Method for generating superhelical circular DNA in vitro

Abstract

Present methods for the production of microrings are slow, expensive and difficult to carry out under GMP conditions, because in most cases, the product is derived from bacteria. In contrast, synthetic circular superhelix DNA production based on HTLA and CHTLA can be completed completely in a test tube, using chemically or enzymatically synthesized oligonucleotides, long single-stranded DNA, and / or double-stranded DNA.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 498,886, entitled “A METHOD TO PRODUCE SUPERCOILED CIRCULAR DNA IN VITRO,” filed April 28, 2023, in the names of Xiang LI and Charles J. BIEBERICH, which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0002] The present invention relates to methods of producing supercoiled plasmid- free circular DNA from two or more precursor DNA fragments, such as single-stranded DNA, double-stranded DNA, and / or oligonucleotides. BACKGROUND

[0003] Advances in our understanding of the molecular basis of disease have driven an explosive growth in new gene-based treatments, also known as gene therapy. Gene therapy is the deliberate genetic modification of DNA in a patient’s cells to achieve a specific therapeutic goal. After three decades of development, the technology has matured in recent years, and pharmaceutical companies have invested heavily in developing new therapies. Currently, there are over 800 clinical trials in the United States targeting a variety of single-gene genetic disorders as well as acquired diseases including cancer and rheumatoid arthritis. To date, five gene therapies have been approved by the FDA: ZYNTEGLO® for the treatment of beta thalassemia; KYMRIAH™, a CAR T cell therapy for relapsed or refractory leukemias and lymphomas; LUXTURNA™ for a genetic retinal disorder; SKYSONA® for cerebral adrenoleukodystrophy; and ZOLGENSMA® for spinal muscular atrophy. In 2023, the global gene therapy market was valued at $9 billion and is projected to reach $46 billion by 2030.

[0004] Historically, gene therapy has heavily relied on viruses as gene delivery vectors, and all five FDA-approved biologics are based on lentiviruses or adeno-associated viruses. During manufacturing, the therapeutic virus is rendered unable to replicate, thus preventing infection by the manufacturing virus in patients. While the approved virus-based gene therapies have a good safety profile, there are safety concerns that the therapeutic virus may recombine with other viruses during manufacturing or in patients, creating new viruses with unknown and potentially harmful properties, including replication. Furthermore, viruses have an inherent preference for infecting specific cell types, severely limiting the range of diseases that can be targeted. This concern, along with others including high manufacturing costs and labor-intensive Good Manufacturing Practice (GMP) workflows, has driven the search for non-viral alternatives to genetically modify patient cells. For example, naked DNA (i.e., not packaged within a viral capsid) can be used to deliver therapeutic genes, but delivering naked DNA into cells requires lipid-based delivery systems, ultrasound, electroporation, or ballistic (i.e., gene gun) methods using DNA-coated gold particles. Alternatively, non-viral gene delivery methods based on circular DNA, known as plasmids, generated in bacteria have been developed. However, plasmids inevitably carry bacterial DNA sequences, which trigger an immune response in patients, limiting their effectiveness.

[0005] To address this problem, small circular DNAs known as "microloops" have been developed, which are completely free of bacterial DNA. Microloops contain only the therapeutic gene and the DNA elements necessary to support its expression; all or almost all other bacterial-derived DNA, including antibiotic resistance genes, is removed, for example, through engineered recombination processes occurring within bacterial cells. Microloops have proven to be a powerful gene delivery platform, outperforming plasmids and producing higher and more durable therapeutic gene expression. However, microloops still require purification to remove other bacterial components, including the original plasmids from which they originated. This process is highly complex, and a single production run can take over 270 days. Enzymatic production of microloop-like DNA (i.e., canine bone DNA) in vitro reduces production time to 50 days, but significant challenges remain with GMP production.

[0006] There remains a need for improved methods to scale up the production of circular DNA (i.e., antibiotic resistance genes, origin of replication) in a GMP-friendly manner. This paper describes a new technique with the potential to produce and scale up circular supercoiled DNA in a fully synthetic manner. The technique utilizes a process called “heteroduplex thermostable ligase assembly,” in which single-stranded or double-stranded DNA precursors are denatured and annealed under specific conditions in the presence of thermostable DNA ligases, with or without thermostable type II topoisomerases, to produce circular, primarily supercoiled DNA molecules, or “synthetic circular supercoiled DNA.” SCS DNA). Summary of the Invention

[0007] In one aspect, a method for forming a sticky end block (SEB) having a 5' or 3' protruding end is described, the method comprising: At least two precursor DNA fragments are introduced into a buffer medium containing a thermostable DNA ligase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Apply heat to a first temperature to denature the at least two precursor DNA fragments; and The temperature is lowered to a second temperature for annealing in the presence of the thermostable DNA ligase, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions, a portion of which has a single-stranded 5' overhang and a portion of which has a 3' overhang.

[0008] In another aspect, a method for producing synthetic circular supercoiled DNA is described. SCS A method for (DNA) comprising: At least two precursor DNA fragments are introduced into a buffer medium containing a thermostable DNA ligase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Apply heat to a first temperature to denature the at least two precursor DNA fragments; and The temperature is lowered to a second temperature for annealing in the presence of the thermostable DNA ligase, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions. A portion of the heteroduplex has a single-stranded 5' overhang, and a portion has a single-stranded 3' overhang. The heteroduplex produces a product when the intramolecular 5' overhangs on the heteroduplex molecules are complementary and ligated onto the two DNA strands. SCS DNA, and when the 3' intramolecular overhangs on the heteroduplex molecule are complementary and join together on the two DNA strands, it produces SCS DNA.

[0009] In another aspect, a method for producing substantially supercoiled DNA is described, the method comprising: At least two precursor DNA fragments are introduced into a buffer medium containing a thermostable DNA ligase and a thermostable type II topoisomerase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Heat is applied to a first temperature to denature the at least two precursor DNA fragments; The temperature is lowered to a second temperature for (i) annealing in the presence of the thermostable DNA ligase, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions, a portion of which has a single-stranded 5' overhang and a portion of which has a 3' overhang, wherein when the intramolecular 5' overhangs on the heteroduplex molecule are complementary and ligated on the two DNA strands, a heteroduplex is produced. SCS DNA, and when the 3' intramolecular overhangs on the heteroduplex molecule are complementary and join together on the two DNA strands, it produces SCS DNA; and (ii) in the presence of a thermostable type II topoisomerase. SCS DNA is essentially supercoiled.

[0010] In another aspect, a method for producing substantially supercoiled DNA is described, the method comprising: At least two precursor DNA fragments are introduced into a buffer medium containing a thermostable DNA ligase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Heat is applied to a first temperature to denature the at least two precursor DNA fragments; The temperature is lowered to a second temperature for annealing in the presence of the thermostable DNA ligase, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions. A portion of the heteroduplex has a single-stranded 5' overhang, and a portion has a single-stranded 3' overhang. The heteroduplex produces a product when the intramolecular 5' overhangs on the heteroduplex molecules are complementary and ligated onto the two DNA strands. SCS DNA, and when the 3' intramolecular overhangs on the heteroduplex molecule are complementary and join together on the two DNA strands, it produces SCS DNA; and The temperature was lowered to a third temperature and a large dose of type II topoisomerase was added to initiate the process in the presence of the type II topoisomerase. SCS DNA is essentially supercoiled.

[0011] In another aspect, a synthetic circular supercoiled DNA ( SCS DNA), wherein SCS The DNA does not contain any DNA of bacterial or viral origin.

[0012] Other aspects, features, and embodiments of the invention will become more apparent from the following disclosure and claims. Attached Figure Description

[0013] Figure 1 Two-segment HTLA generates supercoil SCS DNA.

[0014] Figure 2 Generated by CHTLA SCS The DNA is supercoiled. Two plasmids, pMax-GFP... SCS The DNA precursor was prepared by digesting the plasmid with KpnI or XhoI. The digested DNA was purified to remove restriction enzymes and mixed in a 1:1 ratio. pMax-GFP was generated in 10 cycles of the CHTLA reaction. SCS DNA. Unreacted precursors were removed by treating the reaction mixture with T5 exonuclease. Analysis was performed on an agarose gel without ethidium bromide. SCS DNA. After electrophoresis, the gel was stained with ethidium bromide. P, plasmid pMax-GFP DNA prepared from bacteria; V, SCS DNA; L, 1-kb DNA ladder.

[0015] Figure 3 CHTLA synthesis of circular DNA using single-stranded oligonucleotides as precursors. Six overlapping 80-mer nucleotides (40 bases each) were phosphorylated at 37°C (via T4 polynucleotide kinase) and subjected to CHTLA reactions with or without HiFi-Taq ligase (left panel, lanes 2 and 3) or (right panel, lanes 4 and 5). The reaction products were then treated with T5 exonuclease at 37°C for 1 hour and analyzed on a gel containing ethidium bromide. The yellow box highlights the exonuclease-resistant circular DNA product generated in the CHTLA reaction with HiFi-Taq ligase (lane 3), but which was not generated in the control CHTLA reaction without ligase (lane 5). Lane 1, DNA ladder.

[0016] Figure 4 . SCS DNA was synthesized from a linear precursor via CHTLA. The plasmid-derived DNA precursor (restriction fragments, white arrows, lanes 1 and 4) was ligated in 10 cycles of CHTLA reaction. CHTLA converted the precursor DNA into relaxed circular (lane 2, blue arrow) and supercoiled (lane 2, yellow arrow) DNA. The precursor (lanes 4-7) or CHTLA product (lanes 8-10) was treated with T5 exonuclease (units shown in blue boxes). T5 treatment completely eliminated the linear DNA precursor (lanes 5-7), but… SCS The DNA CHTLA product is T5 exonuclease resistant (lanes 9-11).

[0017] Figure 5A Generated by CHTLA SCS DNA is negatively supercoiled. To prepare CHTLA precursors for production...SCS DNA was digested with plasmid pBluescript-SK(-) using BamHI or KpnI. The digested DNA was purified using a microcentrifuge column to remove restriction enzymes. The purified DNA was mixed in a 1:1 ratio and then ligated using HiFi-Taq DNA ligase in 10 cycles of CHTLA reaction. Unreacted precursors were removed using T5 exonuclease.

[0018] Figure 5B Analysis was performed using agarose gel electrophoresis with or without chloroquine, or with 2.5 μg / ml chloroquine. SCS DNA. After electrophoresis, the gel was stained with ethidium bromide. Yellow boxes indicate DNA with varying degrees of negative supercoil twisting. SCS The location of the DNA. The green arrows on the chloroquine-free side indicate the location of highly negatively supercoiled bacterial plasmid DNA. The red arrows indicate the location after chloroquine insertion. SCS The DNA has changed location, having collapsed into a single substance. The green box indicates the location of the bacterial-derived plasmid DNA after chloroquine insertion. Note the presence of multiple substances, indicating that the negative supercoiling underwent varying degrees of relaxation, producing a series of bands with different degrees of supercoiling distortion. Pr, linearized precursor DNA; VL, produced via CHTLA. SCS DNA; PL, pBluescript-SK(-) purified from bacteria; L, 1 kb DNA ladder; EB, ethidium bromide; after run, after electrophoresis. Detailed Implementation

[0019] Although the claimed subject matter will be described according to certain embodiments, other embodiments, including those that do not provide all the benefits and features set forth herein, are also within the scope of this disclosure. Various structural and parameter changes may be made without departing from the scope of this disclosure.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, this document (including the definitions) shall prevail. Preferred methods and materials are described below, although similar or equivalent methods and materials may be used in practice or testing in accordance with this disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0021] "Approximately" and "about" are used to provide flexibility for the endpoints of a numerical range, specifying that a given value can be "slightly above" or "slightly below" the endpoints without affecting the desired result, such as + / - 5%.

[0022] The phrases “in one embodiment” or “in some embodiments” as used herein do not necessarily refer to the same embodiment, although they may refer to the same embodiment. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to different embodiments, although it may refer to different embodiments. Therefore, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0023] The terms “comprising,” “including,” “having,” “may,” “containing,” and variations thereof, as used herein, are intended as open-ended transitional phrases, terms, or words that do not exclude the possibility of other actions or structures. Unless the context clearly specifies otherwise, no specific number of references includes plural references. This disclosure also contemplates other embodiments that include, constitute, and substantially constitute the embodiments or elements described herein, whether or not explicitly stated.

[0024] As used in this article, the term "heteroduplex" DNA molecule refers to a double-stranded molecule in which the first strand is derived from a double-stranded or single-stranded precursor DNA molecule and the second strand is derived from a different double-stranded or single-stranded DNA molecule, wherein the first and second strands are linked by Watson-Crick base pairing during annealing or hybridization, which is referred to as complementary DNA strands (i.e., the first and second strands).

[0025] As used herein, the terms “ligase” and “ligation reagent” are used interchangeably and refer to a variety of different enzymes or non-enzymatic reagents capable of ligating DNA molecules, for example, between two or more adjacent heteroduplexes with annealed, compatible single-stranded ends, or within a single heteroduplex molecule with compatible single-stranded ends, to form a circular molecule by establishing new bonds. In some embodiments, a ligase is an enzyme ligation reagent that, under appropriate conditions, forms a phosphodiester bond between the 3′-OH and 5′-phosphate of adjacent nucleotides in a DNA molecule, RNA molecule, oligonucleotide, or hybrid. Temperature-sensitive ligases include, but are not limited to, bacteriophage T4 ligase and Escherichia coli ligase. Thermostable ligases include, but are not limited to, Afu ligase, Taq ligase, Tfl ligase, Tth ligase, Tth HB8 ligase, Thermus AK16D ligase and Pfu ligase, HiFi Taq ligase, or Ampilgase. Those skilled in the art will understand that many different thermostable ligases, including DNA ligases and RNA ligases, can be obtained from certain species of thermophilic or hyperthermophilic organisms such as eubacteria and archaea; and that such ligases can be used in the disclosed methods and kits.

[0026] As used in this article, the term "overlapping sequence" refers to a sequence that is complementary in two polynucleotides, wherein the first polynucleotide contains an overlapping single-stranded (ss) sequence and can hybridize with a second polynucleotide containing a complementary ss sequence.

[0027] As used herein, the term "protrusion" refers to the single-stranded region at the end of double-stranded (ds) DNA, which can be classified as 5' or 3' type due to the inherent orientation of DNA. Protrusions of various lengths are typically produced 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 deoxynucleotidyl transferases). In some embodiments, the length of the protrusion ranges from 2 to 1000 base pairs.

[0028] As used herein, the term "double-stranded DNA" or "dsDNA" refers to an oligonucleotide or polynucleotide having 3' overhangs, 5' overhangs, and / or blunt ends, comprising two single strands that are wholly or partially complementary to each other. Therefore, dsDNA may contain single-stranded regions at one or both ends and may be of synthetic origin or naturally derived from cells or tissues. In one embodiment, dsDNA is a product of PCR (polymerase chain reaction) or a fragment produced from genomic DNA, plasmids, or vectors through physical or enzymatic treatment.

[0029] As used herein, the term "buffer" refers to a reagent that allows a solution to resist pH changes when an acid or base is added. Examples of suitable non-naturally occurring buffers that can be used in the compositions, kits, and methods described herein include HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), TAPS (tris(hydroxymethyl)methylamino]propanesulfonic acid), tricine (N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), phosphates, citrates, ammonium salts, acetates, carbonates, 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-acetamido)-iminodiacetic acid (ADA), piperazine- N,N′-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), choline chloride, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl]amino]ethanesulfonic acid (TES), acetamylglycine, glycine, and bicine (2-(bis(2-hydroxyethyl)amino)acetic acid) buffer. It should be understood that the buffer may further comprise at least one additional substance, such as: electrolytes, such as MgCl2, NaCl and KCl; metal ions; type II topoisomerases (e.g. DNA gyrase); single-stranded DNA-binding proteins or heat-stable single-stranded binding proteins; congesting agents (e.g. polyethylene glycol); redox agents, such as dithiothreitol (DTT); nicotinamide adenine dinucleotide (NAD); detergents; and nonionic surfactants, such as TRITON™ X-100 (octylphenol decaethylene glycol ether).

[0030] As used herein, the terms “DNA” or “RNA” are defined as “polynucleotides” and may encompass primers, oligonucleotides, nucleic acid chains, etc. DNA or RNA can be single-stranded, double-stranded, or a mixture thereof. Such DNA or RNA polynucleotides can be synthetic, for example, synthesized in a DNA synthesizer, or naturally occurring, for example, extracted from natural sources, or derived from cloned or amplified material. Polynucleotides as referred to herein may contain modified bases. Furthermore, DNA or RNA sequences may contain one or more random or variable nucleotides. The use of randomized nucleotides may also include sequence restriction regions, where sequence restriction means limiting variation at a position to two or three nucleotide choices (i.e., A or C; A, G, or C, etc.) rather than all four nucleotide choices (ATGC). Typically, polynucleotides contain a 5′ phosphate at one end of the strand (“5′ end”) and a 3′ hydroxyl group at the other end (“3′ end”).

[0031] The nucleic acids used in this article can be any type of nucleic acid, such as human nucleic acid, bacterial nucleic acid, or viral nucleic acid. Nucleic acid samples can be, for example, nucleic acid samples from one or more cells, tissues, or body fluids (e.g., blood, urine, semen, lymph, cerebrospinal fluid, or amniotic fluid), or other biological samples such as tissue culture cells, buccal swabs, mouthwash, feces, tissue sections, biopsy aspirates, and archaeological samples (e.g., bone or mummified tissue). Nucleic acids can be, for example, DNA, RNA, or DNA products that have undergone reverse transcription of RNA. Nucleic acids can originate 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, biopsy, needle aspiration biopsy, cancer, tumors, tissues, cells, cell lysates, crude cell lysates, tissue lysates, tissue culture cells, buccal swabs, mouthwash, feces, mummified tissue, forensic sources, autopsy sources, archaeological sources, infections, nosocomial infections, production sources, pharmaceutical preparations, biomolecular production, protein preparations, lipid preparations, carbohydrate preparations, inanimate objects, air, soil, tree sap, metals, fossils, excavated materials, and / or other terrestrial or extraterrestrial materials and sources. In some embodiments, the nucleic acids do not include bacterial or viral nucleic acids.

[0032] The terms "Taq ligase" and "thermally stable ligase" are synonyms used in this article.

[0033] The term "synthetic circular supercoiled DNA" used in this article or SCS The DNA aims to capture products consisting solely of synthetic circular DNA, as well as products containing a portion of synthetic circular DNA and synthetic supercoiled DNA, wherein the aforementioned... SCS The DNA precursor can be derived partially or entirely from a natural source (e.g., plasmids). In some embodiments, the synthetic supercoiled DNA is substantially supercoiled.

[0034] The synthetic circular supercoiled DNA and covalently closed circular DNA used in this paper are understood to be substantially exonuclease resistant. Any exonucleases used in this paper are understood to be lacking or having minimal endonuclease activity when using double-stranded DNA as a substrate.

[0035] The term “supercoiling” or “supercoiling” as defined in this article is understood as the overall twisting of circular DNA. Supercoiling is understood as the sum of “twist” and “tumble,” where twist is the number of turns of the helix in the DNA, and tumble is the number of times the double helix crosses itself. Positive and negative supercoiling are understood by those skilled in the art, but in short, positive supercoiling includes extra or additional helical twist relative to the relaxed state (i.e., over-winding), while negative supercoiling includes less or reduced helical twist relative to the relaxed state (i.e., under-winding). Most organisms’ DNA is negatively supercoiled, although a certain amount of positive supercoiling is also known. Negative supercoiling advantageously allows processes such as transcription, DNA replication, and recombination. Another common term for supercoiling is “ring number” or “Lk,” which is understood as the sum of twist (T) and tumble (W). Ring number is useful because it is an indicator of changes in DNA topology (e.g., as a result of enzymatic breakage and rejoining events). Of interest is ΔLk, which is expressed by the formula ΔLk = Lk - Lk m To determine, where Lk m ΔLk is the number of loops in the relaxed circular DNA, and Lk is the number of loops in the circular DNA that has undergone supercoiling. It is worth noting that before calculating ΔLk, Lk and Lk... m Rounded to the nearest integer. For circular DNA that has undergone negative supercoiling, ΔLk is negative.

[0036] The “precursor” DNA fragment as defined herein includes dsDNA molecules (i.e., PCR products, restriction enzyme fragments, chemically or enzymatically generated DNA), single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. The nucleotide bases within the precursor DNA fragment may be in the form of natural adenine, guanine, cytosine, thymine, or any chemically modified form thereof, which may be incorporated into the DNA molecule through chemical synthesis or enzymatic action (i.e., DNA polymerase), or may appear chemically or enzymatically in one or more bases after synthesis. The length of the precursor DNA can range from about 20 nucleotides to several thousand to 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, as those skilled in the art will understand, a substantially “perfect” blunt-ended DNA precursor can be obtained for use by “polishing” the ends of the precursor DNA fragment with an IIS-type restriction enzyme (i.e., MlyI) before use in an HTLA or CHTLA reaction. In some embodiments, the precursor DNA fragments are generated using the method described in U.S. Provisional Patent Application No. 63 / 554752, filed February 16, 2024, entitled “Method for generating circular DNA using heteroduplex thermostable ligation assembly of precursors created by rolling circle amplification,” filed by Xiang LI and Charles J. BIEBERICH, the entire provisional patent application of which is incorporated herein by reference.

[0037] It is well known to those skilled in the art that each nucleotide in a dsDNA molecule will pair with its Watson-Crick counterpart (also referred to as a "complementary" nucleotide). Furthermore, it should be understood that a dsDNA sequence is represented by an upper strand or first (sense) strand sequence in the direction from its 5′ end to its 3′ end; therefore, a complementary sequence is a lower strand or second (antisense) strand sequence in the same direction as the upper strand. When DNA sequences are stated as complementary, it is understood that when they anneal or hybridize, a double-stranded DNA with antiparallel strands is formed. It can be further understood that an annealed complementary DNA sequence may include one or more non-canonical (i.e., Watson-Crick) base pairs or modified nucleotides that pair with multiple other nucleotides (e.g., deoxyinosine may also pair with three other DNA bases (deoxythymidine (dT), dA, and dG)).

[0038] As mentioned above, a powerful gene delivery platform has recently emerged, namely covalently closed circular DNA (e.g., microcircles) that is completely free of bacterial DNA, but there are significant problems with GMP production. The inventors previously developed an improved DNA assembly method called heteroduplex thermostable ligase assembly (HTLA™), which enables the complete in vitro construction and commercial production of large linear DNA molecules, as described in International Patent Application No. PCT / US2023 / 064977, filed March 27, 2023, in the name of Charles J. Bieberich and Xiang Li, entitled "Heteroduplextherometic ligation assembly (HTLA) and / or cyclic heteroduplex thermostable ligation assembly (CHTLA) for generating double-stranded DNA fragments with single-stranded sticky ends," which is incorporated herein by reference in its entirety. HTLA is a straightforward assembly platform that generates user-defined lengths of ready-to-connect single-stranded overhangs to produce sticky end blocks (SEBs) for assembly into higher-order linear or circular structures. The starting materials for HTLA or CHTLA are dsDNA or oligonucleotide precursors, which are capable of precise self-assembly to produce much longer DNA. As described in International Patent Application No. PCT / US2023 / 064977, at least three precursors are used as reactants.

[0039] In short, the HTLA process is a highly efficient DNA assembly process that generates ready-to-link, user-defined heteroduplex DNA molecules ranging from 1 to several thousand (or more) nucleotides in length, containing 5' or 3' single-stranded overhangs or "sticky ends" that can be joined to form closed circular DNA molecules from double-stranded or single-stranded DNA precursor molecules (see, for example, [link to other documentation]). Figure 1 When this process is repeated once, it is called HTLA, and when it is repeated more than once, the process is called cyclic heteroduplex thermostable ligase assembly (CHTLA). As described in International Patent Application No. PCT / US2023 / 064977, at least three precursors are used as reactants.

[0040] Surprisingly, in addition to preparing linear DNA, HLTA and CHTLA can also be used to generate closed circular DNA starting from as few as two precursors, such as dsDNA precursors or readily available and inexpensive DNA oligonucleotides (oligomers). Furthermore, it has been unexpectedly found that circular and supercoiled DNA can be obtained efficiently without using precursors containing intentionally nicked areas and without adding bending proteins (such as Abf2p or HMGB1). As described herein, the method involves providing at least two user-designed DNA precursors to generate DNA sequences, wherein portions of the first and second strands of said DNA sequences overlap each other, while generating complementary “sticky” ends that can complement each other and join to form a covalently closed circular loop. This procedure is illustrated in... Figure 1 In the process, the dsDNA (or oligomer) precursor mixture is heated and cooled in a buffer solution to form heteroduplex molecules I and II, and DNA ligase is used to ligate the heteroduplex molecules to form covalently closed circular, plasmid-free DNA molecules (synthesizing circular supercoiled DNA). SCS DNA). In some implementations, performing multiple rounds of heating and cooling (i.e., CHTLA) improves... SCS DNA production. As described above, the method includes forming an intermediate heteroduplex with a sticky end block (SEB), wherein a portion of the heteroduplex has a 5' overhang and a portion of the heteroduplex 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, such that after the 5' end of each strand is joined to its own 3' end, the heteroduplex circularizes (i.e., self-closes). 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, such that after the 5' end of each strand is joined to its own 3' end, the heteroduplex circularizes (i.e., self-closes). In some embodiments, the circular and supercoiled DNA described herein is produced without a plasmid vector and therefore does not contain bacterial DNA.

[0041] Even more surprisingly, the HTLA and CHTLA processes are used to form supercoiled DNA, since DNA ligases are known to lack topoisomerase activity. DNA supercoilization is known to be important for DNA packaging in all cells. Without supercoilization to reduce DNA space, it is impossible to package DNA into cells. Furthermore, transfecting supercoiled DNA into cells (i.e., genetically modifying cells for some purpose) is substantially more efficient than transfecting the same DNA in a linear or open (relaxed) circular form. In one embodiment, after denaturation, annealing, and ligation, some… SCSDNA undergoes negative supercoiling in the presence of DNA ligase. In some embodiments, a type II topoisomerase can be introduced into the... scs In DNA (e.g., by making thermostable type II topoisomerase present in the formation of DNA) scs In the DNA environment, or by adding type II topoisomerase to form DNA at the appropriate time and temperature. scs In the DNA environment), to deliver the scs DNA is essentially supercoiled.

[0042] For the purposes of this application, any enzyme that behaves similarly to a type II topoisomerase capable of inducing DNA supercoiling is permitted. In some embodiments, the type II topoisomerase is a DNA gyrase or topoisomerase IV. DNA gyrases have previously been reported to act via transient double-strand breaks rather than nicks in DNA, and to change the number of rings in DNA in two steps (PO Brown and NR Cozzarelli, Science, 1979, 206(4422), 1081-1083). As discussed herein, the CHTLA reaction occurs in the absence of a type II topoisomerase. scs Any supercoiling that can occur in DNA is prevented by the presence of type II topoisomerases such as DNA gyrase. scs DNA is essentially supercoiled. Therefore, for the purposes of this application, "essentially supercoiled" or "essentially supercoiled" corresponds to a supercoil induced by type II topoisomerase, greater than that produced during the CHTLA reaction in the absence of type II topoisomerase. SCS The degree of supercoiling in DNA. Relating this to the number of rings, ΔLk can be calculated for the purposes of this application. ss = Lk ss – Lk x Lk ss It is essentially supercoiled in the presence of type II topoisomerase. scs The number of DNA rings, Lk, is determined in the absence of type II topoisomerase. scs The number of DNA rings, where ΔLk is calculated. ss (For example, -2, -4, -6, -8, -10, etc.) correspond to essentially superspiraling.

[0043] Those skilled in the art will understand that some type II topoisomerases are thermostable at a temperature of at least about 95°C, while others are not. In some embodiments, the thermostable type II topoisomerase varieties are thermostable for about 30 seconds to about 10 minutes, or about 1 minute to about 5 minutes, at temperatures ranging from about 37°C to 100°C, or about 60°C to 100°C, or about 80°C to 100°C. Therefore, when "thermostable type II topoisomerase" is used hereinafter, it should be understood that the type II topoisomerase can survive at higher temperatures during denaturation / unwinding processes while retaining some or all of its activity. In some embodiments, the thermostable type II topoisomerase is a thermostable DNA gyrase. Thermostable DNA gyrases are known in the art and can be engineered to be thermostable within preferred temperature ranges.

[0044] In a first aspect, a method for forming a sticky end block (SEB) having a 5' or 3' protruding end is described, the method comprising: At least two precursor DNA fragments are introduced into a buffer medium containing a thermostable DNA ligase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Apply heat to a first temperature to denature the at least two precursor DNA fragments; and The temperature is lowered to a second temperature for annealing in the presence of the thermostable DNA ligase, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions, a portion of which has a single-stranded 5' overhang and a portion of which has a 3' overhang.

[0045] In some embodiments, the method for forming SEB is a one-pot process. In some embodiments, the second temperature is lower than the first temperature. In some embodiments, the at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, only two precursor DNA fragments are present, which, after denaturation, annealing, and ligation, form two heteroduplexes, for example, Figure 1 As shown in the image.

[0046] In some embodiments, the desired SEB product of the first aspect can be purified from the precursor DNA fragment, for example by agarose gel purification or any other separation method, for subsequent production in the second and / or third reactions. SCS DNA and / or supercoiled.

[0047] In some embodiments, the buffer medium used for the first aspect comprises a buffer solution to maintain pH. In some embodiments, the buffer solution comprises a combination of ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, DTT, NAD, ATP, and Triton™ X-100 to maintain pH at about 4 to about 12, or about 6 to 10, or about 7.5 to about 9. The denaturation (also referred to as “unwinding” or the 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, for a time in the range of about 0.1 minutes to about 60 minutes, or about 1 minute to about 5 minutes. The annealing is performed by lowering the temperature (i.e., the second temperature) to about 5°C to about 60°C below the denaturation temperature or to about 10°C to about 40°C below the denaturation temperature for a time in the range of about 0.1 minutes to about 60 minutes or about 4 minutes to 6 minutes.

[0048] In some implementations... Figure 1 The precursor DNA fragments described herein and all precursors are user-defined and user-designed. For example, consider the simplified design of two double-stranded precursors (I and II) required to generate a 100-base-pair SEB heteroduplex I (where 94 base pairs overlap and are double-stranded with 5' overhangs at both ends). In this example, the 5' overhangs (i.e., GGGGGG and CCCCCC) are complementary. An inverse heteroduplex II with complementary 3' overhangs is also formed. Precursor I can be a double-stranded molecule consisting of base pairs 1 through 100. Precursor II can be a double-stranded molecule consisting of base pairs 7 through 100 followed by 1 through 6 (see, for example, the green 1 through 6 in precursor II). In this example, the first strand of nucleotides 1 through 100 in precursor I and the second strand of nucleotides 7 through 100 followed by 1 through 6 in precursor II are complementary to each other in bases 7 through 100. After denaturation and annealing to form heteroduplex I, the first strand of nucleotides 7-100 of precursor I and the second strand of nucleotides 7-100 of precursor II, followed by nucleotides 1-6, anneal to produce a double-stranded region of 94 base pairs. Since nucleotides 1-6 of precursor I (see, for example, the green 1-6 in precursor I) have no complementary bases in precursor II, they remain single-stranded. Similarly, nucleotides 1-6 of precursor II have no complementary bases in precursor I, and therefore remain single-stranded. That is, if the precursors are designed such that the 5' overhangs of heteroduplex I are complementary, they will join during ligation to form a 100-base-pair region. SCSDNA. The same applies to heteroduplex II, which contains a 3' overhang. It goes without saying that, in addition to forming the SEB product, the two precursors I and II can also be reformed by re-annealing their complementary strands. Those skilled in the art will understand that the overhangs can be more or less than the six base pairs shown in this example, for example, from 2 to 1000 base pairs, and can contain any combination of nucleotides as long as they are complementary to each other, so that a covalently closed loop can be formed after the complementary overhangs are joined.

[0049] In the second aspect, a method for producing synthetic circular supercoiled DNA is described. SCS A method for (DNA) comprising: At least two precursor DNA fragments are introduced into a buffer medium containing a thermostable DNA ligase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Apply heat to a first temperature to denature the at least two precursor DNA fragments; and The temperature is lowered to a second temperature for annealing in the presence of the thermostable DNA ligase, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions. A portion of the heteroduplex has a single-stranded 5' overhang, and a portion has a single-stranded 3' overhang. The heteroduplex produces a product when the intramolecular 5' overhangs on the heteroduplex molecules are complementary and ligated onto the two DNA strands. SCS DNA, and when the 3' intramolecular overhangs on the heteroduplex molecule are complementary and join together on the two DNA strands, it produces SCS DNA.

[0050] In some embodiments, the method for producing synthetic circular supercoiled DNA is a one-pot process. In some embodiments, the second temperature is lower than the first temperature. In some embodiments, the process of applying heat to the first temperature for denaturation and then reducing to the second temperature for annealing is repeated in cycles. In some embodiments, the number of cycles ranges from 1 to 100 cycles. In some embodiments, the at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, only two precursor DNA fragments are present, which, after denaturation, annealing, and ligation, form two heteroduplexes, for example, such as... Figure 1 As shown in the diagram. In some embodiments, a portion of the description... SCS DNA undergoes negative supercoiling in the presence of the thermostable DNA ligase. In some embodiments, a single-stranded DNA-binding protein or a thermostable single-stranded DNA-binding protein is present in the reaction, for example, added to the buffer medium or added to the environment at a point before annealing, to promote the annealing of the DNA strands.

[0051] In some embodiments of the second aspect, the buffer medium containing the thermostable DNA ligase comprises a buffer solution to maintain pH. In some embodiments, the buffer solution comprises a combination of ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, DTT, NAD, and Triton™ X-100 to maintain pH at about 4 to about 12, or about 6 to 10, or about 7.5 to about 9. The denaturation (also referred to as “unwinding” or the 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, for a time in the range of about 0.1 minutes to about 60 minutes, or about 1 minute to about 5 minutes. The annealing is performed by lowering the temperature (i.e., the second temperature) to about 5°C to about 60°C below the denaturation temperature or to about 10°C to about 40°C below the denaturation temperature for a time in the range of about 0.1 minutes to about 60 minutes or about 4 minutes to 6 minutes. Therefore, in some embodiments, the second temperature is in the range of about 25°C to about 85°C, or about 25°C to about 70°C, or about 25°C to about 65°C, or about 37°C to about 65°C, or about 50°C to about 70°C, and the time is in the range of about 0.1 minutes to about 60 minutes, or about 4 minutes to 6 minutes. As described above, the denaturation / annealing process can be repeated in cycles of about 2 to about 100 times, wherein each cycle increases... SCS DNA production. Therefore, in some embodiments, the nucleic acid ligation scheme involves temperature cycling from, for example, a lower temperature of about 80°C to 100°C to about 40°C to about 70°C for 2 to 100 cycles.

[0052] In some embodiments, the production of synthetic circular supercoiled DNA ( SCS DNA-based methods include: At least two precursor DNA fragments are introduced into a buffer medium having a pH of about 7.5 to about 9 and containing ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, NAD, DTT, Triton X-100 and at least one thermostable ligase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Heat is applied at a first temperature to denature 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 about 37°C to 100°C, lasting for a time ranging from about 0.1 minutes to about 60 minutes; and The temperature is lowered to a second temperature for annealing in the presence of the thermostable DNA ligase, wherein the second temperature is 10°C to 40°C lower than the first temperature, for a duration ranging from approximately 4 minutes to approximately 10 minutes, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions. A portion of the heteroduplex has a single-stranded 5' overhang, and a portion has a single-stranded 3' overhang. This process occurs when the intramolecular 5' overhangs on the heteroduplex molecule are complementary and ligated onto the two DNA strands. SCS DNA, and when the 3' intramolecular overhangs on the heteroduplex molecule are complementary and join together on the two DNA strands, it produces SCS DNA.

[0053] In some embodiments, the process of applying heat to a first temperature for denaturation and then reducing to a second temperature for annealing is repeated in cycles. In some embodiments, the number of cycles ranges from 2 to 100 cycles. In some embodiments, the at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, only two precursor DNA fragments are present, which, after denaturation, annealing, and ligation, form two heteroduplexes, for example, as... Figure 1 As shown in the diagram. In some embodiments, a portion of the description... SCS DNA undergoes negative supercoiling in the presence of the thermostable DNA ligase. In some embodiments, a single-stranded DNA-binding protein or a thermostable single-stranded DNA-binding protein is present in the reaction, for example, added to the buffer medium or added to the environment at a point before annealing, to promote the annealing of the DNA strands.

[0054] In a third aspect, a method for producing substantially supercoiled DNA is described, the method comprising: At least two precursor DNA fragments are introduced into a buffer medium containing a thermostable DNA ligase and a thermostable type II topoisomerase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Heat is applied to a first temperature to denature the at least two precursor DNA fragments; The temperature is lowered to a second temperature for (i) annealing in the presence of the thermostable DNA ligase, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions, a portion of which has a single-stranded 5' overhang and a portion of which has a 3' overhang, wherein when the intramolecular 5' overhangs on the heteroduplex molecule are complementary and ligated on the two DNA strands, a heteroduplex is produced. SCS DNA, and when the 3' intramolecular overhangs on the heteroduplex molecule are complementary and join together on the two DNA strands, it producesSCS DNA; and (ii) in the presence of a thermostable type II topoisomerase. SCS DNA is essentially supercoiled.

[0055] In some embodiments, the method for producing substantially supercoiled DNA is a one-pot process. In some embodiments, the second temperature is lower than the first temperature. In some embodiments, the process of applying heat to the first temperature for denaturation and then reducing to the second temperature for annealing / ligation / supercoilation is repeated in cycles. In some embodiments, the number of cycles ranges from 2 to 100 cycles. In some embodiments, the at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, the thermostable type II topoisomerase includes DNA gyrase or topoisomerase IV. In some embodiments, only two precursor DNA fragments are present, which, after denaturation, annealing, and ligation, form two heteroduplexes, for example, Figure 1 As shown in the diagram. In some embodiments, as those skilled in the art will understand, there are 3, 4, 5, 6, 7, 8, 9, or 10 or more precursor DNA fragments. In some embodiments, a single-stranded DNA-binding protein or a heat-stable single-stranded DNA-binding protein is present in the reaction, for example, added to the buffer medium or added to the environment at a point before annealing to promote the annealing of the DNA strands.

[0056] In some embodiments of the third aspect, the buffer medium comprising the thermostable DNA ligase and the thermostable type II topoisomerase contains a buffer solution to maintain pH. In some embodiments, the buffer solution comprises a combination of ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, DTT, NAD, and Triton™ X-100 to maintain pH at about 4 to about 12, or about 6 to 10, or about 7.5 to about 9. The denaturation (also referred to as “unwinding” or the 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, for a 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 is performed by lowering the temperature (i.e., the second temperature) to about 5°C to about 60°C below the denaturation temperature or to about 10°C to about 40°C below the denaturation temperature for a time in the range of about 0.1 minutes to about 60 minutes or about 4 minutes to 6 minutes. Therefore, in some embodiments, the second temperature is in the range of about 25°C to about 85°C, or about 25°C to about 70°C, or about 25°C to about 65°C, or about 37°C to about 65°C, or about 50°C to about 70°C, and the time is in the range of about 0.1 minutes to about 60 minutes, or about 4 minutes to 6 minutes. As described above, the process of heating to the first temperature and cooling to the second temperature can be repeated approximately 2 to about 100 times, wherein each cycle increases the yield of supercoiled DNA molecules and the degree of supercoilability of individual molecules. Therefore, in some embodiments of the third aspect, the supercoilation scheme is a temperature cycle from, for example, a first temperature of about 80°C to 100°C to a second temperature of about 40°C to about 70°C, lasting 2 to 100 cycles.

[0057] In some embodiments, the method for producing substantially supercoiled DNA includes: At least two precursor DNA fragments are introduced into a buffer medium having a pH of about 7.5 to about 9 and containing ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, NAD, DTT, Triton X-100, at least one thermostable ligase and a thermostable type II topoisomerase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Heat is applied at a first temperature to denature 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, lasting for a time ranging from about 0.1 minutes to about 60 minutes; and The temperature is lowered to a second temperature for (i) annealing in the presence of the thermostable DNA ligase, wherein the second temperature is 10°C to 40°C lower than the first temperature, for a duration ranging from approximately 4 minutes to approximately 10 minutes, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions, a portion of which has a single-stranded 5' overhang and a portion of which has a single-stranded 3' overhang, wherein when the intramolecular 5' overhangs on the heteroduplex molecule are complementary and ligated on the two DNA strands, a heteroduplex is produced. SCS DNA, and when the 3' intramolecular overhangs on the heteroduplex molecule are complementary and join together on the two DNA strands, it produces SCS DNA; and (ii) in the presence of the thermostable type II topoisomerase. SCS DNA is essentially supercoiled.

[0058] In some embodiments, the method for producing essentially supercoiled DNA is a one-pot process. In some embodiments, the process involves repeatedly applying heat to a first temperature for denaturation and then cooling to a second temperature for annealing and production. SCS DNA and supercoiling. In some embodiments, the number of cycles ranges from 2 to 100 cycles. In some embodiments, the at least two precursor DNA fragments are selected from dsDNA molecules, single-stranded DNA molecules, DNA oligonucleotides, or mixtures thereof. In some embodiments, the thermostable type II topoisomerase includes DNA gyrase. In some embodiments, only two precursor DNA fragments are present, which, after denaturation, annealing, and ligation, form two heteroduplexes, for example, such as... Figure 1 As shown in the diagram. In some embodiments, as those skilled in the art will understand, there are 3, 4, 5, 6, 7, 8, 9, or 10 or more precursor DNA fragments. In some embodiments, a single-stranded DNA-binding protein or a heat-stable single-stranded DNA-binding protein is present in the reaction, for example, added to the buffer medium or added to the environment at a point before annealing to promote the annealing of the DNA strands.

[0059] In the fourth aspect, a method for producing substantially supercoiled DNA is described, the method comprising: At least two precursor DNA fragments are introduced into a buffer medium containing a thermostable DNA ligase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Heat is applied to a first temperature to denature the at least two precursor DNA fragments; The temperature is lowered to a second temperature for annealing in the presence of the thermostable DNA ligase, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions. A portion of the heteroduplex has a single-stranded 5' overhang, and a portion has a single-stranded 3' overhang. The heteroduplex produces a product when the intramolecular 5' overhangs on the heteroduplex molecules are complementary and ligated onto the two DNA strands. SCS DNA, and when the 3' intramolecular overhangs on the heteroduplex molecule are complementary and join together on the two DNA strands, it produces SCS DNA; and The temperature was lowered to a third temperature and a large dose of type II topoisomerase was added to initiate the process in the presence of the type II topoisomerase. SCS DNA is essentially supercoiled.

[0060] 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 process of applying heat to the first temperature for denaturation, decreasing to the second temperature for annealing, and further decreasing to the third temperature for supercoiling is repeated in cycles. In some embodiments, the number of cycles ranges from 2 to 100 cycles. In some embodiments, the 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, only two precursor DNA fragments are present, which, after denaturation, annealing, and ligation, form two heteroduplexes, for example, such as... Figure 1 As shown in the diagram. In some embodiments, as those skilled in the art will understand, there are 3, 4, 5, 6, 7, 8, 9, or 10 or more precursor DNA fragments. In some embodiments, a single-stranded DNA-binding protein or a heat-stable single-stranded DNA-binding protein is present in the reaction, for example, added to the buffer medium or added to the environment at a point before annealing to promote the annealing of the DNA strands.

[0061] In some embodiments of the third aspect, the buffer medium containing the thermostable DNA ligase comprises a buffer solution to maintain pH. In some embodiments, the buffer solution comprises a combination of ATP, Tris-HCl, MgCl2, KCl, NaCl, β-mercaptoethanol, DTT, NAD, and Triton™ X-100 to maintain pH at about 4 to about 12, or about 6 to 10, or about 7.5 to about 9. The denaturation (also referred to as “unwinding” or the 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, for a time in the range of about 0.1 minutes to about 60 minutes, or about 1 minute to about 5 minutes. The annealing is performed by lowering the temperature (i.e., the second temperature) to about 5°C to about 60°C below the denaturation temperature or to about 10°C to about 40°C below the denaturation temperature for a time in the range of about 0.1 minutes to about 60 minutes or about 4 minutes to 6 minutes. Therefore, in some embodiments, the second temperature is in the range of about 25°C to about 85°C, or about 25°C to about 70°C, or about 25°C to about 65°C, or about 37°C to about 65°C, or about 50°C to about 70°C, and the time is in the range of about 0.1 minutes to about 60 minutes, or about 4 minutes to 6 minutes. Those skilled in the art will understand that the second temperature is higher than the third temperature. The third temperature depends on the nature of the type II topoisomerase; the third temperature is selected to ensure that the type II topoisomerase does not undergo thermal degradation and can be used to... SCS DNA supercoiling. As described above, the denaturation / annealing / supercoiling process can be repeated approximately 2 to approximately 100 times, with each cycle increasing the yield of supercoiled DNA molecules and the degree of supercoiling of each molecule. Therefore, in some embodiments, the nucleic acid supercoiling scheme involves cycling from a first temperature of, for example, about 80°C to 100°C, to a second temperature of about 40°C to about 70°C, and then to a third temperature below the second temperature, for 2 to 100 cycles.

[0062] refer to Figure 1 It can be noted that heteroduplexes I and II can and do connect to each other to form tandem strands. However, without being bound by theory, intramolecular (or self-) linkage to cyclization is actually more preferred than intermolecular linkage to form tandem strands.

[0063] In some implementations, in generating SCSAfter DNA and / or supercoiled DNA are obtained, tandem and typically tangled DNA, as well as precursors, can be removed by treatment with a 5' to 3' or 3' to 5' exonuclease (e.g., T5 or T7 exonuclease). The exonuclease can be degraded by proteinase K digestion, precipitating the DNA product from solution. Alternatively, column-based methods for deproteinizing and purifying DNA from samples are known in the art, as those skilled in the art will understand.

[0064] As discussed above, in some embodiments, the 5' protrusion of one strand of a heteroduplex is complementary to the 5' protrusion of the opposite strand of the same heteroduplex, causing the heteroduplex to circularize (i.e., self-close) after annealing at the protrusion. Connecting the 5' end of one strand to the now-juxtaposed 3' end of the same strand creates a complete phosphodiester backbone on one strand. The same applies to the opposite strands, resulting in covalently closed circular double-stranded DNA. Furthermore, in some embodiments, the 3' protrusion of one strand of a heteroduplex is complementary to the 3' protrusion of the opposite strand of the same heteroduplex, causing the heteroduplex to circularize (i.e., self-close) after annealing at the protrusion. Connecting the 3' end of one strand to the now-juxtaposed 5' end of the same strand creates a complete phosphodiester backbone on one strand. The same applies to the opposite strands, resulting in covalently closed circular double-stranded DNA.

[0065] In some embodiments, the circular and supercoiled DNAs described herein are generated without plasmid vectors and therefore do not contain bacterial DNA. Advantageously, because the DNA precursor fragment is user-designed, it can be part of a designed vector (plasmid, granule, BAC, YAC, etc.) to release the precursor fragment from the vector (e.g., by digestion with restriction endonucleases), and because complementary sticky ends are formed in the heteroduplex molecule, fully closed circular vectors can be obtained using the methods described herein, wherein the circular and supercoiled DNA products do not contain bacterial DNA.

[0066] In some embodiments, the circular and supercoiled DNAs described herein are generated using plasmid (or cloning vector, BAC, YAC, etc.) vectors and the DNA sequence of interest, thus having the structure of a conventional cloning vector plus an insert fragment, but synthesized entirely through a process of HTLA or CHTLA.

[0067] Therefore, in the fifth aspect, a synthetic circular supercoiled DNA ( SCS DNA), wherein SCS The DNA does not contain any DNA of bacterial or viral origin. In some embodiments, the... SCSThe DNA is a plasmid-free vector. In some embodiments, the fifth aspect... SCS DNA is essentially supercoiled. SCS The DNA sequence can be fully user-defined (i.e., traceless), generated in a one-pot process (such as any of the methods described herein in the first, second, third, or fourth aspects), and produced in quantities suitable 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, the... SCS The DNA is the vaccine, and the precursor DNA fragment is designed by the user to guide the expression of the vaccine antigen. The precursor DNA fragment is mass-produced and then undergoes HTLA or CHTLA as described herein to produce a plasmid-free vector that guides the expression of the vaccine antigen. SCS DNA. In another example, SCS DNA is used in gene therapy settings to carry payload genes, as naked DNA, or in formulations designed to enhance cellular uptake in a systemic or cellular- or tissue-targeted manner. In another example, the... SCS DNA encodes one or more components required to produce lentiviruses, adeno-associated viruses, or other viruses containing specific payloads for gene therapy in living animals, including humans, or for the modification of cultured cells. In another example, SCS DNA has the same structure as conventional plasmids (or granules, BAC, YAC, etc.), but is produced entirely synthetically for use, for example, in producing large quantities of low-copy conventional vectors with insert fragments. Example Example 1 - Production of CHTLA by SCS DNA is partially supercoiled

[0068] To demonstrate that CHTLA produces circular products with varying degrees of de novo supercoiling, plasmid pMaxGFP was digested with KpnI or XhoI, resulting in a 700 bp shift of linearly overlapping DNA fragments, ensuring complete homology of the shifted regions. The digested DNA was purified to remove restriction enzymes, and approximately 5 μg of each fragment was mixed (1:1) in 50 μL 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 44 nM HiFi Taq ligase (New England Biolabs). The mixture was subjected to 10 heating and cooling cycles from 95°C to 60°C in a standard thermal cycler to produce… SCSDNA. After 10 cycles of the CHTLA reaction, aliquots of the product were treated with 10 units of T5 exonuclease at 37°C for one hour. To demonstrate that the CHTLA product was supercoiled, it was analyzed on an agarose gel without ethidium bromide and compared with the plasmid pMaxGFP purified from bacteria. After electrophoresis, the gel was stained with ethidium bromide. Analysis of the resulting gel showed a series of bands in the lanes containing the CHTLA product treated with T5 exonuclease (see [link to article]). Figure 2 (lane "V"). The bands correspond to closed circular CHTLA product DNA with varying degrees of supercoil twisting, including fractions supercoiled to the same degree of maximal distortion seen in the bacterial-derived plasmid pMaxGFP. Example 2 - CHTLA synthesis of circular DNA using single-stranded oligonucleotides as precursors

[0069] To demonstrate that CHTLA converts single-stranded oligonucleotides into closed circular products, CHTLA was performed on six overlapping 40-nucleotide 5'-phosphorylated 80-mer oligonucleotides. The oligonucleotides were mixed at a final concentration of 2 μmol each in a 50 μL 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 heating and cooling cycles from 95°C to 60°C in a standard thermal cycler. Parallel and identical reactions were performed, but without HiFi Taq ligase. After completing the 10 cycles of CHTLA, aliquots of the product were treated with 10 units of T5 exonuclease at 37°C for one hour. Then, CHTLA products with and without T5 exonuclease exposure were analyzed on an agarose gel containing ethidium bromide. (As in...) Figure 3 As seen in the diagram, a large portion of the CHTLA products are resistant to T5 exonuclease digestion, indicating that they are in a closed circular conformation. Example 3 - Supercoiling SCS CHTLA synthesis of DNA from linear dsDNA precursors

[0070] To demonstrate that CHTLA converts linear dsDNA precursors into closed circular and supercoiled circular products, plasmid pBluescript–SK(-) was digested with restriction enzymes BamHI and KpnI, yielding two overlapping linear DNA molecules offset by 62 bp, ensuring complete homology between the offset regions. Approximately 5 μg of each fragment was mixed (1:1) in 50 μL 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 heating and cooling cycles from 95°C to 60°C in a standard thermal cycler. To demonstrate that the CHTLA product is almost entirely 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 one hour. Individual aliquots of the linear CHLTA precursor were also treated in parallel with T5 exonuclease. Then, as... Figure 4 As shown, the products of the T5 exonuclease reaction were analyzed on an agarose gel containing ethidium bromide (in the gel). Ethidium bromide is intercalated into double-stranded DNA and induces negative supercoiling of some, but not all, closed circular DNA during electrophoretic separation. Analysis of the resulting gel images showed that although all linear precursor molecules were completely digested by the T5 exonuclease, approximately 90% of the CHTLA products were resistant to T5 exonuclease digestion, indicating that they were in a closed circular conformation. This data demonstrates that dual-fragment HLTA using dsDNA precursors efficiently produces circular DNA molecules, and a high proportion of the products take supercoiling form. SCS In the form of DNA. Furthermore, as a result of ethidium bromide intercalation during electrophoresis, the closed circular CHTLA product becomes more supercoiled. Example 4 - Production of CHTLA by SCS DNA is negatively supercoiled

[0071] To prove that it was generated by CHTLA SCS DNA is negatively supercoiled, which means that... SCS DNA and plasmid pBluescript-SK(-) were linearized with BamHI or KpnI to produce overlapping DNA molecules with a 62 bp offset (see [link to documentation]). Figure 5AApproximately 5 μg of each fragment was mixed in a 50 μL 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 HiFiTaq ligase (New England Biolabs), and subjected to 10 heating and cooling cycles from 95°C to 60°C in a standard thermal cycler. After completing the 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 separated on agarose gels with or without chloroquine. Chloroquine intercalates into DNA and induces positive helix twisting. Neither gel was ethidium bromide-free. After electrophoresis, both gels were stained with ethidium bromide. In chloroquine-free gels, rings representing varying degrees of supercoil twisting were observed. SCS Multiple bands of DNA (see) Figure 5B (VL column, yellow box). Upon exposure to chloroquine, these multiple bands collapse "upward" into a single substance (see [link]). Figure 5B (See the red arrow in the image). Similarly, bacterial-derived negative supercoiled plasmid DNA also migrates upwards after exposure to chloroquine (see [link]). Figure 5B (The green arrows, as shown, indicate the movement from the side without chloroquine to the side with chloroquine), and various substances were observed to exhibit different degrees of supercoil distortion. Therefore, in the presence of chloroquine, SCS Both DNA and bacterial-derived negatively supercoiled plasmid DNA migrated in the same manner, indicating that... SCS DNA is also negatively supercoiled, although to a lesser extent overall than bacterial plasmid DNA.

Claims

1. A method for producing synthetic circular supercoiled DNA ( SCS A method for (DNA) comprising: At least two precursor DNA fragments are introduced into a buffer medium containing a thermostable DNA ligase, wherein the precursor DNA fragments will be correctly assembled to produce a defined DNA sequence. Heat is applied to a first temperature to denature the at least two precursor DNA fragments; and The temperature is lowered to a second temperature for (i) annealing in the presence of the thermostable DNA ligase, thereby producing a heteroduplex of double-stranded DNA formed by base pairing of complementary regions, a portion of which has a single-stranded 5' overhang and a portion of which has a 3' overhang, wherein when the intramolecular 5' overhangs on the heteroduplex molecule are complementary and ligated on the two DNA strands, a heteroduplex is produced. SCS DNA, and when the 3' intramolecular overhangs on the heteroduplex molecule are complementary and join together on the two DNA strands, it produces SCS DNA; and (ii) in the presence of thermostable type II topoisomerases SCS DNA is essentially supercoiled.

2. The method according to claim 1, wherein the production SCS The DNA approach 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 one of the preceding claims, wherein two precursor DNA fragments are used.

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

6. The method according to any one 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 about 100°C.

7. The method of claim 6, wherein the application time of the first temperature is in the range of about 30 seconds to about 10 minutes, preferably about 1 minute to about 5 minutes.

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

9. The method of claim 8, wherein the duration of the second temperature is in the range of about 0.1 minutes to about 60 minutes.

10. The method according to any one of the preceding claims, wherein applying heat to a first temperature for denaturation and reducing to a second temperature for annealing are repeatedly performed in a cycle.

11. The method of 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 a heteroduplex double-stranded DNA sequence is generated during annealing by base pairing of complementary regions, wherein the heteroduplex comprises a 5' or 3' overhang.

13. The method of claim 12, wherein the 5' or 3' protrusions are complementary when the... SCS DNA.

14. The method according to any one of the preceding claims, wherein the buffer comprises a combination of ATP, Tris-HCl, MgCl2, KCl, NaCl, DTT, β-mercaptoethanol, NAD, and TRITON X-100.

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

16. The method of claim 4, wherein a portion thereof... SCS DNA undergoes negative supercoiling in the presence of the thermostable DNA ligase.

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

18. The method of claim 17, wherein... SCS The DNA was essentially supercoiled.

19. The method according to claim 17 or 18, wherein the type II topoisomerase is thermostable, and the thermostable type II isomerase is capable of cycling through the denaturation and annealing processes while retaining some or all of its activity.

20. The method of claims 17 and 18, wherein the type II topoisomerase is thermostable or not thermostable, and the method further comprises lowering the temperature to a third temperature and adding a large dose of type II topoisomerase to initiate the process in the presence of the type II topoisomerase. SCS DNA is essentially supercoiled.

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

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

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

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

25. The method according to any one of the preceding claims, wherein the produced SCS 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. A synthetic circular supercoiled DNA ( SCS DNA), which is produced by the method according to any one of claims 1-27.

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