treatment times of 10 seconds or more, 20 seconds or more, 30 seconds or more, 40 seconds or more, 50 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, or 1 hour or more, and / or 24 hours or less, 12 hours or less, 6 hours or less, 3 hours or less, or 2 hours or less, for example, 10 seconds to 24 hours, 20 seconds to 12 hours, 25 seconds to 6 hours, 30 seconds to 3 hours, 40 seconds to 2 hours, 50 seconds to 1 hour, 1 minute to 30 minutes, or 2 minutes to 5 minutes can be used.

[0091] DNA cleaved intracellularly by protelomerase or an active fragment thereof and / or endonuclease or an active fragment thereof is extracted from the cells as needed. The extraction method may be a conventional method known in the art. For example, the DNA may be prepared according to the method described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Alternatively, the DNA may be prepared using a commercially available DNA extraction kit.

[0092] In yet another embodiment, cleavage with a protelomerase or an active fragment thereof may be performed intracellularly, followed by cleavage with an endonuclease or an active fragment thereof in a cell-free system (in vitro cell-free method). For example, as described above, expression of a protelomerase or an active fragment thereof may be induced in a transformed cell, cleavage with a protelomerase or an active fragment thereof may be performed, and then DNA may be extracted from the transformed cell using a conventional method known in the art or a commercially available DNA extraction kit, and the extracted DNA may be cleaved with an endonuclease or an active fragment thereof according to the cleavage conditions in a cell-free system described below.

[0093] (2) When cleavage with protelomerase is performed in a cell-free system In one embodiment, in this step, cleavage with protelomerase or an active fragment thereof can be performed in a cell-free system. In this case, cleavage with an endonuclease or an active fragment thereof can also be performed simultaneously in a cell-free system.

[0094] The conditions for cleaving a double-stranded circular DNA vector or a linear double-stranded DNA with a protelomerase or an active fragment thereof and / or an endonuclease or an active fragment thereof in a cell-free system are not particularly limited, as long as the double-stranded circular DNA vector or the linear double-stranded DNA is sufficiently cleaved by these two types of cleavage enzymes. The time for the cleavage reaction is, for example, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 6 hours or more, 12 hours or more, or 24 hours or more, and the temperature for the cleavage reaction may be, for example, 20°C or more, 25°C or more, 28°C or more, 30°C or more, or 31°C or more, and / or 42°C or less, 40°C or less, 39°C or less, 38°C or less, 37°C or less, 36°C or less, 35°C or less, 34°C or less, 33°C or less, 32°C or less, or 31°C or less, for example, 26 to 38°C, 27 to 37°C, 28 to 36°C, 29 to 35°C, 30 to 34°C, 30 to 33°C, 30 to 32°C, or 30 to 31°C. For example, a temperature range between the optimal temperature for protelomerase and the optimal temperature for endonuclease can also be used.

[0095] In this step, the double-stranded circular DNA vector or linear double-stranded DNA is cleaved by protelomerase or an active fragment thereof and endonuclease or an active fragment thereof to produce a linear covalently closed DNA containing the target nucleic acid sequence, and also to produce, as by-products, a DNA fragment containing a functional sequence and not containing hairpin structures at either end, and a looped fragment, as described below.

[0096] (Dissociation step) In this embodiment, the "dissociation step" refers to a step of dissociating the linear covalently closed DNA containing the target nucleic acid sequence obtained after the generation step, and the DNA fragment containing the functional sequence and not containing hairpin structures at both ends, into single strands. By this step, the linear covalently closed DNA is dissociated into a single-stranded circular DNA.

[0097] In this step, any known method capable of dissociating double-stranded DNA into single strands can be used. Typical methods for dissociating double-stranded DNA into single strands include alkali denaturation and heat denaturation, and either method may be used in this step.

[0098] In the dissociation method using alkaline denaturation, the linear covalently closed DNA and DNA fragments described above can be dissociated into single strands by adding an alkaline aqueous solution. The pH conditions for alkaline denaturation based on the addition of an alkaline aqueous solution are, for example, pH 10 or higher, preferably pH 11 or higher, and may be pH 11 to pH 14 or pH 12 to pH 13. Specific examples of alkaline aqueous solutions include aqueous sodium hydroxide, aqueous potassium hydroxide, aqueous disodium hydrogen carbonate, aqueous sodium dihydrogen carbonate, aqueous sodium acetate, and aqueous ammonium hydroxide.

[0099] Alkali denaturation may be carried out in the presence of a surfactant. The surfactant may be cationic, anionic, zwitterionic, or nonionic. Examples of surfactants include, but are not limited to, sodium dodecyl sulfate (SDS), CHAPS, Tween 20, etc. The concentration of the surfactant used is not particularly limited, but is, for example, 0.005 to 5% (w / v), preferably 0.01 to 2% (w / v).

[0100] In the dissociation method using thermal denaturation, the above-mentioned linear covalently closed DNA and DNA fragments can be dissociated into single strands by heating the solution containing them to a temperature equal to or higher than their melting temperature. Specific temperature conditions are, for example, 80°C or higher or 90°C or higher, preferably 95°C or higher, for example, 95°C to 100°C or 98°C to 100°C.

[0101] (Hybridization Step) In this embodiment, the "hybridization step" refers to a step of (self-)hybridizing or annealing the single-stranded circular DNA derived from the linear covalently closed DNA obtained after the dissociation step. The method for hybridizing the single-stranded circular DNA in this step may be appropriately selected depending on the type of dissociation method used in the dissociation step. For example, hybridization can be achieved by neutralization in the case of alkali denaturation, or by cooling in the case of heat denaturation.

[0102] Neutralization after alkali denaturation can be carried out by adding an acidic aqueous solution as a neutralizing solution. The pH condition for neutralization based on the addition of the acidic aqueous solution is, for example, pH 10 or less, preferably pH 9 or less, or pH 8 or less, and may be pH 8 to pH 10 or pH 8.5 to pH 9.5. Specific examples of acids used in the acidic aqueous solution include hydrochloric acid, acetic acid, and citric acid.

[0103] Cooling after thermal denaturation may be performed to a temperature below the melting temperature of the linear covalently closed DNA. Specific temperature conditions include, for example, 10°C or below or 5°C or below, preferably 4°C or below, and may be, for example, −20°C to 4°C or 0°C to 2°C. The cooling method is not particularly limited, and cooling on ice may also be used.

[0104] In this step, it is preferable to rapidly perform neutralization or cooling so that the single-stranded circular DNA derived from the linear covalently closed DNA hybridizes while the DNA fragments containing the functional sequence dissociated into single strands do not hybridize and become insoluble fractions. The time for neutralization or cooling in this step is, for example, 60 seconds or less, preferably 30 seconds or less or 20 seconds or less, and more preferably 0 to 10 seconds or 2 to 5 seconds. For example, the neutralization can be rapid neutralization, and the cooling can be rapid cooling.

[0105] The methods for extracting DNA from cells based on alkaline denaturation and heat denaturation are known as the alkaline extraction method and the boiling method, respectively. Therefore, in the production method of this embodiment, any known conditions for the alkaline extraction method (e.g., the alkaline-SDS method) and the boiling method may be used in the dissociation step and the hybridization step.

[0106] (Separation Step) In this embodiment, the "separation step" refers to a step of separating the linear covalently closed DNA after the hybridization step.

[0107] The purpose of this step is to separate the linear covalently closed DNA that formed double strands in the hybridization step and to remove other components, such as single-stranded DNA derived from DNA fragments containing functional sequences and / or looped fragments. Note that in this method, it is sufficient to remove functional sequences containing antibiotic resistance genes, etc., and it is not necessarily necessary to remove looped fragments that do not contain gene sequences from the final product. However, in order to obtain linear covalently closed DNA containing the target nucleic acid sequence with higher purity, it is more preferable to also remove the looped fragments.

[0108] As used herein, the term "looped fragment" refers to a double-stranded DNA in which only one end is closed with a hairpin structure. In the method of this embodiment, in a double-stranded circular DNA vector or a linear double-stranded DNA, the sequence between the protelomerase recognition sequence and the endonuclease recognition sequence (i.e., the above-mentioned spacer sequence) is generated by cleaving the protelomerase recognition sequence at one end to form a hairpin structure and cleaving the other end at the endonuclease recognition sequence.

[0109] The method for removing single-stranded DNA and / or looped fragments derived from the DNA fragments in this step is not particularly limited and can be selected appropriately. For example, centrifugation, filtration, precipitation, and / or sedimentation of the insoluble fraction containing single-stranded DNA derived from the DNA fragments, and / or ultrafiltration of the looped fragments can be used. The molecular weight cutoff of the ultrafiltration membrane can be, for example, less than 3 kDa, less than 10 kDa, or less than 100 kDa.

[0110] The linear, covalently closed DNA isolated by this step may be further purified by, for example, a method using a commercially available kit, gel extraction, cation chromatography, anion chromatography, size exclusion chromatography, or separation using a filter or ultrafiltration membrane.

[0111] 4-3. Effects According to the production method of this embodiment, sequences that become unnecessary when producing linear covalently closed DNA can be removed as an insoluble fraction, making it possible to simply and efficiently produce highly pure linear covalently closed DNA.

[0112] In one embodiment of the method of this aspect, the production step is carried out in cells, followed by DNA extraction using alkaline extraction or boiling, which removes unnecessary sequences as an insoluble fraction, allowing linear covalently closed DNA to be isolated simultaneously with DNA extraction from cells.

[0113] In another embodiment of the method of this aspect, after the cleavage reaction in the production step is carried out in a cell-free system, unnecessary sequences can be removed as an insoluble fraction by performing alkaline denaturation and neutralization in a cell-free system.

[0114] In this embodiment of the production method, unnecessary sequences generated as by-products in the production of linear covalently closed DNA can be removed very simply and efficiently, thereby enabling the mass production of linear covalently closed DNA to be achieved at low cost.

[0115] 5. Method for Removing Functional Sequences 5-1. Overview A fifth aspect of the present invention is a method for removing functional sequences contained in linear covalently closed DNA. The removal method of this aspect includes, as essential steps, a cleavage step, a dissociation step, an insoluble fraction formation step, and a removal step, and can efficiently remove functional sequences contained in linear covalently closed DNA. The removal method of this aspect can remove unnecessary sequences contained in specific linear covalently closed DNAs from a mixture containing two or more linear covalently closed DNAs.

[0116] 5-2. Method Hereinafter, each step in the removal method of this embodiment will be specifically explained.

[0117] (Cleavage step) In this embodiment, the "cleavage step" refers to a step of cleaving a linear covalently closed DNA (hereinafter referred to as "first linear covalently closed DNA" in this embodiment) containing two or more endonuclease recognition sequences and a functional sequence located between the two or more endonuclease recognition sequences, with an endonuclease or an active fragment thereof.

[0118] In one embodiment, the cleavage in this step can be performed intracellularly, for example, by inducing expression of an endonuclease or an active fragment thereof in the transformed cell in which the first linear covalently closed DNA was produced.

[0119] In another embodiment, the cleavage in this step can be carried out in a cell-free system. Since the cleavage in a cell-free system can be carried out in accordance with the conditions described in "(2) When cleavage with protelomerase is carried out in a cell-free system" in the fourth aspect, detailed explanation of the conditions will be omitted here.

[0120] In this step, the first linear covalently closed DNA is cleaved by the endonuclease or an active fragment thereof to generate a DNA fragment containing a functional sequence and having no hairpin structures at either end, and a looped fragment.

[0121] (Dissociation step) In this embodiment, the "dissociation step" refers to a step of dissociating the DNA fragments containing the functional sequence obtained after the cleavage step into single strands by alkali denaturation or heat denaturation. The alkali denaturation and heat denaturation in this step can be carried out in accordance with the method described in the "dissociation step" of the fourth embodiment, and therefore detailed description thereof will be omitted here.

[0122] (Insoluble fraction formation step) In this embodiment, the "insoluble fraction formation step" refers to a step of forming an insoluble fraction by neutralizing or cooling single-stranded DNA derived from DNA fragments obtained by alkali denaturation or heat denaturation, respectively. This step can be carried out in accordance with the method described in the "hybridization step" of the fourth embodiment, and therefore a detailed description thereof will be omitted here.

[0123] (Removal step) In this embodiment, the "removal step" refers to a step of removing the insoluble fraction. This step can be performed in accordance with the method described in the "separation step" of the fourth embodiment, and therefore a detailed description thereof will be omitted here.

[0124] In one embodiment, in the method of this aspect, the cleavage step is carried out in the presence of yet another linear covalently closed DNA that does not contain an endonuclease recognition sequence (hereinafter referred to as the "second linear covalently closed DNA" in this aspect). It is preferable that the second linear covalently closed DNA does not contain a sequence recognized by the endonuclease or an active fragment thereof used in the cleavage step of this aspect. In this embodiment, the cleaved first linear covalently closed DNA is removed in the removal step, while the second linear covalently closed DNA that is not cleaved in the cleavage step is recovered without being removed.

[0125] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0126] Example 1: Construction of vectors for preparing linear covalently closed DNA (Objective) As plasmid vectors for preparing linear covalently closed DNA, a plasmid vector containing one I-SceI recognition sequence (hereinafter referred to as "S1 vector"; Figure 2A) and a plasmid vector containing two I-SceI recognition sequences (hereinafter referred to as "S2 vector"; Figure 2B) are prepared.

[0127] (Method and Results) S1 vector and S2 vector having the following structures were constructed. The S1 vector is a plasmid vector with a total length of 9,312 bp, and includes the following: an EGFP (fluorescent protein) gene sequence (positions 1,341 to 2,057) as the target gene sequence, one of a pair of protelomerase recognition sequences being used as the reference (position 0); the other protelomerase recognition sequence (positions 2,814 to 2,869); an araC gene sequence (positions 2,870 to 3,784); an arabinose-inducible promoter (positions 3,775 to 4,059) and a TelN gene sequence (positions 4,108 to 6,003); an arabinose-inducible promoter (positions 6,004 to 6,288) and an I-SceI gene sequence (positions 6,337 to 7,044); a replication origin (positions 7,184 to 7,736); a kanamycin resistance gene sequence (positions 8,279 to 9,094); and an I-SceI recognition sequence (position 9,241) (FIG. 2A).

[0128] The S2 vector is a plasmid vector with a total length of 9,336 bp, and contains, with one of a pair of protelomerase recognition sequences as the reference (position 0), the EGFP gene sequence (positions 1,341 to 2,057); the other protelomerase recognition sequence (positions 2,814 to 2,869); the I-SceI recognition sequence (position 2,884); the araC gene sequence (positions 2,894 to 3,772); an arabinose-inducible promoter (positions 3,799 to 4,083) and the TelN gene sequence (positions 4,132 to 6,027); an arabinose-inducible promoter (positions 6,028 to 6,312) and the I-SceI gene sequence (positions 6,361 to 7,068); a replication origin (positions 7,172 to 7,760); a kanamycin resistance gene sequence (positions 8,303 to 9,118); and the I-SceI recognition sequence (position 9,265) (FIG. 2B).

[0129] In the S1 and S2 vectors, the TelN protelomerase gene sequence (TelN gene sequence) and the I-SceI gene sequence are placed under the control of an arabinose-inducible promoter. Hereinafter, the region from the araC gene sequence to the kanamycin resistance gene sequence will be referred to as the "functional sequence."

[0130] Example 2: Removal by alkaline denaturation and neutralization of DNA fragments containing functional sequences generated by protelomerase and endonuclease cleavage in transformed cells (Objective) E. coli transformed with the S1 vector and S2 vector prepared in Example 1 were cultured, and the expression of the TelN gene and I-SceI gene was induced to cleave the vectors, followed by alkaline denaturation and neutralization to verify whether the functional sequences could be removed as an insoluble fraction. Additionally, it was verified whether the desired linear, covalently closed DNA could be recovered as a soluble fraction.

[0131] (Method and Results) (1) Obtaining Transformed Cells Containing Plasmid Vectors E. coli was transformed with the S1 or S2 vector prepared in Example 1. 25 μL of competent cell solution of E. coli DH10B strain was mixed with a solution containing each vector and allowed to stand on ice for 30 minutes. After standing for 30 minutes, the mixture was incubated at 42°C for 45 seconds (heat shock) and then allowed to stand on ice for 2 minutes. 225 μL of SOC medium was then added. E. coli was plated on LB agar medium (1% tryptone, 0.5% dry yeast extract, 1% sodium chloride, 0.005% kanamycin sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.)). Strains that grew after static culture at 37°C for 1 day were selected, and E. coli containing the introduced plasmid vector was obtained.

[0132] (2) Culturing of transformed cells E. coli into which the S1 vector or S2 vector had been introduced was inoculated into 2 mL of Plusgrow II medium (4% Plusgrow II (Nacalai Tesque), 0.005% kanamycin sulfate), and this was cultured with shaking at 37°C for 6 hours to obtain a preculture solution. 200 μL of the preculture solution was inoculated into 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% kanamycin sulfate) in a flask, and this was cultured with shaking at 37°C for 16 hours.

[0133] (3) Cleavage by protelomerase and endonuclease in transformed cells. 100 mL of Plusgrow II medium (4% Plusgrow II, 0.005% kanamycin sulfate) at pH 7.0 and 2 mL of arabinose solution (10% arabinose) were added to the culture medium after culturing, and expression of protelomerase (TelN) and endonuclease (I-SceI) was induced in the E. coli cells. Linear, covalently closed DNA containing the nucleic acid sequence of interest and DNA fragments containing functional sequences were generated by protelomerase and endonuclease in the E. coli cells. After expression induction, the cells were further cultured with shaking at 37°C for 6 hours, and the cells were collected by centrifugation.

[0134] (4) Alkaline Denaturation and Neutralization Treatment (DNA Extraction from Transformed Cells) The recovered cells were subjected to alkali denaturation and neutralization treatment using a QIAprep Spin Miniprep Kit (QIAGEN), and DNA was extracted. Specifically, cells recovered from 1 mL of culture medium were suspended in 250 μL of Buffer P1. Next, 250 μL of Buffer P2 (NaOH / SDS solution) was added and mixed by inversion, and double-stranded DNA was dissociated into single strands by alkali denaturation (pH 12-13). After standing for 2 minutes, 350 μL of Buffer N3 was added and immediately mixed by inversion to neutralize the solution (pH 7-9), and single-stranded DNA was hybridized into double strands. The resulting solution was centrifuged at 14,000 rpm for 10 minutes to remove the insoluble fraction. The supernatant was added to a QIAprep spin column and centrifuged at 14,000 rpm for 2 minutes to adsorb the DNA to the membrane. Next, 750 μL of Buffer PE was added to the QIAprep spin column and centrifuged twice at 14,000 rpm for 2 minutes to wash the membrane. Finally, 50 μL of Buffer EB was added to the QIAprep spin column, allowed to stand at room temperature for 2 minutes, and then centrifuged at 14,000 rpm to obtain an eluate containing linear covalently closed DNA containing the target EGFP gene sequence.

[0135] (5) Electrophoresis: The extracted DNA was evaluated by electrophoresis. Specifically, a mixture of 5 μL of the DNA solution obtained in (4) and 1 μL of Gel Loading Dye, Purple (6X) (New England Biolabs) was loaded onto a 1% agarose gel prepared with TAE buffer, and electrophoresis was performed at 100 V for 40 minutes. GelGreen (Biotium) was used as the staining reagent, and detection was performed by UV irradiation.

[0136] The electrophoresis results are shown in Figures 2A and 2B. In the S1 vector, in addition to a band (approximately 2.8 kb) of linear covalently closed DNA containing the EGFP gene sequence, a band (approximately 6.4 kb) corresponding to double-stranded DNA containing the functional sequence was detected (Figure 2A). This result indicates that the double-stranded DNA containing the functional sequence was not removed during the alkaline denaturation and neutralization treatments (Figure 1A).

[0137] In contrast, in the S2 vector, a band corresponding to the linear covalently closed DNA containing the EGFP gene (approximately 2.8 kb) was detected at a similar level, but a band corresponding to the size of the double-stranded DNA containing the functional sequence (approximately 6.4 kb) was barely detected (Fig. 2B).This result indicates that the double-stranded DNA containing the functional sequence was removed as an insoluble fraction during the alkaline denaturation and neutralization treatments (Fig. 1B).

[0138] The results of this example demonstrate that the functional sequence can be removed by inducing the expression of protelomerase and endonuclease in E. coli cells, cleaving a double-stranded circular DNA vector with the protelomerase and endonuclease, and then performing alkaline denaturation and neutralization treatment.

[0139] Example 3: Removal of DNA fragments containing target sequences generated by endonuclease cleavage in a cell-free system by alkaline denaturation and neutralization treatment (Purpose) To verify whether linear covalently closed DNA having two endonuclease recognition sequences is cleaved with an endonuclease in a cell-free system and then subjected to alkaline denaturation and neutralization treatment, the sequence between the two endonuclease recognition sequences (referred to as the "target sequence" in this example) is removed.

[0140] (Method and Results) (1) Construction of Linear Covalently Closed DNA In this example, a 2.9 kb linear covalently closed DNA was used, which had been previously isolated by anion column purification. This linear covalently closed DNA contains HindIII recognition sequences near both covalently closed ends. In this example, the 2.8 kb sequence (target sequence) between these two HindIII recognition sequences was targeted for removal.

[0141] (2) Removal of target sequence by endonuclease cleavage in a cell-free system, alkaline denaturation, and neutralization treatment. The linear covalently blocked DNA (50 μg) described in (1) above was cleaved using 100 U of HindIII-HF (New England Biolabs) in 500 μL of rCutSmart Buffer (New England Biolabs) at 37 °C for 1 hour. Subsequently, alkaline denaturation and neutralization treatment were performed using a QIAprep Spin Miniprep Kit (QIAGEN). Specifically, 100 μL of the HindIII-HF reaction solution was mixed with 250 μL of Buffer P1. Next, 250 μL of Buffer P2 (NaOH / SDS solution) was added and mixed by inversion. The double-stranded DNA was dissociated into single strands by alkaline denaturation (pH 12-13). After standing for 2 minutes, 350 μL of Buffer N3 was added and immediately mixed by inversion to neutralize the solution (pH 7-9), allowing the single-stranded DNA to hybridize into double strands. The resulting solution was centrifuged at 14,000 rpm for 10 minutes to remove the insoluble fraction. The supernatant was added to a QIAprep spin column and centrifuged at 14,000 rpm for 2 minutes to adsorb the DNA to the membrane. Next, 750 μL of Buffer PE was added to the QIAprep spin column, and the membrane was washed by centrifuging twice at 14,000 rpm for 2 minutes. Finally, 50 μL of Buffer EB was added to the QIAprep spin column, and the column was left to stand at room temperature for 2 minutes, followed by centrifugation at 14,000 rpm to obtain an eluate.

[0142] (3) Removal of target sequence by alkaline denaturation and neutralization treatment without endonuclease cleavage in a cell-free system (control experiment) The linear covalently blocked DNA (50 μg) described in (1) above was subjected to alkaline denaturation and neutralization treatment in the same manner as in (2) above, without cleavage with HindIII-HF, to obtain an eluate.

[0143] (4) Digestion with exonuclease 20 U of exonuclease was added to 250 μL of the HindIII-HF reaction solution obtained in (2) above, and digestion was carried out at 37° C. for 1 hour.

[0144] (5) Electrophoresis: The reaction mixtures after each reaction and the eluate after elution were evaluated by electrophoresis. Specifically, a mixture of 5 μL of the eluate obtained in (3) and the reaction mixture obtained in (4) and 1 μL of Gel Loading Dye, Purple (6X) (New England Biolabs) was loaded onto a 1% agarose gel prepared with TAE buffer and electrophoresed at 100 V for 40 minutes. GelGreen (Biotium) was used as the staining reagent, and detection was performed by UV irradiation.

[0145] The electrophoresis results are shown in Figures 3A and 3B. The fourth lane from the left in Figure 3A shows that almost all DNA was digested after exonuclease digestion in (4) above, indicating that cleavage by HindIII-HF proceeded completely. The fifth lane from the left shows that linear, covalently blocked DNA that was not cleaved by HindIII-HF in (3) above was not removed by alkaline denaturation and neutralization (Figure 3B, top). The sixth lane from the left shows that the target sequence that was cleaved by HindIII-HF in (2) above and lacked covalently blocked termini on both ends was removed as an insoluble fraction after alkaline denaturation and neutralization (Figure 3B, bottom).

[0146] The results of this example demonstrate that, similar to the case of protelomerase and endonuclease cleavage in transformed cells, endonuclease cleavage, alkaline denaturation, and neutralization treatment can remove the target sequence in a cell-free system.

[0147] Example 4: Removal by alkaline denaturation and neutralization of DNA fragments containing functional sequences generated by protelomerase and endonuclease cleavage in a cell-free system (Purpose) To verify whether functional sequences are removed from linear, covalently closed DNA generated by the protelomerase reaction when protelomerase cleavage, endonuclease cleavage, alkaline denaturation, and neutralization are all performed in a cell-free system.

[0148] (Method and Results) (1) Preparation of Two Linear Covalently Closed DNAs by Protelomerase in a Cell-Free System The S1 vector (2.2 μg) and S2 vector (1.4 μg) prepared in Example 1 were cleaved with 5 U of TelN protelomerase (New England Biolabs) in 100 μL of rCutSmart Buffer (New England Biolabs) at 30° C. for 1 hour to prepare a linear covalently closed DNA containing the EGFP gene sequence as the target gene sequence, and a linear covalently closed DNA containing a functional sequence (hereinafter, sometimes referred to as “two linear covalently closed DNAs”) from each of the S1 vector and the S2 vector.

[0149] (2) Cleavage with I-SceI in a cell-free system, alkaline denaturation, and neutralization: 5 U of I-SceI was further added to the reaction solution containing the two linear covalently closed DNAs derived from the S1 vector or S2 vector obtained in (1) above, and the reaction was allowed to proceed at 37°C for 1 hour. Subsequently, alkaline denaturation and neutralization were carried out using a QIAprep Spin Miniprep Kit (manufactured by QIAGEN). Specifically, the entire enzyme reaction solution was mixed with 250 μL of Buffer P1. Next, 250 μL of Buffer P2 (NaOH / SDS solution) was added and mixed by inversion, and the double-stranded DNA was dissociated into single strands by alkaline denaturation (pH 12-13). After standing for 2 minutes, 350 μL of Buffer N3 was added and immediately mixed by inversion to neutralize the solution (pH 7-9), allowing the single-stranded DNA to hybridize to double strands. The resulting solution was centrifuged at 14,000 rpm for 10 minutes to remove the insoluble fraction. The supernatant was added to a QIAprep spin column and centrifuged at 14,000 rpm for 2 minutes to adsorb the DNA to the membrane. Next, 750 μL of Buffer PE was added to the QIAprep spin column, and the membrane was washed by centrifugation twice at 14,000 rpm for 2 minutes. Finally, 50 μL of Buffer EB was added to the QIAprep spin column, allowed to stand at room temperature for 2 minutes, and then centrifuged at 14,000 rpm to obtain the eluate.

[0150] (3) Alkaline denaturation and neutralization treatment without cleavage with I-SceI The two linear covalently closed DNAs derived from the S1 vector or S2 vector prepared in (1) above were subjected to alkaline denaturation and neutralization treatment in the same manner as in (2) above, without cleavage with I-SceI, to obtain an eluate.

[0151] (4) Electrophoresis: The reaction solution after each reaction and the eluate after elution were evaluated by electrophoresis. Specifically, a mixture of 5 μL of the eluate obtained in (3) and 1 μL of Gel Loading Dye, Purple (6X) (New England Biolabs) was loaded onto a 1% agarose gel prepared with TAE buffer and electrophoresed at 100 V for 40 minutes. GelGreen (Biotium) was used as the staining reagent, and detection was performed by UV irradiation.

[0152] The electrophoresis results are shown in Figure 4. The "M" in the first and fifth lanes from the left in Figure 4 indicates a 1 kb DNA ladder. The second lane from the left in Figure 4 shows that the two linear, covalently closed DNA fragments derived from the S1 vector prepared in (1) above were subjected to alkali denaturation and neutralization without cleavage with I-SceI, resulting in the removal of neither of the two linear, covalently closed DNA fragments. The sixth lane from the left in Figure 4 shows that the two linear, covalently closed DNA fragments derived from the S1 vector prepared in (1) above were subjected to alkali denaturation and neutralization, resulting in the removal of neither the linear, covalently closed DNA fragments containing the EGFP gene sequence nor the DNA fragments containing the functional sequence. The fourth lane from the left in Figure 4 shows that the two linear covalently closed DNAs derived from the S2 vector prepared in (1) above were subjected to alkali denaturation and neutralization without cleavage with I-SceI, resulting in the removal of neither of the two linear covalently closed DNAs. The eighth lane from the left in Figure 4 shows that the two linear covalently closed DNAs derived from the S2 vector prepared in (1) above were cleaved with I-SceI and then subjected to alkali denaturation and neutralization, resulting in the removal of the linear covalently closed DNA containing the EGFP gene sequence as the target gene sequence, but the removal of the DNA fragment containing the functional sequence as an insoluble fraction.

[0153] The results of this example demonstrate that functional sequences are removed when protelomerase cleavage, endonuclease cleavage, alkaline denaturation, and neutralization treatment are all performed in a cell-free system. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

1. A method for making linear covalently closed DNA, comprising: (a) a pair of protelomerase recognition sequences; a first region that constitutes one of two regions that constitute a double-stranded circular DNA vector between the pair of protelomerase recognition sequences, and that includes two or more endonuclease recognition sequences and a functional sequence disposed between the two or more endonuclease recognition sequences; and a second region that constitutes the other of the two regions and contains the nucleic acid sequence of interest but does not contain the endonuclease recognition sequence; or (b) two or more protelomerase recognition sequences that contain a nucleic acid sequence of interest therebetween; and Two or more endonuclease recognition sequences with a functional sequence between them A linear double-stranded DNA comprising with the protelomerase or an active fragment thereof and the endonuclease or an active fragment thereof to generate a linear covalently closed DNA containing the nucleic acid sequence of interest and a DNA fragment containing the functional sequence; a dissociation step of dissociating the linear covalently closed DNA and the DNA fragment into single strands; a hybridization step of hybridizing the single-stranded circular DNA derived from the linear covalently closed DNA obtained after the dissociation step; a separation step of separating the linear covalently closed DNA after the hybridization step; The method comprising: 　 The method of claim 1, wherein the hybridization step is performed in 30 seconds or less. 　 The method of claim 1 , wherein the dissociation step and the hybridization step use alkaline denaturation and neutralization, respectively. 　 The method according to claim 3, wherein the alkaline denaturation is carried out by adding an alkaline aqueous solution at a pH of 11 or higher. 　 The method according to claim 4 , wherein the alkaline denaturation is carried out in the presence of a surfactant.

6. The method according to any one of claims 3 to 5, wherein the neutralization is carried out by addition of an aqueous acidic solution to a pH of 9 or less.

2. The method of claim 1, wherein the dissociation step and the hybridization step involve heating to 95°C or higher and cooling to 4°C or lower, respectively. 　 The method of claim 1 , wherein the separation step removes single-stranded DNA and / or looped fragments derived from the DNA fragments. 　 The removing centrifugation, filtration, precipitation, and / or sedimentation of the insoluble fraction containing single-stranded DNA derived from said DNA fragments, and / or Ultrafiltration of the looped fragments The method of claim 8, wherein 　 2. The method of claim 1, wherein the producing step uses a transformed cell that contains the double-stranded circular DNA vector and expresses the protelomerase or an active fragment thereof. 　 The method of claim 10 , wherein the transformed cell further expresses the endonuclease or an active fragment thereof. 　 The method according to claim 10, wherein the producing step comprises performing cleavage with the protelomerase or an active fragment thereof in the transformed cell, and then performing cleavage with the endonuclease or an active fragment thereof in a cell-free system. 　 The method of claim 1 , wherein the production step is performed in a cell-free system. 　 the functional sequence is 100 bases or more in length, and / or 2. The method of claim 1, wherein each of the two or more endonuclease recognition sequences is located within 100 base lengths of each of the pair of protelomerase recognition sequences.

1. A method for removing functional sequences contained in linear covalently closed DNA, comprising: the linear covalently closed DNA comprises two or more endonuclease recognition sequences and a functional sequence disposed between the two or more endonuclease recognition sequences; The method comprises: a cleaving step of cleaving the linear covalently closed DNA with the endonuclease or an active fragment thereof; a dissociation step in which the DNA fragment containing the functional sequence obtained after the cleavage step is dissociated into single strands by alkali denaturation or heating to 95°C or higher; an insoluble fraction forming step of neutralizing or cooling to 4°C or below the single-stranded DNA derived from the DNA fragment obtained by the alkali denaturation or the heating, respectively, to form an insoluble fraction; and a removing step of removing the insoluble fraction; The method comprising: 　 the cleavage step is carried out in the presence of an additional linear covalently closed DNA that does not contain the endonuclease recognition sequence; and 16. The method of claim 15, wherein the removing step separates the additional linear covalently closed DNA. 　 the functional sequence is 100 bases or more in length, and / or 16. The method of claim 15, wherein each of the two or more endonuclease recognition sequences is located within 100 base lengths of each of the covalently closed ends of the linear covalently closed DNA. 　 A double-stranded circular DNA vector, a pair of protelomerase recognition sequences, a first region constituting one of the two regions constituting the double-stranded circular DNA vector between the pair of protelomerase recognition sequences, the first region comprising two or more endonuclease recognition sequences and a functional sequence disposed between the two or more endonuclease recognition sequences; The double-stranded circular DNA vector, wherein the second region constituting the other of the two regions contains a nucleic acid sequence of interest but does not contain the endonuclease recognition sequence.

19. The double-stranded circular DNA vector of claim 18, wherein the functional sequence comprises a gene sequence and / or an untranslated sequence.

20. The double-stranded circular DNA vector according to claim 19, wherein the gene is a selectable marker gene and / or an enzyme gene.

21. The double-stranded circular DNA vector of claim 20, wherein the selectable marker gene is an antibiotic resistance gene and / or an auxotrophy complementation gene. 　 The double-stranded circular DNA vector according to claim 20, wherein the enzyme gene is a gene sequence encoding the protelomerase or an active fragment thereof and / or a gene sequence encoding the endonuclease or an active fragment thereof, both of which are placed under the control of an expression-controllable promoter.

20. The double-stranded circular DNA vector of claim 19, wherein the non-translated sequence is a nucleic acid sequence encoding a replication origin region and / or a non-translated RNA. 　 The double-stranded circular DNA vector according to any one of claims 18 to 23, wherein the functional sequence is 100 bases or more in length.

19. The double-stranded circular DNA vector according to claim 18, wherein the two or more endonuclease recognition sequences are the same or different from each other.

19. The double-stranded circular DNA vector of claim 18, wherein the nucleic acid sequence of interest comprises a gene sequence of interest encoding a protein of interest or a fragment thereof.

19. The double-stranded circular DNA vector according to claim 18, wherein each of the two or more endonuclease recognition sequences is located within 100 base lengths from each of the pair of protelomerase recognition sequences.

19. The double-stranded circular DNA vector of claim 18, wherein the protelomerase is TelN protelomerase, TelA protelomerase, or TelK protelomerase.

19. The double-stranded circular DNA vector of claim 18, wherein the endonuclease is a homing endonuclease. 　 A single-stranded DNA selected from the two DNA strands that constitute the double-stranded circular DNA vector of claim 18. 　 A transformed cell comprising the double-stranded circular DNA vector of claim 18.

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