Method for producing supercoiled circular DNA in vitro

Abstract

Current methods for generating minicircles are often slow, expensive, and difficult to perform under GMP conditions, due to the bacterial origin of the product. In contrast, the generation of synthetic circular supercoiled DNA based on HTLA and CHTLA can be carried out entirely in vitro using chemically or enzymatically synthesized oligonucleotides, long single-stranded DNA, and / or double-stranded DNA.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 498,886, filed on April 28, 2023, under the names of Xiang LI and Charles J. BIEBERICH, titled "A METHOD TO PRODUCE SUPERCOILED CIRULAR DNA IN VITRO", which is hereby incorporated by reference in its entirety.

[0002] Field The present invention relates to a method for producing supercoiled circular DNA that is not a plasmid vector from two or more precursor DNA fragments, such as single - stranded DNA, double - stranded DNA, and / or oligonucleotides.

Background Art

[0003] Background Advances in our understanding of the molecular basis of disease have facilitated the explosive growth of new gene - based therapies, also known as gene therapy. Gene therapy is the intentional genetic modification of DNA within a patient's cells to achieve specific therapeutic goals. Under three decades of development, this technology has matured in recent years, and pharmaceutical companies are heavily investing in the development of new therapies. More than 800 clinical trials in the United States are currently underway for a wide range of single - gene hereditary diseases, as well as acquired diseases including cancer and rheumatoid arthritis. To date, the following five gene therapies have received FDA approval: ZYNTEGLO® for the treatment of beta - thalassemia, KYMRIAH™, a CAR T - cell therapy for relapsed or treatment - resistant leukemia and lymphoma, LUXTURNA® for hereditary retinal diseases, SKYSONA® for cerebral adrenoleukodystrophy, and ZOLGENSMA® for spinal muscular atrophy. The global gene therapy market was worth $9 billion in 2023 but is expected to reach $46 billion by 2030.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Historically, gene therapy has heavily relied on viruses as gene delivery vehicles, and all five biologics approved by the FDA are based on either lentiviruses or adeno-associated viruses. During manufacturing, the therapeutic virus is made non-replicable, so no proliferative viral infection occurs in patients. Gene therapy based on approved viruses has a good safety profile, but there are safety concerns that the therapeutic virus can potentially recombine with other viruses during manufacturing or within the patient, producing new viruses with unknown and potentially harmful characteristics (including replication). Also, viruses have a bias specific to the particular cell types they infect, severely limiting the range of diseases they can target. This concern and other concerns (including, for example, high manufacturing costs and complex Good Manufacturing Practice (GMP) workflows) have spurred the search for non-viral alternatives for genetically modifying patients' cells. For example, naked DNA (i.e., not encapsulated within a viral capsid) can be used to deliver therapeutic genes, but to get naked DNA into cells, a lipid-based delivery system, sonication, electroporation, or a ballistic method using DNA-coated gold particles (i.e., a gene gun) must be used. Alternatively, non-viral gene delivery methods have been developed based on circular DNA called plasmids that are produced in bacteria. However, plasmids carry bacterial DNA sequences that necessarily trigger the patient's immune response, limiting their effectiveness.

[0005] To address this challenge, small circular DNA molecules called "minicircles" have been developed that contain no bacterial DNA whatsoever. Minicircles contain only the therapeutic gene and the DNA elements necessary to support its expression, while all or almost all other bacterial DNA, including antibiotic resistance genes, is removed, for example, through an engineered recombination process that occurs within the bacterial cell. Minicircles have been demonstrated to be a powerful gene delivery platform that is superior to plasmids and provides higher and more sustained therapeutic gene expression, but they require further purification from other bacterial components (including the original plasmid from which they are derived). This process is so complex that it can take more than 270 days to produce a single batch. Enzymatic synthesis of minicircle-like DNA (i.e., Doggybone DNA) in vitro reduces the production time to 50 days, but GMT production still presents significant challenges.

[0006] There is still a need for improved methods that can scale up the production of circular DNA (i.e., antibiotic resistance genes, origins, or replications) free from bacterial vector DNA in a GMP-compliant manner. This specification describes a novel technique that has the potential to generate circular supercoiled DNA entirely synthetically and scale up the production process. This technique uses a process called "hetero-double-stranded thermostable ligase assembly," in which, under specific conditions, a single-stranded or double-stranded DNA precursor is denatured and annealed in the presence of a thermostable DNA ligase (with or without thermostable type II topoisomerase) to produce a circular, predominantly supercoiled DNA molecule, i.e., "synthetic circular supercoiled DNA." SCS To create "DNA". [Means for solving the problem]

[0007] In one embodiment, a method for forming an adhesive end block (SEB) having a 5' or 3' overhang is described, A step of introducing at least two precursor DNA fragments into a buffer medium containing a heat-stable DNA ligase enzyme, and a step of correctly assembling the precursor DNA fragments to produce a defined DNA sequence. The steps include: applying heat to a first temperature to induce denaturation of the at least two precursor DNA fragments, and The steps include lowering the temperature to a second temperature, annealing in the presence of the heat-stable DNA ligase enzyme, thereby producing a double-stranded DNA heteroduplex formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a 3' overhang; Includes.

[0008] In other embodiments, synthetic cyclic supercoiled DNA ( SCS A method for generating DNA is described, and this method is described as A step of introducing at least two precursor DNA fragments into a buffer medium containing a heat-stable DNA ligase enzyme, and a step of correctly assembling the precursor DNA fragments to produce a defined DNA sequence. The steps include: applying heat to a first temperature to induce denaturation of the at least two precursor DNA fragments, and The step of lowering the temperature to a second temperature and annealing in the presence of the heat-stable DNA ligase enzyme, thereby producing a double-stranded DNA heteroduplex formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a single-stranded 3' overhang, wherein the 5' intramolecular overhangs on the heteroduplex molecule are complementary and ligation occurs in both DNA strands, SCS When DNA is generated and the 3' intramolecular overhangs on the heteroduplex molecule are complementary, and ligation occurs in both DNA strands, SCS The step in which DNA is generated, Includes.

[0009] In yet another embodiment, a method for generating substantially supercoiled DNA is described, the method is described as follows: A step of introducing at least two precursor DNA fragments into a buffer medium containing a heat-stable DNA ligase enzyme and a heat-stable type II topoisomerase, wherein the precursor DNA fragments are correctly assembled to produce a defined DNA sequence. The steps include: applying heat to a first temperature to induce denaturation of the at least two precursor DNA fragments, and The following is a step to lower the temperature to a second temperature: (i) Annealing in the presence of the heat-stable DNA ligase enzyme to produce a double-stranded DNA heteroduplex formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a 3' overhang, where the 5' intramolecular overhangs on the heteroduplex molecule are complementary and ligation occurs in both DNA strands, SCS When DNA is generated and the 3' intramolecular overhangs on the heteroduplex molecule are complementary, and ligation occurs in both DNA strands, SCS DNA is generated, and (ii) In the presence of thermally stable type II topoisomerase, SCS To essentially supercoil DNA, Includes.

[0010] In another embodiment, a method for generating substantially supercoiled DNA is described, the method is described as follows: A step of introducing at least two precursor DNA fragments into a buffer medium containing a heat-stable DNA ligase enzyme, and a step of correctly assembling the precursor DNA fragments to produce a defined DNA sequence. The steps include: applying heat to a first temperature to induce denaturation of the at least two precursor DNA fragments, and Lower the temperature to a second temperature and anneal in the presence of the thermostable DNA ligase enzyme, thereby creating a heteroduplex of double-stranded DNA formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a single-stranded 3' overhang, where the 5' intramolecular overhangs on the heteroduplex molecules are complementary and when ligation occurs on both DNA strands, SCS DNA is produced and the 3' intramolecular overhangs on the heteroduplex molecules are complementary and when ligation occurs on both DNA strands, <000...​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The SCSDNA produced by CHTLA is supercoiled. Two plasmid pMax-GFP SCSDNA precursors were prepared by digesting the plasmid with either KpnI or XhoI. The digested DNA was purified, restriction enzymes were removed, and the mixture was mixed in a 1:1 ratio. pMax-GFP SCSDNA was produced in a 10-cycle CHTLA reaction. Unreacted precursors were removed by treating the reaction mixture with T5 exonuclease. The SCSDNA was analyzed on an ethidium bromide-free agarose gel. After electrophoresis was complete, the gel was stained with ethidium bromide. P: pMax-GFP DNA plasmid prepared from bacteria, V: SCSDNA, L: 1-kb DNA ladder. [Figure 3] CHTLA synthesis of circular DNA using single-stranded oligonucleotides as precursors. Six 80-base-long duplicate nucleotides, each with 40 bases duplicated, were phosphorylated at 37°C (by T4 polynucleotide kinase) and subjected to the CHTLA reaction in the presence (left figure, lanes 2 and 3) or absence (right figure, lanes 4 and 5) of HiFi-Taq ligase. Subsequently, the reaction products were treated with T5 exonuclease at 37°C for 1 hour and analyzed on a gel containing ethidium bromide. The yellow box highlights exonuclease-resistant circular DNA products that were produced in the CHTLA reaction in the presence of HiFi-Taq ligase (lane 3) but not in the control CHTLA reaction in the absence of ligase (lane 5). Lane 1, DNA ladder. [Figure 4]CHTLA synthesis of SCSDNA from linear precursors. Plasmid-derived DNA precursors (restriction fragments, white arrows in lanes 1 and 4) were ligated in a 10-cycle CHTLA reaction. CHTLA converted the precursor DNA into relaxed circular DNA (lane 2, blue arrow) and supercoiled DNA (lane 2, yellow arrow). The precursors (lanes 4-7) or CHTLA products (lanes 8-10) were treated with T5 exonuclease (units shown in blue boxes). T5 treatment completely eliminated the linear DNA precursors (lanes 5-7), but the CHTLA products of SCSDNA were resistant to T5 exonuclease (lanes 9-11). [Figure 5A] SCSDNA produced by CHTLA is negatively supercoiled. To prepare the CHTLA precursor for SCSDNA production, the pBluescript-SK (-) plasmid was digested with either BamHI or KpnI. The digested DNA was purified using a minispin column to remove restriction enzymes. The purified DNA was mixed in a 1:1 ratio and subsequently ligated in a 10-cycle CHTLA reaction using HiFi-Taq ligase. Unreacted precursor was removed using T5 exonuclease. [Figure 5B]SCSDNA was analyzed using agarose gel electrophoresis in gels containing or without chloroquine. After electrophoresis, the gels were stained with ethidium bromide. The yellow boxes indicate the locations of SCSDNA with varying degrees of negative supercoiling. The green arrows on the chloroquine-free side indicate the locations of plasmid DNA from highly negatively supercoiled bacterial sources. The red arrows indicate the locations of SCSDNA that have shifted due to chloroquine intercalation, where the SCSDNA has broken down into a single species. The green boxes indicate the locations of plasmid DNA from bacterial sources intercalated with chloroquine. Note the multiple species currently present, which show that the negative supercoiling undergoes varying degrees of loosening, resulting in a series of bands with varying degrees of supercoiling. Pr: Linearization precursor DNA, VL: SCSDNA produced by CHTLA, PL: pBluescript-SK (-) purified from bacteria, L: 1 kb DNA ladder, EB: Ethidium bromide, post-run: after electrophoresis. [Modes for carrying out the invention]

[0014] While the claimed subject matter is described in relation to specific embodiments, other embodiments (including those that do not provide all of the advantages and characteristics described herein) are also within the scope of this disclosure. Various structural and parameter modifications can be made without departing from the scope of this disclosure.

[0015] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those widely understood by those ordinarily skilled in the art. In case of any conflict, this specification (including definitions) shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials may also be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. The materials, methods, and examples disclosed herein are for illustrative purposes only and are not intended to limit the scope of use.

[0016] "About" and "approximately" are used to provide flexibility to numerical range endpoints by specifying that a given value may be "slightly above" or "slightly below" the endpoint without affecting the desired result, for example, + / - 5%.

[0017] The phrases "in one embodiment" or "in some embodiments" as used herein do not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, the phrase "in other embodiments" as used herein does not necessarily refer to a different embodiment, but may refer to a different embodiment. Accordingly, various embodiments of the present invention can be easily combined without departing from the scope or spirit of the invention, as described below.

[0018] The terms “comprise,” “include,” “having,” “has,” “can,” “contain,” and their variations as used herein are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “(a),” “and,” and “the” include plural references unless the context clearly indicates otherwise. This disclosure also contemplates other embodiments that “include,” “consist of,” and “essentially consist of,” the embodiments or elements presented herein, whether expressly described or not.

[0019] As used herein, the term “heteroduplex” DNA molecule refers to a double-stranded molecule in which the first strand originates from one double-stranded or single-stranded precursor DNA molecule, and the second strand originates from a different double-stranded or single-stranded DNA molecule, and the first and second strands are linked by Watson-Crick base pairing in a process known as complementary DNA strand annealing or hybridization.

[0020] As used herein, the terms “ligase” and “ligating agent” are used interchangeably and refer to any enzymatic or non-enzymatic agent capable of ligating DNA molecules, e.g., between two or more adjacent heteroduplexes having annealed, compatible single-stranded ends, or within heteroduplex molecules having compatible single-stranded ends, to form a cyclic molecule by establishing a new bond. In some embodiments, the ligase is an enzymatic ligating agent that, under suitable conditions, forms phosphodiester bonds between the 3′-OH and 5′-phosphate of adjacent nucleotides in DNA molecules, RNA molecules, oligonucleotides, or hybrids. Examples of temperature-sensitive ligases include, but are not limited to, bacteriophage T4 ligases and Escherichia coli ligases. Examples of heat-stable ligases include, but are not limited to, Afu ligase, Taq ligase, Tfl ligase, Tth ligase, Tth HB8 ligase, AK16D ligase of Thermus species, and Pfu ligase, HiFi Taq ligase, or Ampilgase. Those skilled in the art will understand that any heat-stable ligase (including DNA ligases and RNA ligases) can be obtained from thermophilic or hyperthermophilic organisms, such as certain species of eubacteria and archaea, and that such ligases can be utilized in the disclosed methods and kits.

[0021] As used herein, the term “duplication sequence” refers to sequences that are complementary in two polynucleotides, where the first polynucleotide contains a single-stranded (ss) duplication sequence and can hybridize to a second polynucleotide containing a complementary single-stranded sequence.

[0022] As used herein, the term “overhang” refers to the single-stranded region at the end of double-stranded (ds) DNA, which is either 5' or 3' due to the inherent directivity of DNA. Overhangs are generally produced in various lengths by treating double-stranded DNA with restriction enzymes or exonucleases and / or by adding appropriate dNTPs (e.g., dATP, dTTP, dCTP, dGTP) through the action of enzymes, i.e., terminal deoxynucleotide transferases. In some embodiments, overhangs are in the range of 2 to 1000 base pairs in length.

[0023] As used herein, the term “double-stranded DNA” or “dsDNA” means an oligonucleotide or polynucleotide having 3' overhangs, 5' overhangs, and / or blunt ends, comprising two single strands, all or part of which are complementary to each other, and thus dsDNA may contain single-stranded regions at one or both ends, and may be synthetic or of natural origin derived from cells or tissues. In one embodiment, dsDNA is a product of PCR (polymerase chain reaction), or a fragment of genomic DNA or plasmid or vector prepared by physical or enzymatic processing.

[0024] As used herein, the term “buffering agent” refers to a substance that makes a solution resistant to changes in pH when an acid or alkali is added to it. Examples of suitable non-natural source buffers that may be used in the compositions, kits, and methods described herein include HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TAPS (tris(hydroxymethyl)methylamino]propanesulfonic acid), Trisine (N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), phosphate, citrate, ammonium, acetate, carbonate, tris(hydroxymethyl)aminomethane (TRIS), TRIS-HCl, 3-(N-morpholino)propanesulfonic acid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), 2-(N-morpholino)ethanesulfonic acid (MES), N-(2-acetamide)-iminodiacetic acid (ADA), and piperazine-N,N′-bis(2-ethanesulfonic acid). Examples include buffers for (PIPES), N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), coramine chloride, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), acetamidoglycine, glycinamide, and bicine (2-(bis(2-hydroxyethyl)amino)acetic acid). It should be understood that the buffering agent may further include at least one additional species, such as electrolytes, e.g., MgCl2, NaCl, and KCl; metal ions; type II topoisomerases (e.g., DNA gyrase); single-strand DNA binding proteins or thermostable single-strand binding proteins; crowding agents (e.g., polyethylene glycol); redox agents, e.g., dithiothreitol (DTT), nicotinamide adenine dinucleotide (NAD); surfactants; and nonionic surfactants, e.g., TRITON® X-100 (octylphenol decaethylene glycol ether).

[0025] As used herein, the terms “DNA” or “RNA” are defined as “polynucleotides” and may include primers, oligonucleotides, nucleic acid chains, etc. DNA or RNA may be single-stranded, double-stranded, or a mixture thereof. Such polynucleotides of DNA or RNA may be synthetic, e.g., synthesized in a DNA synthesizer, or of natural origin, e.g., extracted from natural sources or derived from cloned or amplified material. Polynucleotides as shown herein may contain modified bases. Additionally, the sequences of DNA or RNA may contain one or more random or variable nucleotides. The use of randomized nucleotides may also include sequences restricted, where sequences restricted means that the variation at a single position is limited to a selection of two or three nucleotides (i.e., A or C, A, G, or C, etc.) rather than all four (ATGC). Typically, a polynucleotide contains a 5' phosphate group at one end of the chain ("5' end") and a 3' hydroxyl group at the other end ("3' end").

[0026] The nucleic acids used herein may be any nucleic acids, for example, human nucleic acids, bacterial nucleic acids, or viral nucleic acids. Nucleic acid samples may be, for example, one or more cells, tissues, or bodily fluids, such as blood, urine, semen, lymph, cerebrospinal fluid, or amniotic fluid, or other biological samples, such as tissue culture cells, oral swabs, mouthwash, stool, tissue sections, biopsy aspirates, and archaeological samples, such as nucleic acid samples derived from bone or mummified tissue. Nucleic acids may be, for example, DNA, RNA, or DNA products of RNA subjected to reverse transcription. Nucleic acids may be derived from any source, including, but not limited to, eukaryotes, plants, animals, vertebrates, fish, mammals, humans, non-humans, bacteria, microorganisms, viruses, biological sources, serum, plasma, blood, urine, semen, lymph, cerebrospinal fluid, amniotic fluid, biopsies, biopsy needle aspirates, cancer, tumors, tissues, cells, cell lysates, crude cell lysates, tissue lysates, tissue culture cells, oral swabs, mouthwashes, feces, mummified tissue, forensic sources, anatomical specimens, archaeological sources, infectious materials, hospital-acquired infectious materials, production sources, drug preparations, biological molecular products, protein preparations, lipid preparations, carbohydrate preparations, inanimate objects, air, soil, sap, metals, fossils, excavated materials, and / or other earthly or extraterrestrial matter and its derivatives. In some embodiments, the nucleic acids do not contain bacterial or viral nucleic acids.

[0027] As used herein, "Taq ligase" and "thermal-stable ligase" are synonymous.

[0028] As used herein, "synthetic cyclic supercoiled DNA" or SCS DNA is intended to encompass products consisting solely of synthesized circular DNA, and products containing both synthesized circular DNA and a portion of synthesized supercoiled DNA, where, SCS The DNA precursor that generates the DNA may be derived in part or entirely from a natural source (e.g., a plasmid). In some embodiments, the synthesized supercoiled DNA is substantially supercoiled.

[0029] As used herein, synthetic circular supercoiled DNA and covalently closed circular DNA are understood to be substantially exonuclease-resistant. As used herein, either exonuclease is understood to lack endonuclease activity or have minimal endonuclease activity using double-stranded DNA as a substrate.

[0030] As defined herein, “supercoil” or “supercoiling” is understood as the overall contortion of circular DNA. Supercoiling is understood as a combination of “twist” and “writhe,” where twist is the number of helical turns in DNA and writhe is the number of times the double helix crosses itself. Positive and negative supercoiling are understood by those skilled in the art, but simply put, positive supercoiling involves extra or additional helical twists relative to a loose state (i.e., over-twisting), while negative supercoiling involves fewer or subtractive helical twists relative to a loose state (i.e., under-twisting). It is understood that the DNA of most organisms is negatively supercoiled, but a certain amount of positive supercoiling also exists. Negative supercoiling advantageously enables processes such as transcription, DNA replication, and recombination. Another term common to supercoilation is "linking number" or "Lk," which is understood as a combination of twist (T) and kinks (W). The linking number is useful as an indicator for identifying phenomena resulting from DNA topological changes, such as enzymatic cleavage and rejoining. Of particular note is ΔLk, where ΔLk = Lk - Lk m It is determined by the following formula, where Lk mLk is the number of entangles for loose circular DNA, and Lk is the number of entangles for supercoiled circular DNA. Notably, Lk and Lk m This is rounded to the nearest integer before calculating ΔLk. For negatively supercoiled circular DNA, ΔLk can be negative.

[0031] As defined herein, “precursor” DNA fragments include dsDNA molecules (i.e., PCR products, restriction enzyme fragments, chemically or enzymatically produced DNA), single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. The nucleotide bases in the precursor DNA fragment may be natural adenine, guanine, cytosine, thymine, or any chemically modified form thereof, which can be incorporated into the DNA molecule by chemosynthesis or enzymatic action, i.e., by DNA polymerase, or can be made to appear in the bases or groups of bases chemically or enzymatically after synthesis. The length of the precursor DNA may range from about 20 nucleotides to several thousand or several million nucleotides, more preferably about 200 to 10,000 nucleotides for double-stranded precursors and 30 to 200 nucleotides for single-stranded precursors. In some embodiments, substantially “perfect” blunt-ended DNA precursors are obtained for use by “polishing” the ends of the precursor DNA fragment using an IIS-type restriction enzyme (i.e., MlyI) before using it in an HTLA or CHTLA reaction, as will be understood by those skilled in the art. In some embodiments, the precursor DNA fragments are prepared using the method described in U.S. Provisional Patent Application No. 63 / 554,752, filed on 16 February 2024 in the names of Xiang LI and Charles J. BIEBERICH, entitled “Method for generating circular DNA using heteroduplex thermostable ligation assembly of precursors created by rolling circle amplification” (which is incorporated herein by reference in its entirety).

[0032] It is known to those skilled in the art that each nucleotide within a dsDNA molecule can be paired with a Watson-Crick base pair, also known as a "complementary" nucleotide. Furthermore, it is understood that a dsDNA sequence is represented by the sequence of the upper or first (sense) strand in the direction from its 5' end to its 3' end, and therefore the complementary sequence is the sequence of the lower or second (antisense) strand in the same direction as the upper strand. When DNA sequences are described as complementary, it is understood that when they anneal or hybridize, they form a double-stranded DNA with antiparallel strands. Furthermore, it is understood that an annealed complementary DNA sequence may contain one or more non-classical (i.e., Watson-Crick) base pairs, or modified nucleotides that pair with several other nucleotides (for example, deoxyinosine may also pair with three other DNA bases (deoxythymidine (dT), dA, and dG)).

[0033] As described above in this specification, covalently closed circular DNA (e.g., minicircles) that do not contain any bacterial DNA have recently emerged as a powerful gene delivery platform, but they present many problems for GMP manufacturing. The inventors have previously developed an improved DNA assembly process called heteroduplex thermostable ligase assembly (HTLA®), which enables the construction and commercialization of large linear DNA molecules entirely in vitro, as described in International Patent Application No. PCT / US2023 / 064977, filed on 27 March 2023 under the names of Charles J. Bieberich and Xiang Li, and titled "HETERODUPLEX THEROMSTABLE LIGATION ASSEMBLY (HTLA) AND / OR CYCLIC HETERODUPLEX THERMOSTABLE LIGATION ASSEMBLY (HTLA) FOR GENERATING DOUBLE-STRANDED DNA FRAGMENTS WITH SINGLE-STRANDED STICKY ENDS" (the entirety of which is incorporated herein by reference). HTLA is a simple assembly platform for creating ligable single-stranded overhangs of user-defined length, generating sticky-end blocks (SEBs), and assembling them into higher-order linear or cyclic structures. Starting materials for HTLA or CHTLA are dsDNA or oligonucleotide precursors that precisely self-assemble to create very long DNA. At least three precursors were used as reactants, as described in International Patent Application PCT / US2023 / 064977.

[0034] In simple terms, the HTLA process is an efficient DNA assembly process for producing user-defined heteroduplex DNA having a nucleotide length of 1 to several thousand (or more), ligationable, and wherein the heteroduplex DNA includes a 5' or 3' single-stranded overhang, or "sticky end," that can be ligated from a double-stranded or single-stranded DNA precursor molecule to form a closed-circular DNA molecule (see, for example, Figure 1). When such a process is performed in one cycle, it is called HTLA; for more than one cycle, the process is called Cyclic Heteroduplex Thermostable Ligase Assembly (CHTLA). As described in International Patent Application PCT / US2023 / 064977, at least three precursors were used as reactants.

[0035] Surprisingly, in addition to producing linear DNA, HLTA and CHTLA can also be used to produce closed circular DNA starting from just two precursors, e.g., dsDNA precursors or readily available and inexpensive DNA oligonucleotides (oligonucleotides). Furthermore, it was unexpectedly discovered that circular and supercoiled DNA can be efficiently obtained without using precursors containing intentional nicks and without adding bending proteins (e.g., Abf2p or HMGB1). As described herein, the method comprises the step of providing at least two DNA precursors designed by the user and generating a DNA sequence in which portions of the first and second strands of the DNA sequence overlap each other and, at the same time, are complementary to each other and complementary "sticky" ends are created that can ligate and form a covalently closed ring. This procedure is shown in Figure 1. A mixture of dsDNA (or oligo) precursors is heated and cooled in a buffer to form heteroduplex molecules I and II, and DNA ligase links the heteroduplex molecules to form a covalently closed circular DNA molecule that is not a plasmid vector (synthetic circular supercoiled DNA). SCSTo form DNA. In some embodiments, heating and cooling are performed multiple times (i.e., CHTLA). SCS The method increases the yield of DNA. As described, the method involves the formation of an intermediate heteroduplex having a sticky-end block (SEB), where one portion of the heteroduplex has a 5' overhang and the other portion has a 3' overhang. In some embodiments, the 5' overhang on one strand of the heteroduplex is complementary to the 5' overhang on the other strand of the same heteroduplex, resulting in the heteroduplex becoming circular (i.e., self-closing) by ligation of the 5' end of each strand with its own 3' end. In some embodiments, the 3' overhang on one strand of the heteroduplex is complementary to the 3' overhang on the other strand of the same heteroduplex, resulting in the heteroduplex becoming circular (i.e., self-closing) by ligation of the 5' end of each strand with its own 3' end. In some embodiments, the circular and supercoiled DNA described herein is produced without a plasmid vector and therefore does not contain bacterial DNA.

[0036] Since DNA ligases are not known to possess topoisomerase activity, the formation of supercoiled DNA using the HTLA and CHTLA steps was even more unexpected. Supercoilation of DNA is known to be important for accommodating DNA within any cell. Without supercoilation, which reduces the volume of DNA, it would be impossible to accommodate DNA within a cell. Furthermore, transfection of supercoiled DNA into cells (i.e., genetically modifying cells for certain purposes) is far more efficient than transfection of the same DNA in its linear or open (loose) circular form. In one embodiment, after the denaturation, annealing, and ligation steps, the resulting supercoiled DNA is produced. SCS A portion of the DNA is negatively supercoiled in the presence of DNA ligase. In some embodiments, type II topoisomerase, SCSIt is introduced into DNA (for example, SCS The environment in which DNA is formed contains a thermostable type II topoisomerase, or SCS (By adding type II topoisomerase at the appropriate time and temperature to the environment in which DNA is being formed), SCS It can effectively supercoil DNA.

[0037] For the purposes of this application, any enzyme that behaves like a type II topoisomerase capable of inducing DNA supercoilation is acceptable. In some embodiments, the type II topoisomerase is DNA gyrase or topoisomerase IV. DNA gyrase acts via transient double-strand breaks in DNA rather than nicks, and it has been previously reported that DNA gyrase alters the number of DNA bonds by two (PO Brown and NR Cozzarelli, Scince, 1979, 206(4422), 1081-1083). As discussed herein, the presence of a type II topoisomerase, e.g., DNA gyrase, during the CHTLA reaction in the absence of type II topoisomerase is beneficial. SCS For any supercoilation that can occur in DNA, SCS This substantially supercoils the DNA. Therefore, for the purposes of this application, "substantially supercoiled" or "substantially supercoiled" means the degree of supercoilation induced by type II topoisomerase that is produced during the CHTLA reaction in the absence of type II topoisomerase. SCS This corresponds to a degree superior to supercoilation in DNA. In relation to this and the number of entanglements, for the purposes of this application, ΔLk ss = Lk ss - Lk x The following can be calculated, and here, Lk ss In the presence of type II topoisomerase, it is substantially supercoiled. SCS Lk is the number of DNA entanglements, and Lk is the number in the absence of type II topoisomerase. SCS This is the number of DNA entanglements, and the calculated ΔLk ss(For example, -2, -4, -6, -8, -10, etc.) are equivalent to effective supercoilization.

[0038] It should be understood by those skilled in the art that some type II topoisomerases are thermally stable at temperatures of at least about 95°C, while others are not. In some embodiments, thermally stable type II topoisomerase species are thermally stable at temperatures in the range of about 37°C to 100°C, or about 60°C to 100°C, or about 80°C to 100°C, for a period of time in the range of about 30 seconds to about 10 minutes or about 1 minute to about 5 minutes. Therefore, when “thermally stable type II topoisomerase” is used hereafter, it should be understood that the type II topoisomerase can withstand the high temperatures of the denaturation / melting process while maintaining some or all of its activity. In some embodiments, the thermally stable type II topoisomerase is a thermally stable DNA gyrase. Thermally stable DNA gyrarases are known in the art and can be manipulated to be thermally stable over a preferred temperature range.

[0039] In a first embodiment, a method for forming an adhesive end block (SEB) having a 5' or 3' overhang is described, A step of introducing at least two precursor DNA fragments into a buffer medium containing a heat-stable DNA ligase enzyme, and a step of correctly assembling the precursor DNA fragments to produce a defined DNA sequence. The steps include: applying heat to a first temperature to induce denaturation of the at least two precursor DNA fragments, and The steps include lowering the temperature to a second temperature, annealing in the presence of the heat-stable DNA ligase enzyme, thereby producing a double-stranded DNA heteroduplex formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a 3' overhang; Includes. In some embodiments, the method for forming SEB is a one-pod method. In some embodiments, the second temperature is lower than the first temperature. In some embodiments, at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, for example, as shown in Figure 1, only two precursor DNA fragments are present, which undergo denaturation, annealing, and ligation to form two heteroduplex species.

[0040] In some embodiments, the desired SEB product of the first embodiment is obtained from the precursor DNA fragment by, for example, agarose gel purification or any other separation method, in the subsequent second and / or third reactions. SCS It can be purified for the production of DNA and / or supercoils.

[0041] In some embodiments, the buffer medium for the first embodiment includes a buffer for maintaining pH. In some embodiments, the buffer includes a combination of ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, DTT, NAD, ATP, and Triton® X-100, and maintains the pH at about 4 to about 12, or about 6 to 10, or about 7.5 to about 9. The temperature of the denaturation step (also referred to as the “melting” temperature or first temperature) may be in the range of about 37°C to 100°C, or about 60°C to 100°C, or about 80°C to 100°C, over a period of time ranging from about 0.1 minutes to about 60 minutes or about 1 minute to about 5 minutes. The annealing step is performed by lowering the temperature to a second temperature (i.e., a temperature approximately 5°C to 60°C lower than the temperature of the denaturation step) or to a second temperature approximately 10°C to 40°C lower than the temperature of the denaturation step, over a period of time ranging from approximately 0.1 minutes to approximately 60 minutes or approximately 4 minutes to 6 minutes.

[0042] In some embodiments, the precursor DNA fragments of Figure 1 and all precursors described herein are specifically defined and designed by the user. For example, consider a simple case for designing two double-stranded precursors (I and II) required to create a 100-base-pair SEB heteroduplex I, where 94 base pairs are duplicated to form a double strand and both ends have 5' overhangs. In this example, the 5' overhangs (i.e., GGGGGG and CCCCCC) are complementary. An opposite heteroduplex II with complementary 3' overhangs is also formed. Precursor I may be a double-stranded molecule consisting of 1 to 100 base pairs. Precursor II may be a double-stranded molecule consisting of 1 to 6 base pairs 7 to 100 followed by nucleotides 1 to 6 (see, for example, green 1 to 6 in Precursor II). In this example, the first strand of nucleotides 1 to 100 of Precursor I and the second strand of nucleotides 7 to 100 followed by nucleotides 1 to 6 of Precursor II are complementary at bases 7 to 100. When denaturation and annealing occur to form heteroduplex I, the first strand of nucleotides 7-100 of precursor I and the second strand of nucleotides 1-6 following nucleotides 7-100 of precursor II anneal to produce a 94-base pair double-stranded region. Nucleotides 1-6 of precursor I (see green 1-6 in precursor I) do not have complementary bases in precursor II, and therefore remain single-stranded. Similarly, nucleotides 1-6 in precursor II do not have complementary bases in precursor I, and therefore remain single-stranded. That is, if the precursors are designed so that the 5' overhangs of heteroduplex I are complementary, they will ligate together to form a 100-base pair region. SCSDNA can be formed. The same applies to heteroduplex II, which is composed of a 3' overhang. In addition to the formation of the SEB product, it is obvious that the two precursors I and II can also be reformed by the re-annealing of their complementary strands. Those skilled in the art will understand that the overhangs may be more or less than the 6 base pairs shown in this example, for example, 2 to several thousand base pairs, and may contain any combination of nucleotides as long as they are complementary to each other and can form a covalently closed ring by ligation of the complementary overhangs.

[0043] In a second embodiment, synthetic circular supercoiled DNA ( SCS A method for generating DNA is described, and this method is described as A step of introducing at least two precursor DNA fragments into a buffer medium containing a heat-stable DNA ligase enzyme, and a step of correctly assembling the precursor DNA fragments to produce a defined DNA sequence. The steps include: applying heat to a first temperature to induce denaturation of the at least two precursor DNA fragments, and The step of lowering the temperature to a second temperature and annealing in the presence of the heat-stable DNA ligase enzyme, thereby producing a double-stranded DNA heteroduplex formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a single-stranded 3' overhang, wherein the 5' intramolecular overhangs on the heteroduplex molecule are complementary and ligation occurs in both DNA strands, SCS When DNA is generated and the 3' intramolecular overhangs on the heteroduplex molecule are complementary, and ligation occurs in both DNA strands, SCS The step in which DNA is generated, Includes. In some embodiments, the method for generating synthetic cyclic supercoiled DNA is a one-pod method. In some embodiments, the second temperature is lower than the first temperature. In some embodiments, the steps of heating to the first temperature to denature and then lowering to the second temperature to anneal are repeated in cycles. In some embodiments, the number of cycles ranges from 1 to 100 cycles. In some embodiments, at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, for example, as shown in Figure 1, only two precursor DNA fragments are present, which undergo denaturation, annealing, and ligation to form two heteroduplex species. In some embodiments, SCS A portion of the DNA is negatively supercoiled in the presence of a heat-stable DNA ligase enzyme. In some embodiments, a single-stranded DNA-binding protein or a heat-stable single-stranded DNA-binding protein is present during the reaction, for example, at some point before the annealing step, either by being added to the buffer medium or the environment, to facilitate the annealing of the DNA strands.

[0044] In some embodiments of the second embodiment, the buffer medium containing the heat-stable DNA ligase enzyme includes a buffer for maintaining pH. In some embodiments, the buffer includes a combination of ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, DTT, NAD, and Triton® X-100, and maintains the pH at about 4 to about 12, or about 6 to 10, or about 7.5 to about 9. The temperature of the denaturation step (also referred to as the “melting” temperature or first temperature) may be in the range of about 37°C to 100°C, or about 60°C to 100°C, or about 80°C to 100°C, over a period of time ranging from about 0.1 minutes to about 60 minutes or about 1 minute to about 5 minutes. The annealing step is performed by lowering the temperature to a second temperature (i.e., a temperature about 5°C to 60°C lower than the temperature of the denaturation step) or to a temperature about 10°C to 40°C lower than the temperature of the denaturation step, over a period of time ranging from about 0.1 minutes to about 60 minutes or from about 4 minutes to 6 minutes. Therefore, in some embodiments, the second temperature is in the range of about 25°C to 85°C, or about 25°C to 70°C, or about 25°C to 65°C, or about 37°C to 65°C, or 50°C to 70°C, over a period of time ranging from about 0.1 minutes to about 60 minutes or from about 4 minutes to 6 minutes. As described, the denaturation / annealing process may be cycled for about 2 to about 100 times, where each cycle is: SCS This increases the yield of DNA. Therefore, in some embodiments, the nucleic acid ligation scheme is a temperature cycle from approximately 80°C to 100°C to approximately 40°C to approximately 70°C, for example, over 2 to 100 cycles.

[0045] In some embodiments, synthetic cyclic supercoiled DNA ( SCS Methods for generating DNA include: A step of introducing at least two precursor DNA fragments into a buffer medium having a pH of approximately 7.5 to approximately 9 and containing ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, NAD, DTT, Triton X-100, and at least one heat-stable ligase, and a step of correctly assembling the precursor DNA fragments to produce a defined DNA sequence. A step of applying heat at a first temperature to induce denaturation of the at least two precursor DNA fragments, wherein the first temperature is determined by the size of the DNA sequence and can be in the range of approximately 37°C to 100°C for a time period of approximately 0.1 minutes to approximately 60 minutes, and The step of lowering the temperature to a second temperature and annealing in the presence of the heat-stable DNA ligase enzyme, wherein the second temperature is 10°C to 40°C lower than the first temperature for a period of time ranging from about 4 minutes to about 10 minutes, and this step produces a heteroduplex of double-stranded DNA formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a single-stranded 3' overhang, where the 5' intramolecular overhangs on the heteroduplex molecule are complementary and ligation occurs in both DNA strands, SCS When DNA is generated and the 3' intramolecular overhangs on the heteroduplex molecule are complementary, and ligation occurs in both DNA strands, SCS The step in which DNA is generated. In some embodiments, the steps of heating to a first temperature to denature and then lowering to a second temperature to anneal are carried out in repeated cycles. In some embodiments, the number of cycles ranges from 2 to 100. In some embodiments, at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, for example, as shown in Figure 1, only two precursor DNA fragments are present, which undergo denaturation, annealing, and ligation to form two heteroduplex species. In some embodiments,SCS A portion of the DNA is negatively supercoiled in the presence of a heat-stable DNA ligase enzyme. In some embodiments, a single-stranded DNA-binding protein or a heat-stable single-stranded DNA-binding protein is present during the reaction, for example, at some point before the annealing step, either by being added to the buffer medium or the environment, to facilitate the annealing of the DNA strands.

[0046] In a third embodiment, a method for generating substantially supercoiled DNA is described, the method is A step of introducing at least two precursor DNA fragments into a buffer medium containing a heat-stable DNA ligase enzyme and a heat-stable type II topoisomerase, wherein the precursor DNA fragments are correctly assembled to produce a defined DNA sequence. The steps include: applying heat to a first temperature to induce denaturation of the at least two precursor DNA fragments, and The following is a step to lower the temperature to a second temperature: (i) Annealing in the presence of the heat-stable DNA ligase enzyme to produce a double-stranded DNA heteroduplex formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a 3' overhang, where the 5' intramolecular overhangs on the heteroduplex molecule are complementary and ligation occurs in both DNA strands, SCS When DNA is generated and the 3' intramolecular overhangs on the heteroduplex molecule are complementary, and ligation occurs in both DNA strands, SCS DNA is generated, and (ii) In the presence of thermally stable type II topoisomerase, SCS To essentially supercoil DNA, Includes. In some embodiments, the method for generating substantially supercoiled DNA is a one-pod method. In some embodiments, the second temperature is lower than the first temperature. In some embodiments, the steps of heating to the first temperature to denature and then lowering to the second temperature to anneal / ligate / supercoil are carried out in repeated cycles. In some embodiments, the number of cycles ranges from 2 to 100. In some embodiments, at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, the heat-stable type II topoisomerase includes DNA gyrase or topoisomerase IV. In some embodiments, for example, as shown in Figure 1, only two precursor DNA fragments are present, which undergo denaturation, annealing, and ligation to form two heteroduplex species. In some embodiments, as understood by those skilled in the art, 3, 4, 5, 6, 7, 8, 9, or 10 or more precursor DNA fragments are present. In some embodiments, a single-stranded DNA-binding protein or a heat-stable single-stranded DNA-binding protein is present during the reaction, for example, at some point before the annealing step, either by being added to the buffer medium or the environment, to facilitate the annealing of the DNA strands.

[0047] In some embodiments of the third aspect, the buffer medium containing the heat-stable DNA ligase enzyme and the heat-stable type II topoisomerase includes a buffer for maintaining pH. In some embodiments, the buffer includes a combination of ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, DTT, NAD, and Triton® X-100, and maintains the pH at about 4 to about 12, or about 6 to 10, or about 7.5 to about 9. The temperature of the denaturation step (also referred to as the “melting” temperature or first temperature) may be in the range of about 37°C to 100°C, or about 60°C to 100°C, or about 80°C to 100°C, over a period of time in the range of about 0.1 minutes to about 60 minutes or about 1 minute to about 5 minutes. The annealing / ligation / supercoiling step is carried out by lowering the temperature to a second temperature (i.e., a temperature about 5°C to 60°C lower than the temperature of the denaturation step) or to a temperature about 10°C to 40°C lower than the temperature of the denaturation step, over a period of time ranging from about 0.1 minutes to about 60 minutes or from about 4 minutes to 6 minutes. Thus, in some embodiments, the second temperature is in the range of about 25°C to 85°C, or about 25°C to 70°C, or about 25°C to 65°C, or about 37°C to 65°C, or 50°C to 70°C, over a period of time ranging from about 0.1 minutes to about 60 minutes or from about 4 minutes to 6 minutes. As described, the step or the step of raising the temperature to the first temperature and the step of lowering the temperature to the second temperature may be cycled for about 2 to about 100 times, where each cycle increases the yield of supercoiled DNA molecules and the degree of supercoiling of individual molecules. Accordingly, in some embodiments of the third aspect, the supercoilation scheme is, for example, a temperature cycle of about 80°C to 100°C to a second temperature of about 40°C to 70°C over 2 to 100 cycles.

[0048] In some embodiments, a method for generating substantially supercoiled DNA includes the following: The steps include introducing at least two precursor DNA fragments into a buffer medium having a pH of approximately 7.5 to 9 and containing ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, NAD, DTT, Triton X-100, at least one heat-stable ligase, and a heat-stable type II topoisomerase, and correctly assembling the precursor DNA fragments to produce a defined DNA sequence. A step of applying heat at a first temperature to induce denaturation of the at least two precursor DNA fragments, wherein the first temperature for denaturation is determined by the size of the DNA sequence and can be in the range of about 37°C to 100°C for a time period of about 0.1 minutes to about 60 minutes, and The following is a step to lower the temperature to a second temperature: (i) Annealing in the presence of the heat-stable DNA ligase enzyme, wherein the second temperature is 10°C to 40°C lower than the first temperature for a period of time ranging from about 4 minutes to about 10 minutes, and this step produces a heteroduplex of double-stranded DNA formed by base pairing of complementary regions, a portion of the heteroduplex having a single 5' overhang, and a portion of the heteroduplex having a single 3' overhang, where the 5' intramolecular overhangs on the heteroduplex molecule are complementary and ligation occurs in both DNA strands, SCS When DNA is generated and the 3' intramolecular overhangs on the heteroduplex molecule are complementary, and ligation occurs in both DNA strands, SCS DNA is generated, and (ii) In the presence of the aforementioned thermally stable type II topoisomerase, SCS The process of essentially supercoiling DNA. In some embodiments, the method for generating substantially supercoiled DNA is the one-pod method. In some embodiments, the steps involve heating to a first temperature to denature the DNA, and then lowering the temperature to a second temperature to anneal it. SCSThe steps for generating DNA and supercoils are carried out in repeated cycles. In some embodiments, the number of cycles ranges from 2 to 100. In some embodiments, at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, the thermally stable type II topoisomerase includes DNA gyrase. In some embodiments, as shown in Figure 1, for example, only two precursor DNA fragments are present, which undergo denaturation, annealing, and ligation to form two heteroduplex species. In some embodiments, as understood by those skilled in the art, three, four, five, six, seven, eight, nine, or ten or more precursor DNA fragments are present. In some embodiments, a single-stranded DNA-binding protein or a thermally stable single-stranded DNA-binding protein is present during the reaction, for example, at some point before the annealing step, either added to the buffer medium or added to the environment, to facilitate the annealing of the DNA strands.

[0049] In a fourth embodiment, a method for generating substantially supercoiled DNA is described, the method is A step of introducing at least two precursor DNA fragments into a buffer medium containing a heat-stable DNA ligase enzyme, and a step of correctly assembling the precursor DNA fragments to produce a defined DNA sequence. The steps include: applying heat to a first temperature to induce denaturation of the at least two precursor DNA fragments, and The step of lowering the temperature to a second temperature and annealing in the presence of the heat-stable DNA ligase enzyme, thereby producing a double-stranded DNA heteroduplex formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a single-stranded 3' overhang, wherein the 5' intramolecular overhangs on the heteroduplex molecule are complementary and ligation occurs in both DNA strands, SCSWhen DNA is generated and the 3' intramolecular overhangs on the heteroduplex molecule are complementary, and ligation occurs in both DNA strands, SCS The step of generating DNA, and The temperature is lowered to a third temperature, and a bolus of type II topoisomerase is added, in the presence of the type II topoisomerase. SCS A step to initiate the effective supercoilation of DNA, Includes. In some embodiments, the second temperature is lower than the first temperature. In some embodiments, the third temperature is lower than the second temperature. In some embodiments, the steps of heating to the first temperature to denature, lowering to the second temperature to anneal, and further lowering to the third temperature to supercoil are carried out in repeated cycles. In some embodiments, the number of cycles ranges from 2 to 100 cycles. In some embodiments, at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, the type II topoisomerase includes DNA gyrase or topoisomerase IV. In some embodiments, for example, as shown in Figure 1, only two precursor DNA fragments are present, which undergo denaturation, annealing, and ligation to form two heteroduplex species. In some embodiments, as understood by those skilled in the art, 3, 4, 5, 6, 7, 8, 9, or 10 or more precursor DNA fragments are present. In some embodiments, a single-stranded DNA-binding protein or a heat-stable single-stranded DNA-binding protein is present during the reaction, for example, at some point before the annealing step, either by being added to the buffer medium or the environment, to facilitate the annealing of the DNA strands.

[0050] In some embodiments of the third embodiment, the buffer medium containing the heat-stable DNA ligase enzyme includes a buffer for maintaining pH. In some embodiments, the buffer includes a combination of ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, DTT, NAD, and Triton® X-100, and maintains the pH at about 4 to about 12, or about 6 to 10, or about 7.5 to about 9. The temperature of the denaturation step (also referred to as the “melting” temperature or first temperature) may be in the range of about 37°C to 100°C, or about 60°C to 100°C, or about 80°C to 100°C, over a period of time ranging from about 0.1 minutes to about 60 minutes or about 1 minute to about 5 minutes. The annealing step is carried out by lowering the temperature to a second temperature (i.e., a temperature about 5°C to 60°C lower than the temperature of the denaturation step) or to a temperature about 10°C to 40°C lower than the temperature of the denaturation step, over a period of time ranging from about 0.1 minutes to about 60 minutes or from about 4 minutes to 6 minutes. Thus, in some embodiments, the second temperature is in the range of about 25°C to 85°C, or about 25°C to 70°C, or about 25°C to 65°C, or about 37°C to 65°C, or 50°C to 70°C, over a period of time ranging from about 0.1 minutes to about 60 minutes or from about 4 minutes to 6 minutes. It should be understood by those skilled in the art that the second temperature is higher than the third temperature. The third temperature depends on the properties of the type II topoisomerase and that the type II topoisomerase is not thermally decomposed. SCS The DNA is selected to ensure it is available for supercoilation. As described, the denaturation / annealing / supercoilation process may be cycled for about 2 to about 100 times, where each cycle increases the yield of supercoiled DNA molecules and the degree of supercoilation of individual molecules. Thus, in some embodiments, the nucleic acid supercoilation scheme is a temperature cycle over 2 to 100 cycles, for example, from a first temperature of about 80°C to 100°C to a second temperature of about 40°C to about 70°C, and then to a third temperature lower than the second temperature.

[0051] Referring to Figure 1, it is shown that heteroduplexes I and II are ligable to each other and can ligate to form concatemers. However, without wanting to get bogged down in theory, intramolecular (or self) ligation for cyclization is considerably preferable to intermolecular ligation for concatemer formation.

[0052] In some embodiments, SCS After the generation of DNA and / or supercoiled DNA, concatemers and tangled DNA, as well as precursors, can be removed by treatment with a 5'-3' or 3'-5' exonuclease, such as a T5 exonuclease or a T7 exonuclease. The exonuclease can be degraded by proteinase K digestion, and the DNA product precipitates from the solution. Alternatively, column-based methods for deproteinizing a sample and purifying DNA are known in the art, as understood by those skilled in the art.

[0053] As discussed above in this specification, in some embodiments, the 5' overhang of one strand of a heteroduplex is complementary to the 5' overhang of the opposite strand of the same heteroduplex, resulting in the heteroduplex becoming circular (i.e., self-closing) through annealing of the overhangs. Ligation of the 5' end of one strand with the 3' end just adjacent to it on the same strand results in a complete phosphodiester skeleton on one strand. If the same thing happens on the opposite strand, a covalently closed circular double-stranded DNA is obtained. Furthermore, in some embodiments, the 3' overhang of one strand of a heteroduplex is complementary to the 3' overhang of the opposite strand of the same heteroduplex, resulting in the heteroduplex becoming circular (i.e., self-closing) through annealing of the overhangs. Ligation of the 3' end of one strand with the 5' end just adjacent to it on the same strand results in a complete phosphodiester skeleton on one strand. If the same thing happens on the opposite strand, it results in a circular double-stranded DNA molecule closed by covalent bonds.

[0054] In some embodiments, the circular and supercoiled DNA described herein is generated without a plasmid vector and therefore does not contain bacterial DNA. Advantageously, since the DNA precursor fragment is designed by the user, the DNA precursor fragment can be part of a designed vector (plasmid, cosmid, BAC, YAC, etc.) so that it is liberated from the vector (e.g., by restriction endonuclease digestion), and complementary sticky ends are formed in the heteroduplex molecule, so a fully closed circular vector can be obtained using the method described herein, where the circular and supercoiled DNA product does not contain bacterial DNA.

[0055] In some embodiments, the circular and supercoiled DNA described herein is generated using a plasmid (or cosmid, BAC, YAC, etc.) vector and the DNA sequence of interest, and thus has the structure of a conventional cloning vector and insert, but is entirely synthesized and produced through an HTLA or CHTLA process.

[0056] Therefore, in the fifth embodiment, synthetic cyclic supercoiled DNA ( SCS DNA is described here, SCS The DNA does not include any DNA of bacterial or viral origin. In some embodiments, SCS DNA is not a plasmid vector. In some embodiments, the fifth embodiment SCS DNA is essentially in a supercoiled state. SCS DNA sequences are entirely user-defined (i.e., scarless) and produced in a one-pot method (e.g., any of the first, second, third, or fourth embodiments described herein) and can be generated in quantities useful for gene therapy, cell engineering (i.e., CAR-T therapy), vaccines, and genome engineering in bacteria, yeast, or other organisms. For example, in some embodiments, SCSThe DNA is a vaccine, and the precursor DNA fragment is designed by the user to be directed to the expression of the vaccine antigen, the precursor DNA fragment is produced in large quantities, and the precursor DNA fragment is subsequently directed to express the vaccine antigen via HTLA or CHTLA as described herein, although it is not a plasmid vector. SCS It generates DNA. In other examples, SCS DNA is used to carry payload genes in gene therapy, either as naked DNA or in formulations designed to enhance cellular uptake by conventional or cell- or tissue-targeted methods. In other examples, SCS DNA encodes one or more components required to produce lentiviruses, adeno-associated viruses, or other viruses containing specific payloads for gene therapy or modification of cells in culture in living organisms (including humans). In other examples, SCS The DNA has the same structure as conventional plasmids (or cosmids, BACs, YACs, etc.), but is entirely synthesized, allowing for the mass production of, for example, low-copy conventional vectors with inserts. [Examples]

[0057] (Example 1) Generated by CHTLA SCS DNA is partially supercoiled. To demonstrate that CHTLA produces cyclic products with varying degrees of de novo supercoilation, pMaxGFP plasmids were digested with either KpnI or XhoI, resulting in 700 bp linearly overlapping DNA fragment offsets with completely homologous offset regions. The digested DNA was purified, restriction enzymes were removed, and approximately 5 μg of each fragment was mixed in a 1:1 ratio in 50 μl of buffer containing 20 mM Tris-HCl, 150 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM (nicotinamide adenine dinucleotide) NAD, 0.1% Triton™ X-100 (pH 8.5 at 25°C), and 1 μL of 44 nM HiFi Taq ligase (New England Biolabs). The mixture was then subjected to 10 cycles of heating from 95°C to 60°C and cooling in a standard thermal cycler. SCS DNA was prepared. Ten cycles of the CHTLA reaction were completed, and aliquots of the product were treated with 10 units of T5 exonuclease at 37°C for 1 hour. To demonstrate that the CHTLA product is supercoiled, the CHTLA product was analyzed on an ethidium bromide-free agarose gel and compared with pMaxGFP plasmid purified from bacteria. After electrophoresis, the gel was stained with ethidium bromide. Analysis of the resulting gel revealed a series of bands in the lane containing the CHTLA product treated with T5 exonuclease (see Figure 2, lane "V"). The bands correspond to closed-circular CHTLA product DNA with varying degrees of superhelical twisting, and include supercoiled fractions similar to those seen in bacterial pMaxGFP plasmids.

[0058] (Example 2) CHTLA synthesis of circular DNA using single-stranded oligonucleotide as a precursor To demonstrate that CHTLA converts single-stranded oligonucleotides into closed-cyclic products, six 80-nucleotide-long oligonucleotides with 40 nucleotide duplicates and phosphorylated at the 5' position were subjected to CHTLA. The oligonucleotides were mixed at their final concentrations of 2 micromolar in 50 μl of buffer containing 20 mM Tris-HCl, 150 mM KCl, 10 mM MgCl2, 10 mM DTT, 1 mM NAD, 0.1% Triton™ X-100 (pH 8.5 at 25°C), and 1 μL of 44 nM HiFi Taq ligase (New England Biolabs), and subjected to 10 cycles of heating from 95°C to 60°C and cooling in a standard thermal cycler. In parallel, the same reaction was carried out without the HiFi-Taq ligase. Ten cycles of the CHTLA reaction were completed, and aliquots of the product were treated with 10 units of T5 exonuclease at 37°C for 1 hour. The CHTLA products exposed to T5 exonuclease and those not exposed were subsequently analyzed on an agarose gel containing ethidium bromide. As can be seen in Figure 3, a significant portion of the CHTLA products were resistant to T5 exonuclease digestion, demonstrating that they possessed a closed cyclic structure.

[0059] (Example 3) From linear dsDNA precursor to supercoiled SCS DNA CHTLA synthesis To demonstrate that CHTLA converts linear dsDNA precursors into closed-cyclic and supercoiled cyclic products, the pBluescript-SK (-) plasmid was digested with restriction enzymes BamHI and KpnI to produce two overlapping linear DNA molecules with a 62 bp offset, such that the offset regions were completely homologous. Approximately 5 μg of each fragment was mixed in a 1:1 ratio in 50 μL of buffer containing 20 mM Tris-HCl, 150 mM KCl, 10 mM MgCl2, 10 mM DTT, 1 mM NAD, 0.1% Triton™ X-100 (pH 8.5 at 25°C), and 1 μL of 44 nM HiFi Taq ligase (New England Biolabs). The mixture was subjected to 10 cycles of heating from 95°C to 60°C and cooling in a standard thermal cycler. To demonstrate that the CHTLA product is a nearly completely covalently closed circular DNA, the CHTLA product was directly treated with 10, 2, or 0.4 units of T5 exonuclease in HiFi-Taq ligase reaction buffer and incubated at 37°C for 1 hour. Aliquots of linear CHLTA precursors were also treated in parallel with T5 exonuclease. The products of the T5 exonuclease reaction were subsequently analyzed on an agarose gel containing ethidium bromide (in the gel), as shown in Figure 4. Ethidium bromide intercalates into the interior of the double-stranded DNA during the electrophoretic separation step, inducing negative supercoilation in some, but not all, of the closed circular DNA. Image analysis of the obtained gel revealed that all of the linear precursor molecules were completely digested by T5 exonuclease, while approximately 90% of the CHTLA product was resistant to T5 exonuclease digestion, indicating that the CHTLA product has a closed cyclic structure. This data suggests that two-fragment HLTA using dsDNA precursors efficiently produces circular DNA molecules, and that a high proportion of the product is supercoiled. SCSThis demonstrates that the DNA form is present. Furthermore, the closed-circular CHTLA product becomes more supercoiled during the electrophoresis process as a result of ethidium bromide interposition.

[0060] (Example 4) Prepared by CHTLA SCS DNA is negatively supercoiled. Fabricated by CHTLA SCS To demonstrate that DNA is negatively supercoiled, SCS DNA and the pBluescript-SK (-) plasmid were linearized using either BamHI or KpnI to produce duplicate DNA molecules with a 62 bp offset (see Figure 5A). Approximately 5 μg of each fragment was mixed in 50 μL of buffer containing 20 mM Tris-HCl, 150 mM KCl, 10 mM MgCl2, 10 mM DTT, 1 mM NAD, 0.1% Triton™ X-100 (pH 8.5 at 25°C), and 1 μL of 44 nM HiFi Taq ligase (New England Biolabs). The mixture was subjected to 10 cycles of heating from 95°C to 60°C and cooling in a standard thermal cycler. After completing 10 cycles of the CHTLA reaction, aliquots of the product were treated with 10 units of T5 exonuclease at 37°C for 1 hour. The resulting T5 exonuclease-resistant products were degraded in agarose gels containing or without chloroquine. Chloroquine intercalates into the DNA, inducing positive helical twisting. Neither gel contained ethidium bromide. After electrophoresis was complete, both gels were stained with ethidium bromide. In the chloroquine-free gel, cyclic DNA with varying degrees of superhelical twisting was observed. SCSMultiple bands representing DNA were observed (yellow box, column VL, see Figure 5B). Upon exposure to chloroquine, these multiple bands "upward" disintegrate into a single species (red arrow, see Figure 5B). Similarly, negatively supercoiled plasmid DNA of bacterial origin also shifts upward upon exposure to chloroquine (see green arrow in Figure 5B, moving from the chloroquine-free side to the chloroquine-containing side), and is observed as multiple species with varying degrees of superhelical twist. Therefore, in the presence of chloroquine, SCS Both DNA and negatively supercoiled plasmid DNA derived from bacteria migrated similarly, although in smaller quantities overall than bacterial plasmid DNA. SCS DNA has also been shown to be negatively supercoiled.

Claims

1. Synthetic circular supercoiled DNA ( SCS A method for generating DNA, wherein the method is A step of introducing at least two precursor DNA fragments into a buffer medium containing a heat-stable DNA ligase enzyme, and a step of correctly assembling the precursor DNA fragments to produce a defined DNA sequence. The steps include: applying heat to a first temperature to induce denaturation of the at least two precursor DNA fragments, and The following is a step to lower the temperature to a second temperature: (i) Annealing in the presence of the heat-stable DNA ligase enzyme to produce a double-stranded DNA heteroduplex formed by base pairing of complementary regions, a portion of the heteroduplex having a single-stranded 5' overhang, and a portion of the heteroduplex having a 3' overhang, where the 5' intramolecular overhangs on the heteroduplex molecule are complementary and ligation occurs in both DNA strands, SCS When DNA is generated and the 3' intramolecular overhangs on the heteroduplex molecule are complementary, and ligation occurs in both DNA strands, SCS DNA is generated, and (ii) In the presence of thermally stable type II topoisomerase, SCS To essentially supercoil DNA, Methods that include...

2. SCS The method according to claim 1, wherein the method for generating DNA is a one-pot method.

3. The method according to claim 1 or 2, wherein the at least two precursor DNA fragments are selected from double-stranded (ds) DNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof.

4. The method according to any of the preceding claims, wherein two precursor DNA fragments are used.

5. The method according to any of the preceding claims, wherein the second temperature is lower than the first temperature.

6. The method according to any of the preceding claims, wherein the first temperature is in the range of about 37°C to about 100°C, preferably about 80°C to 100°C.

7. The method according to claim 6, wherein the first temperature is applied for a period of time ranging from about 30 seconds to about 10 minutes, preferably from about 1 minute to about 5 minutes.

8. The method according to any of the preceding claims, wherein the second temperature is about 25°C to about 85°C, preferably 50°C to about 70°C.

9. The method according to claim 8, wherein the second temperature is maintained for a period of time ranging from about 0.1 minutes to about 60 minutes.

10. The method according to any of the preceding claims, wherein the steps of heating to a first temperature to cause denaturation and lowering to a second temperature to cause annealing are carried out in repeated cycles.

11. The method according to claim 10, wherein the number of cycles is in the range of 2 to 100 cycles.

12. The method according to any one of the preceding claims, wherein, during annealing, a heteroduplex double-stranded DNA sequence is produced by base pairing of complementary regions, wherein the heteroduplex includes a 5' or 3' overhang.

13. When the 5' or 3' overhangs are complementary, SCS The method according to claim 12, wherein DNA is produced.

14. The buffer consists of ATP, Tris-HCl, and MgCl. 2 The method according to any of the prior claims, comprising a combination of KCl, NaCl, DTT, β-mercaptoethanol, NAD, and TRITON X-100.

15. The method according to claim 14, wherein the pH is maintained at about 4 to about 12, preferably about 7.5 to about 9.

16. The aforementioned SCS The method according to claim 4, wherein a portion of the DNA is negatively supercoiled in the presence of the thermally stable DNA ligase enzyme.

17. The method according to any one of the preceding claims, wherein the buffer medium further comprises a type II topoisomerase, preferably a heat-stable type II topoisomerase.

18. The foregoing SCS The method according to claim 17, wherein the DNA is substantially supercoiled.

19. The method according to claim 17 or 18, wherein the type II topoisomerase is thermally stable, and the thermally stable type II isomerase can be circulated while maintaining some or all of its activity through the denaturation and annealing steps.

20. The type II topoisomerase is either thermally stable or thermally unstable, and the method comprises the steps of lowering the temperature to a third temperature, and adding a bolus of type II topoisomerase in the presence of the type II topoisomerase. SCS The method according to claims 17 and 18, further comprising the step of initiating substantial supercoilation of DNA.

21. The method according to any one of claims 17 to 20, wherein the type II topoisomerase is a DNA gyrase.

22. The method according to any one of claims 17 to 20, wherein the type II topoisomerase is topoisomerase IV.

23. The method according to any of the preceding claims, wherein the at least two precursor DNA fragments do not contain intentional nicks.

24. The method according to any of the preceding claims, wherein no curving protein is added.

25. generated SCS The method according to any of the prior claims, wherein the DNA does not contain bacterial DNA.

26. The method according to the preceding claim, wherein a single-stranded DNA-binding protein is added.

27. The method according to the preceding claim, wherein a heat-stable single-stranded DNA-binding protein is added.

28. Synthetic circular supercoiled DNA produced according to the method of any one of claims 1 to 27 ( SCS DNA).

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