Cell-free method for producing synthetic cyclic nucleic acids

Abstract

The present disclosure relates to methods of cell-free cyclic nucleic acid amplification using rolling circle amplification.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 398,354, filed Aug. 16, 2022, under 35 U.S.C. § 119(e), the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to methods for amplifying cell-free circular nucleic acids. Background Art

[0004] Since their development, synthetic nucleic acid (NA) molecules have become increasingly useful in biomedical research, medicine, and therapeutics. The use of these molecules has applications in many fields and allows, for example, the ability to combine specific sequences from different organisms to produce constructs that exhibit specific advantages and utilities. For example, certain sequences found in bacteria or viruses can help enhance the expression of mammalian genes. By generating synthetic NA molecules containing such bacterial or viral sequences and mammalian genes on the same construct, it is possible to increase gene expression levels to levels that are not normally achievable in mammalian cells, and even to therapeutic doses that may be possible. In addition, the ability to generate synthetic NA molecules allows for the production of molecules with specific sequences (such as specific gene variants, polymorphisms, or mutations) with excellent precision.

[0005] Current methods for generating useful amounts of synthetic NA molecules (such as for use in the laboratory or as therapeutic agents) require the use of non-mammalian cell systems. However, while these methods allow for the production of sufficient amounts of synthetic NA molecules, the resulting NA molecules typically contain impurities generated by the cell systems that amplify them. In addition, it is difficult to control fidelity and expression, and these methods require expensive specialized equipment. Summary of the Invention

[0006] The present disclosure provides cost-effective methods by which NA can be highly amplified in a synthetic, cell-free environment, enabling strict control of the amplification and processing reactions and eliminating the problems associated with cell-mediated NA amplification.

[0007] In some aspects, provided herein are methods for amplifying synthetic circular nucleic acids. The methods include providing a circular nucleic acid template that comprises: (i) a backbone, wherein the backbone comprises one or more internal endonuclease cleavage sites, and (ii) an insert sequence, wherein the insert sequence comprises endonuclease cleavage sites at its 5' and 3' termini; or providing a circular nucleic acid template that comprises: (i) an insert sequence, wherein the insert sequence is flanked by first endonuclease cleavage sites; and (ii) a backbone, wherein the backbone comprises one or more second internal endonuclease cleavage sites. The methods further include amplifying the circular nucleic acid template to produce an amplification product; contacting the amplification product with a first endonuclease under digestion conditions to produce a first digestion product; adding the first digestion product to a ligation reaction mixture comprising a ligase; and incubating the ligation reaction mixture to produce a circular ligation product, wherein the circular ligation product is at least partially supercoiled.

[0008] In some embodiments, the backbone comprises more than one internal endonuclease cleavage site.

[0009] In some embodiments, the backbone comprises two or more different internal endonuclease cleavage sites.

[0010] In some embodiments, the backbone comprises from 1 to 5 internal endonuclease cleavage sites.

[0011] In some embodiments, the endonuclease cleavage sites flanking the insert sequence are the same endonuclease cleavage sites.

[0012] In some embodiments, the endonuclease cleavage sites flanking the insert sequence are different from each other.

[0013] In some embodiments, the first digestion product is added to the ligation reaction mixture at a rate of about 1% to about 20% of the final ligation reaction volume per hour over a period of 5 - 14 hours.

[0014] In some embodiments, the first digestion product is added to the ligation reaction mixture at a rate of about 3% to about 5% of the final ligation reaction volume per hour over a period of 5 - 14 hours.

[0015] In some embodiments, the first digestion product is added to the ligation reaction mixture at a rate of about 1% to about 20% of the final ligation reaction volume per hour over a period of 10 - 12 hours.

[0016] In some embodiments, the first digestion product is added to the ligation reaction mixture at a rate of about 3% to about 5% of the final ligation reaction volume per hour over a period of 10 - 12 hours.

[0017] In some embodiments, the circular ligation product is contacted with a second endonuclease under digestion conditions to produce a second digestion product, wherein the first endonuclease and the second endonuclease do not have the same recognition site.

[0018] In some embodiments, the method further comprises contacting the circular ligation product or the second digestion product with a topoisomerase under conditions that promote supercoiling to produce a supercoiled product.

[0019] In some embodiments, the method further comprises contacting the ligation product, the second digestion product, or the supercoiled product with an exonuclease that digests single-stranded nucleic acids and open circular nucleic acids to produce a final reaction product comprising a synthetic circular nucleic acid substantially free of linear nucleic acids.

[0020] In some embodiments, the method further comprises purifying the circular ligation product, the second digestion product, the supercoiled product, or the final reaction product. In some embodiments, the final reaction product is purified by chromatography, such as ion exchange chromatography (including anion exchange chromatography), affinity chromatography, reverse phase chromatography, or size exclusion chromatography; isopropanol precipitation, methanol precipitation, ethanol precipitation, solid phase purification, electrophoresis, or a combination thereof. In some embodiments, the final reaction product is purified by anion exchange chromatography.

[0021] In some embodiments, the method further comprises inactivating the first and / or second endonuclease by lowering the pH of the solution to pH 3-5 after digestion by the first endonuclease and / or digestion by the second endonuclease.

[0022] In some embodiments, the nucleic acid template comprises: a nucleic acid fragment to be amplified; two recognition sites of a first endonuclease located at the 5' and 3' ends of the nucleic acid fragment to be amplified, respectively, and a second endonuclease recognition site in the vector backbone. In some embodiments, the nucleic acid template further comprises a vector backbone.

[0023] In some embodiments, phi29 DNA polymerase is used for rolling circle amplification. In some embodiments, bacterial DNA polymerase III is used for rolling circle amplification. In some embodiments, bacterial DNA polymerase I is used for rolling circle amplification. In some embodiments, modified DNA polymerase I is used for rolling circle amplification. In some embodiments, M2 DNA polymerase is used for rolling circle amplification. In some embodiments, B103 DNA polymerase is used for rolling circle amplification. In some embodiments, GA-1 DNA polymerase is used for rolling circle amplification. In some embodiments, phi-PRD1 polymerase is used for rolling circle amplification. In some embodiments, VENT DNA polymerase is used for rolling circle amplification. In some embodiments, DEEP VENT DNA polymerase is used for rolling circle amplification. In some embodiments, KlenTaq DNA polymerase is used for rolling circle amplification. In some embodiments, the Klenow fragment of DNA polymerase I is used for rolling circle amplification. In some embodiments, DNA polymerase III is used for rolling circle amplification. In some embodiments, T3 DNA polymerase is used for rolling circle amplification. In some embodiments, T4 DNA polymerase is used for rolling circle amplification. In some embodiments, T5 DNA polymerase is used for rolling circle amplification. In some embodiments, T7 DNA polymerase is used for rolling circle amplification. In some embodiments, Bst polymerase is used for rolling circle amplification. In some embodiments, rBST DNA polymerase is used for rolling circle amplification. In some embodiments, N29 DNA polymerase is used for rolling circle amplification. In some embodiments, TopoTaq DNA polymerase is used for rolling circle amplification. In some embodiments, T7 RNA polymerase is used for rolling circle amplification. In some embodiments, SP6 RNA polymerase is used for rolling circle amplification. In some embodiments, T3 RNA polymerase is used for rolling circle amplification. In some embodiments, reverse transcriptase is used for rolling circle amplification.

[0024] In some embodiments, the ligase is T4 DNA ligase. In some embodiments, the ligase is T4 RNA ligase. In some embodiments, the ligase is T3 DNA ligase. In some embodiments, the ligase is T7 DNA ligase. In some embodiments, the ligase is Taq DNA ligase. In some embodiments, the ligase is E. coli DNA ligase.

[0025] In some embodiments, the topoisomerase is DNA gyrase. In some embodiments, the DNA gyrase is Escherichia coli DNA gyrase. In some embodiments, the DNA gyrase is Staphylococcus aureus (S. aureus) DNA gyrase. In some embodiments, the DNA gyrase is DNA topoisomerase 2-α. In some embodiments, the DNA gyrase is DNA topoisomerase 2-β.

[0026] In some embodiments, the exonuclease is T5 exonuclease. In some embodiments, the exonuclease is exonuclease I. In some embodiments, the exonuclease is exonuclease II. In some embodiments, the exonuclease is exonuclease III. In some embodiments, the exonuclease is exonuclease IV. In some embodiments, the exonuclease is exonuclease V. In some embodiments, the exonuclease is exonuclease VIII. In some embodiments, the exonuclease is exonuclease T. In some embodiments, the exonuclease is λ exonuclease. In some embodiments, the exonuclease is T7 exonuclease.

[0027] In some embodiments, the first endonuclease or the second endonuclease is a type I restriction endonuclease. In some embodiments, the first endonuclease or the second endonuclease is a type II restriction endonuclease. In some embodiments, the first endonuclease or the second endonuclease is a type IIs restriction endonuclease. In some embodiments, the first endonuclease or the second endonuclease is a type III restriction endonuclease. In some embodiments, the first endonuclease or the second endonuclease is endonuclease III. In some embodiments, the first endonuclease or the second endonuclease is endonuclease IV. In some embodiments, the first endonuclease or the second endonuclease is endonuclease V. In some embodiments, the first endonuclease or the second endonuclease is endonuclease VIII. In some embodiments, the first endonuclease or the second endonuclease is T7 endonuclease I. In some embodiments, the first endonuclease or the second endonuclease is T4 endonuclease V. In some embodiments, the first endonuclease or the second endonuclease is T4 endonuclease VII. In some embodiments, the first endonuclease or the second endonuclease is deoxyribonuclease I. In some embodiments, the first endonuclease or the second endonuclease is deoxyribonuclease II. In some embodiments, the first endonuclease or the second endonuclease is deoxyribonuclease III. In some embodiments, the first endonuclease or the second endonuclease is deoxyribonuclease IV. In some embodiments, the first endonuclease or the second endonuclease is an RNA endonuclease. In some embodiments, the first endonuclease or the second endonuclease is an engineered RNA endonuclease with customized sequence specificity. In some embodiments, the first endonuclease or the second endonuclease is an RNA-guided endonuclease. In some embodiments, the first endonuclease or the second endonuclease is a CRISPR / Cas endonuclease. In some embodiments, the first endonuclease or the second endonuclease is Cas9. In some embodiments, the first endonuclease or the second endonuclease is Cpf1.

[0028] On the other hand, provided are synthetic circular nucleic acids produced by the methods provided herein.

[0029] In some embodiments, provided herein are cells comprising synthetic circular nucleic acids produced by the methods provided herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells.

[0030] In some embodiments, provided herein are delivery agents comprising synthetic circular nucleic acids produced by the methods described herein, wherein the delivery agent comprises a lipid, peptide, protein, antibody, carbohydrate, nanoparticle, or microparticle. In some embodiments, the nanoparticle or microparticle is a lipid nanoparticle or lipid microparticle, polymer nanoparticle or polymer microparticle, protein nanoparticle or protein microparticle, or solid nanoparticle or solid microparticle.

[0031] In some embodiments, provided herein are compositions comprising synthetic circular nucleic acids produced by the methods provided herein, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition further comprises an additional reagent. In some embodiments, the additional reagent has a therapeutic effect when administered to a subject. In some embodiments, the additional reagent is a nucleotide, nucleic acid, amino acid, peptide, protein, small molecule, aptamer, lipid, or carbohydrate. In some embodiments, the composition is for preventing or treating a disease or disorder in a subject in need thereof.

[0032] In some embodiments, provided herein are methods of introducing synthetic circular nucleic acids produced by the methods provided herein into cells. In some embodiments, the method comprises introducing the synthetic circular nucleic acid into a subject. In some embodiments, the subject is a human. In some embodiments, the method comprises preventing or treating a disease in a subject in need thereof. These and other aspects of the disclosure are described further below. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following drawings form a part of this specification and are included to further illustrate certain aspects of the disclosure, and certain aspects of the disclosure may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It should be understood that the data shown in the drawings do not limit the scope of the disclosure in any way.

[0034] Figure 1 A step-by-step overview of the NA amplification method described herein is presented, starting with rolling circle amplification (RCA). This is followed by a first endonuclease digestion, self-ligation, and a second endonuclease digestion. In some embodiments, the second endonuclease digestion product is further processed via NA supercoiling, exonuclease digestion, and purification by methods such as column chromatography or isopropanol precipitation.

[0035] Figures 2A - 2B The rolling circle amplification (RCA) step of the method provided herein is shown. Figure 2AA schematic diagram of the RCA method is described, which is the first step of the disclosed method. RCA is performed to amplify a circular NA template containing both the desired expression cassette insertion sequence and the vector backbone, generating long linear concatemers with multiple copies of the insertion sequence. Figure 2B The results of performing RCA on a plasmid for 90 minutes (lane 2) and 16 hours (lane 3) are shown. Lane 1 shows a DNA ladder for size reference. The numbers shown on the left side of the gel represent the length of each corresponding ladder band in base pairs (bp).

[0036] Figures 3A - 3D Shows the first endonuclease digestion ( Figure 3A ) and ligation ( Figure 3B ) of the RCA amplification product ( Figure 3C ) generated from a nucleic acid template ( Figure 3D ). Through these steps, circular NA molecules containing only the expression cassette insertion sequence can be generated, which is the desired final product in some embodiments. Figure 3E shows the products of the first digestion (lane 2) and SCAM ligation (lane 3) run on a DNA gel. Lanes 1 and 2 show linear and supercoiled DNA ladders, respectively, for size reference.

[0037] Figures 4A - 4C Shows the effect of changing the pH value of the general reaction buffer on endonuclease activity. In Figure 4A , lane 1 shows a linear DNA ladder, and lanes 2 - 8 show pH values of 7.8, 7.0, 6.0, 5.0, 4.0, 3.0, and 2.0, respectively. The numbers shown on the left side of the gel represent the length of each corresponding ladder band in base pairs (bp). In Figure 4B , lane 1 shows a linear DNA ladder, lanes 2 - 7 show pH values of 7.8, 7.0, 6.0, 5.0, 4.0, and 3.0, respectively, and lane 8 shows a control where no HindIII enzyme was added to the reaction. The numbers shown on the left side of the gel represent the length of each corresponding ladder band in base pairs (bp). Figure 4C Shows the experimental results of contacting supercoiled DNA (SC) with HindIII inactivated by 3 different methods: heat inactivation (HI), pH inactivation (pH), or a combination of heat and pH (HIpH). Then the solution was added to a ligation reaction containing T4 DNA ligase and incubated overnight at room temperature before running the products on a gel. Lane 1 shows a DNA ladder, lane 2 shows untreated SC DNA as a control, and lanes 3 - 5 show HI, pH, and HIpH samples, respectively. The numbers shown on the left side of the gel represent the length of each corresponding ladder band in kilobases (kb). The numbers corresponding to the experimental bands represent the intensity readings of each band.

[0038] Figures 5A - 5Bshows the beneficial effect of generating a greater amount of circular monomers by the method of continuously adding substrates (SCAM) during the ligation reaction. In Figure 5A , lanes 1 and 2 show linear and supercoiled DNA ladders, respectively. The numbers shown on the left and right sides of the gel represent the length of each corresponding ladder band of the linear (left) and supercoiled (right) ladders in base pairs (bp). Lanes 3, 5, and 7 show the products from conventional ligation reactions loaded in increasing amounts (200 ng, 400 ng, and 800 ng, respectively). Lanes 4, 6, and 8 show the products from SBS ligation loaded in increasing amounts (200 ng, 400 ng, and 800 ng, respectively). In Figure 5B , lanes 1 and 2 show linear and supercoiled DNA ladders, respectively. The numbers shown on the left and right sides of the gel represent the length of each corresponding ladder band of the linear (left) and supercoiled (right) ladder bands in kilobases (kb). Lane 3 shows the ligation product (CTRL) generated by the conventional ligation reaction. Lane 4 shows the ligation product (SCAM) generated by SCAM ligation. The numbers corresponding to the experimental bands represent the intensity readings of each band.

[0039] Figures 6A - 6C Shows the ligation step of the method provided herein. Figure 6A Shows five non-limiting different possible ligation products that can be generated by the method provided herein. Figure 6B Shows the product of the second endonuclease digestion to remove the ligation product containing the vector backbone. Figure 6C Shows a DNA gel electrophoresis comparing the sizes of the ligation products before (lane 2; ligation) and after (lane 3; XhoI digestion) the second digestion. Lane 1 shows the linear DNA ladder for size reference. The numbers shown on the left side of the gel represent the length of each corresponding ladder band in base pairs (bp).

[0040] Figures 7A - 7B Shows the supercoiling step of the method provided herein. Figure 7A Shows how topoisomerase acts on supercoiled NA to produce tightly wound NA molecules that migrate rapidly through the agarose gel due to their increased density after supercoiling. In Figure 7B , lane 2 shows the vector digested with XhoI (XhoI digestion), lane 3 shows the result of supercoiling the digested vector with DNA gyrase for 2 hours (2hr), and lane 4 shows the result of supercoiling the digested vector with DNA gyrase overnight (O / N). Lane 1 shows the linear DNA ladder. The numbers shown on the left side of the gel represent the length of each corresponding ladder band in base pairs (bp).

[0041] Figures 8A - 8BShows the exonuclease step of the method provided herein. Figure 8A Shows the result of contacting the product of a second endonuclease digestion with an exonuclease, which is used to remove linear and open circular NA. This step allows for further purification of the amplified NA product. In Figure 8B , lane 1 shows a linear DNA ladder, lane 2 shows the result of supercoiling the product of a ligation reaction with DNA gyrase (gyrase), and lane 3 shows the result of contacting the supercoiled product with an exonuclease to remove unwanted NA (T5). The numbers shown to the left of the gel indicate each corresponding ladder band (1-14), which matches Figure 7B the length of the DNA ladder bands shown in lane 1.

[0042] Figure 9 Shows the purity of the final NA product produced using the method provided herein. Lanes 1 and 2 show linear and supercoiled DNA ladders, respectively, and lane 3 shows the final NA product (final) after exonuclease digestion and anion exchange chromatography. The numbers shown to the left of the gel indicate the length of each corresponding ladder band in base pairs (bp). Detailed Description

[0043] Traditional cell-based methods for amplifying nucleic acids (NA) are expensive and often error-prone. For example, using bacteria to amplify NA requires growing large amounts of bacteria in expensive fermenters, which need to be kept sterile to prevent contamination of the bacterial culture. The bacteria must also be lysed to release the amplified NA, and the NA must be cleaned and purified to remove other bacterial components that are toxic to mammals, such as endotoxins. Typical methods for purifying NA from bacterial and other cell sources include methods using organic, mutagenic, and toxic compounds (including phenol, ethidium bromide, and cesium chloride) and enzymes (such as lysozyme, proteinase K, and RNase A). All of these compounds, if injected as contaminants in an NA vaccine or other therapeutic NA preparation, can pose potential health hazards.

[0044] In addition to cost issues, the use of bacteria can pose difficulties for the fidelity of the amplification process in many cases. In the complex biochemical environment of bacterial cells, it is difficult to control the quality and yield of the desired NA product. Bacteria may inadvertently alter the sequence of the amplified NA, rendering it unusable for its intended purpose. Recombination events can also cause problems with the NA molecule of interest.

[0045] In contrast, cell-free (synthetic) NA amplification can provide significant cost savings due to streamlined production and simplified purification. In addition, it eliminates impurities typically associated with the conventional methods described herein. In particular, cell-free enzymatic methods for amplifying NA avoid the requirement for host cells, effectively eliminating problems associated with cell-based NA amplification. Provided herein are methods for amplifying circular NA molecules in a synthetic manner, enabling the production of high-purity amplification products in a cost-effective and time-efficient manner.

[0046] Provided herein are methods for amplifying synthetic circular nucleic acid (NA) molecules (e.g., circular NA vectors as described herein). Specifically, the methods provided herein involve in vitro synthesis of NA (e.g., in the absence of cells (i.e., cell-free)), which provides a more pure composition of the resulting NA molecules compared to NA of bacterial or yeast origin and enables faster and more efficient synthesis of NA.

[0047] In some embodiments, amplification of an NA vector using the methods disclosed herein begins with providing a sample comprising circular NA. In some embodiments, the circular NA is generated by self-ligation of linear NA. This can be accomplished by any method known in the art, e.g., contacting linear NA with a ligase under conditions suitable for self-ligation. In some embodiments, the circular NA is DNA. In some embodiments, the circular NA is RNA. In some embodiments, the circular NA comprises: 1) an expression cassette (i.e., an insert sequence) having a heterologous gene, and 2) a backbone (i.e., a scaffold) comprising a nucleotide sequence providing isolation, expression, and / or amplification characteristics. In some embodiments, the circular NA is covalently closed. Circular NA vectors can be synthesized in vitro or obtained from cells using standard NA extraction / isolation techniques known in the art. In some embodiments, linear NA is specifically degraded, e.g., using an exonuclease, to purify the circular NA.

[0048] In some embodiments, the NA vector is single-stranded. In some embodiments, the NA vector is double-stranded. In some embodiments, the NA vector is supercoiled. In some embodiments, the NA vector is monomeric. In some embodiments, the circular NA vector comprises a promoter sequence upstream (in the 5' direction) of one or more heterologous genes. Additionally, or optionally, the circular NA vector can include a polyadenylation site downstream of one or more heterologous genes. Thus, in some embodiments, the circular NA vector comprises the following elements operably linked from 5' to 3' or from 3' to 5': (i) a promoter sequence; (ii) one or more heterologous genes; and (iii) a polyadenylation (poly-A) site (e.g., a site for adding a poly-A tail).

[0049] In some embodiments, the poly-A tail comprises 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, or 400 to 500 nucleotides. In some embodiments, 25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100% of the nucleotides of the poly-A tail are adenosine nucleotides.

[0050] In some embodiments, the NA vector comprises two or more heterologous genes. In some embodiments, the two or more heterologous genes comprise more than one identical gene. As used herein, "identical gene" refers to an NA sequence encoding a protein having the same function and / or structure. In some embodiments, the more than one heterologous gene comprises different genes. As used herein, "different genes" refers to NA sequences encoding proteins having different functions and / or structures. In some embodiments, the different genes encode proteins that interact functionally (e.g., as part of a signaling pathway) or structurally (e.g., by dimerization, e.g., the heavy and light chains of an antibody or a fragment thereof), or proteins that do not interact. In some embodiments, the heterologous gene comprises one or more trans-splicing molecules or portions thereof (e.g., binding domains).

[0051] In some embodiments, the NA vector comprising the expression cassette is amplified in vitro in a cell-free preparation by rolling circle amplification (RCA) by incubating the NA with a polymerase (e.g., a phage polymerase, e.g., Phi29 DNA polymerase), a primer (e.g., a specific primer, a random primer, e.g., a random polymer primer), and a nucleotide mixture (e.g., dNTP or NTP, e.g., dATP, dCTP, dGTP, and dTTP, or ATP, CTP, GTP, and UTP). In some embodiments, the nucleotide mixture is a natural nucleotide mixture (i.e., substantially free of nucleotide analogs). In other embodiments, the nucleotide mixture comprises one or more nucleotide analogs, such as unnatural nucleotides.

[0052] In some embodiments, the nucleotide analogs comprise modified phosphates such that when incorporated into nascent NA, they result in modified internucleotide linkages. The modified phosphates used in the present invention can be, but are not limited to, phosphorothioate (PS), thiophosphate, 5′-O-methylphosphonate, 3′-O-methylphosphonate, 5′-hydroxyphosphonate, hydroxyphosphonate, phosphororoselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinocyclo, triazole ring, borophosphonate (BP), methylphosphonate, and guanidinopropylaminophosphate. In some embodiments, more than one modified phosphate is used.

[0053] In some embodiments, the nucleotide analogs comprise modified sugars. The modified sugars used in the present invention can be, but are not limited to, 2′-deoxyfluoro (2FA), L-adenosine (LA), 2′-deoxyadenosine (dA), locked nucleic acid (LNA), 2′-methoxy (2OMe), 2′-methoxyethoxy (2MOE), 2′-thiogalactose, 2′,3′-dideoxyribose, 2′-amino-2′-deoxyribose, 2′-deoxyribose, 2′-azido-2′-deoxyribose, 2′-fluoro-2′-deoxyribose, 2′-O-methylribose, 2′-O-methyldeoxyribose, 3′-amino-2′,3′-dideoxyribose, 3′-azido-2′,3′-dideoxyribose, 3′-deoxyribose, 3′-O-(2-nitrobenzyl)-2′-deoxyribose, 3′-O-methylribose, 5′-aminoribose, 5′-thiogalactose, 5-nitro-1-indolyl-2′-deoxyribose, 5′-biotin-ribose, 2′-O,4′-C-methylene-linked, 2′-O,4′-C-amino-linked ribose, and 2′-O,4′-C-thio-linked ribose. In some embodiments, more than one modified sugar is used.

[0054] In some embodiments, the nucleotide analogs comprise modified nucleobases. The modified nucleobases used in the present invention can be, but are not limited to, inosine, xanthine, allylaminouridine, allylaminothymidine, hypoxanthine, digoxigenin adenine, digoxigenin cytosine, digoxigenin guanine, digoxigenin uridine, 6-chloropurine riboside, N6-methyladenosine, methylpseudouridine, 2-thiocytosine, 2-thiouridine, 5-methyluridine, 4-thiothymidine, 4-thiouridine, 5,6-dihydro-5-methyluridine, 5,6-dihydrouridine, 5-[(3-indolyl)propionamide-N-allyl]uridine, 5-aminoallylcytosine, 5-aminoallyluridine, 5-bromouridine, 5-bromocytidine, 5-carboxycytosine, 5-carboxymethylesteruridine, 5-carboxyuridine, 5-fluorouridine, 5-formylcytosine, 5-formyluridine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluridine, 5-hydroxyuridine, 5-iodocytidine, 5-iodouridine, 5-methoxycytidine, 5-methoxyuridine, 5-methylcytidine, 5-methyluridine, 5-propynylaminocytosine, 5-propynylaminouridine, 5-propynylcytidine, 5-propynyluridine, 6-azacytidine, 6-azauridine, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deaza-7-propynylaminoadenine, 7-deaza-7-propynylaminoguanine, 8-azaaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, araumacil, biotin-16-7-deaza-7-propynylaminoguanidine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluridine, cyanine3-5-propynylaminocytosine, cyanine3-6-propynylaminouridine, cyanine3-aminoallylcytosine, cyanine5-6-propynylaminocytosine, cyanine5-6-propynylaminouridine, cyanine5-aminoallylcytosine, cyanine5-aminoallyluridine, cyanine7-aminoallyluridine, dabcyl-5-3-aminoallyluridine, desthiobiotin-16-aminoallyluridine, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouridine, N1-methoxymethylpseudouridine, N1-methyladenine, N1-methylpseudouridine, N1-propylpseudouridine, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine, pseudouridine, thiophenocytosine, thiophenoguanine, thiophenouridine, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-diaminoguanine, 5-formyluracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methylthio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycylcarbamoylaniline (g6A), N6-threonylcarbamoylaniline (t6A), 2-methylthio-N6-threonylcarbamoylaniline (ms2t6A), N6-methyl-N6-threonylcarbamoylaniline (m6t6A), N6-hydroxynorvalylcarbamoylaniline (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, more than one modified nucleobase is used.,

[0055] The polymerase amplifies the NA molecule by rolling circle amplification, thereby generating a linear concatemer having multiple copies of the NA molecule. Suitable polymerases include thermophilic polymerases, polymerases having high processive synthesis ability through GC-rich sequences, and polymerases having NA displacement ability.

[0056] The resulting concatemer is digested using an endonuclease (e.g., a restriction endonuclease) to cleave within the concatenated NA molecules, thereby generating unit length linear NA molecules comprising the heterologous gene (if the heterologous gene was present in the NA molecule that underwent the amplification process). Self-ligation of the linear NA molecules (e.g., by addition of a ligase) generates circular synthetic NA molecules, optionally completed with the heterologous gene. Because the replication and amplification of the NA described herein can be performed using a polymerase under cell-free conditions, the synthetic NA molecules can be isolated from the bacterial components of the plasmid in which it was cloned, thereby removing bacterial markers such as bacterial CpG motifs and / or dam or dcm methylation from the isolated vector.

[0057] On the one hand, the present disclosure provides a cell-free method for amplifying isolated NA vectors by: (i) providing a sample comprising a circular NA vector that comprises an expression cassette, wherein the expression cassette comprises (a) a heterologous gene that comprises a first endonuclease cleavage site at the 5' and 3' ends of the expression cassette, and (b) one or more second endonuclease cleavage sites within the vector backbone; (ii) amplifying the expression cassette using polymerase-mediated RCA to generate a linear concatemer; (iii) digesting the linear concatemer with a first endonuclease to separate the expression cassette from the backbone; and (iv) incubating the expression cassette under conditions that permit self-ligation to generate a circular ligation product that comprises the expression cassette. In some embodiments, the method further comprises digesting the ligation product with a second endonuclease selected to cleave the circular NA product that comprises the vector backbone to generate a second digestion product. In some embodiments, the method further comprises contacting the ligation product or the second digestion product with a topoisomerase to supercoil the remaining circular NA product, i.e., the circular NA product that lacks the vector backbone. In some embodiments, the supercoiled NA can be polymeric supercoiled NA. In some embodiments, the supercoiled NA can be monomeric supercoiled NA. In some embodiments, the method further comprises purifying the supercoiled product. In some embodiments, the supercoiled product is purified using column chromatography, electrophoresis, or other methods known in the art. In some embodiments, open relaxed circular NA is separated from supercoiled NA during the purification step.

[0058] Aspects of the methods disclosed herein combine multiple technologies with the goal of producing large amounts of NA for therapeutic, diagnostic, and research applications in an affordable manner. Due to the cell-free nature of the methods disclosed herein, there is no source of endotoxin contamination other than the minimal amount of endotoxin contained in the reagents used. Additional advantages include: the ability to produce large amounts of fermentation-like products in small laboratory flasks; the need for only a minimal number of reagents; the ability to produce large amounts of product in a relatively short period of time; and a streamlined purification procedure.

[0059] Synthetic nucleic acid molecule

[0060] On the one hand, the methods disclosed herein provide isolated circular NA (e.g., circular NA vectors), wherein the circular NA lacks: (a) an origin of replication (e.g., a bacterial origin of replication) and / or a drug resistance gene; and (b) recombination sites. For example, in some embodiments, the circular NA lacks an origin of replication, a drug resistance gene, and recombination sites. In some embodiments, the circular NA comprises one or more heterologous genes. In some embodiments, one or more of the heterologous genes comprise an open reading frame (ORF). In some embodiments, one or more of the heterologous genes encode a protein. In some embodiments, the protein is monomeric (e.g., a monomeric protein having a secondary, tertiary, and / or quaternary structure under physiological conditions). In some embodiments, the protein is a multimer (e.g., a dimeric protein (e.g., a homodimeric protein or a heterodimeric protein), a trimeric protein, etc.). In some aspects, the heterologous gene encodes one or more proteins (e.g., a single protein, two proteins, three proteins, four proteins, etc.). In such embodiments, the heterologous gene sequence can be a polycistronic sequence or a polytranscriptional unit sequence.

[0061] In some embodiments, the heterologous gene comprises a spacer region. As used herein, a spacer region refers to a nucleotide sequence located between coding sequences in a polycistronic locus or polycistronic mRNA to facilitate translation or processing of one or more of the coding sequences into one or more independent proteins. Non-limiting examples of spacer regions are internal ribosome entry sites (IRESs), self-cleaving peptide coding sequences, and nucleotide sequences encoding endogenous protease cleavage sites. In some embodiments, the spacer region is an IRES. As used herein, an IRES refers to a DNA sequence that, once transcribed into RNA, allows translation to initiate from an internal region of the RNA (e.g., mRNA). Translation in eukaryotes typically begins at the 5' cap of the mRNA, such that each mRNA undergoes only one translation event. However, an IRES can initiate translation independently of the 5' cap and serve as an additional ribosome recruitment site, resulting in the co-expression of one or more proteins from a single mRNA.

[0062] In some embodiments, the spacer encodes a self-cleaving peptide, including but not limited to 2A, E2A, F2A, P2A, and T2A self-cleaving peptides. As used herein, a self-cleaving 2A peptide refers to a short oligopeptide (usually 19-22 amino acids) located between two proteins of certain members of the picornavirus family. The 2A self-cleaving peptide can self-cleave through a translational effect called "stop-go" or "stop-carry" to produce mature proteins (Wang et al. (2015), Nature Scientific Reports 5:16237). The term "self-cleaving" is a misnomer because these peptides are thought to act by causing ribosomes to skip the synthesis of the C-terminal peptide bond of the 2A element, resulting in the separation between the end of the 2A sequence and the next peptide downstream. "Cleavage" occurs between the glycine and proline residues found at the C-terminus, meaning that the upstream cistron will add some additional residues at the end, while the downstream cistron will start from proline.

[0063] In some embodiments, the spacer encodes a cleavage site for an endogenous protease of the host cell. Non-limiting examples of proteases are trypsin, elastase, matrix metalloproteinase (MMP), and pepsin.

[0064] In some embodiments, the protein is a therapeutic protein. As used herein, "therapeutic protein" refers to a protein that, when expressed in a subject, prevents, reduces, or alleviates one or more signs or symptoms of a disease, where the subject is, for example, a human subject suffering from a disease or disorder or at risk of developing a disease or disorder. The therapeutic protein can be, for example, an enzyme, a blood coagulation factor, a peptide, an interleukin, an allergen, an interferon, a transcription factor, a growth factor, a cytokine, an anti-apoptotic factor, an anti-diabetic factor, a blood coagulation factor, an enzyme-activated protein, an anti-tumor factor, a pro-apoptotic factor, a chemokine, an antibody (or an antibody fragment thereof), a protein hormone, a signaling protein, a structural protein, or a cell surface receptor encoded by a mutated gene in the subject. As a non-limiting example, a mutation in the gene encoding such a protein can result in a reduced expression level of the protein in one or more cells of the subject. Thus, expression of the therapeutic protein can compensate for the mutation in the gene encoding such a protein in the subject. In some embodiments, the enzyme is an epigenetic regulator. In some embodiments, the epigenetic regulator is a histone methyltransferase, a histone demethylase, a histone acetyltransferase, a DNA methyltransferase, or a DNA demethylase. In some embodiments, the therapeutic protein is an antigen. In some embodiments, the antigen is a tumor antigen, a viral antigen, a microbial antigen, a bacterial antigen, or a plant antigen. In some embodiments, the therapeutic protein is an antibody or a portion, fragment, or variant thereof. The antibodies include fragments capable of binding an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab’, bispecific scFv, single-domain antibody (sdAb), (Fab’)2 (including chemically linked (Fab’)2), chimeric antibodies, and humanized antibodies.

[0065] On the other hand, the methods disclosed herein provide NA vectors comprising one or more non-coding heterologous genes. In some embodiments, the non-coding heterologous gene is a therapeutic NA. As used herein, therapeutic nucleic acid (NA) is a nucleic acid or related compound that modifies gene expression to prevent or treat a disease or disorder. In some embodiments, the therapeutic NA is an antisense oligonucleotide (ASO), a DNA aptamer, an RNA aptamer, a ribozyme, an RNA decoy, siRNA, shRNA, miRNA, gRNA, or a CRISPRi molecule.

[0066] In some embodiments, the heterologous gene contains a reporter sequence in addition to the sequence encoding a protein or therapeutic NA. In some embodiments, the reporter gene is used to verify heterologous gene expression, for example, in specific cells and tissues. Reporter sequences that can be provided in the heterologous gene include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and other genes well known in the art. When combined with regulatory elements driving its expression, the reporter sequence provides a signal that can be detected by conventional methods, including enzymatic, radiographical, colorimetric, fluorescent or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, when the reporter sequence is the LacZ gene, the LacZ expression product produces a detectable signal in the presence of an appropriate reagent. When the reporter sequence expresses green fluorescent protein or luciferase, the presence of green fluorescent protein or luciferase (along with a suitable reagent) produces a detectable signal that can be visually measured by color or light production in a photometer.

[0067] On the other hand, provided herein are NA vectors comprising a promoter sequence upstream (in the 5' direction) of one or more heterologous genes. In some embodiments, the NA vector includes a polyadenylation site downstream of one or more heterologous genes. In some embodiments of any of the above aspects, one or more heterologous genes include a trans-splicing molecule or a portion thereof (e.g., a binding domain). On the other hand, provided herein are isolated circular NA vectors having one or more therapeutic NAs. Such isolated circular NA vectors lack an origin of replication and / or a drug resistance gene and lack recombination sites. In some embodiments, the NA vector contains terminal repeats. In some embodiments, the length of the terminal repeats is at least 10 base pairs (bp). In some embodiments, the NA vector lacks bacterial plasmid DNA. In some embodiments, the NA vector contains one or more unmethylated GATC sequences, one or more unmethylated CCAGG sequences, and / or one or more CCTGG sequences. Additionally, or optionally, the NA vector (a) lacks immunogenic bacterial markers; (b) lacks RNA polymerase termination sites; and / or (c) is substantially free of CpG islands.

[0068] In some embodiments, the NA vectors (e.g., circular NA vectors) used herein comprise conventional control elements that are operably linked to the heterologous gene in a manner that permits transcription, translation, and / or expression in a target cell. Control elements include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly-A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product. A variety of control elements, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized. If a promoter region can affect the transcription of a heterologous gene, then the promoter region is operably linked to the heterologous gene such that the resulting transcript can be translated into the desired protein. Promoters that can be used as part of the NA vectors described herein include constitutive and inducible promoters.

[0069] On the other hand, the present disclosure provides isolated linear NA molecules, optionally produced in a cell-free manner, comprising multiple identical concatemers, wherein each identical concatemer comprises at least one heterologous gene. In some embodiments, the isolated linear NA molecule is DNA. In some embodiments, the isolated linear NA molecule is RNA. In some embodiments, the at least one heterologous gene encodes one or more therapeutic proteins. In some embodiments, the isolated linear NA molecule lacks: (a) an origin of replication (e.g., a bacterial origin of replication) and / or a drug resistance gene; and (b) recombination sites. For example, in some embodiments, the isolated linear NA molecule lacks an origin of replication, a drug resistance gene, and recombination sites. In some embodiments, the isolated linear NA molecule comprises an endonuclease cleavage site. In some embodiments, the endonuclease cleavage site is located at the 5' and 3' ends of the heterologous gene insertion.

[0070] In some embodiments, the circular NA provided herein is >250 base pairs. In some embodiments, the circular NA ranges from about 250 base pairs to about 250,000 base pairs (i.e., 250 bp to 250,000 bp). In some embodiments, the circular NA is 250 to 1000 bp. In some embodiments, the circular NA is 1000 to 10,000 bp. In some embodiments, the circular NA is 10,000 to 25,000 bp. In some embodiments, the circular NA is 25,000 bp to 50,000 bp. In some embodiments, the circular NA is 50,000 bp to 75,000 bp. In some embodiments, the circular NA is 75,000 bp to 100,000 bp. In some embodiments, the circular NA is 100,000 bp to 125,000 bp. In some embodiments, the circular NA is 125,000 bp to 150,000 bp. In some embodiments, the circular NA is 150,000 bp to 175,000 bp. In some embodiments, the circular NA is 175,000 bp to 200,000 bp. In some embodiments, the circular NA is 200,000 bp to 225,000 bp. In some embodiments, the circular NA is 225,000 bp to 250,000 bp.

[0071] In some embodiments, the circular NA is about 500 bp. In some embodiments, the circular NA is about 1,000 bp. In some embodiments, the circular NA is about 1,100 bp. In some embodiments, the circular NA is about 1,200 bp. In some embodiments, the circular NA is about 1,300 bp. In some embodiments, the circular NA is about 1,400 bp. In some embodiments, the circular NA is about 1,500 bp. In some embodiments, the circular NA is about 1,600 bp. In some embodiments, the circular NA is about 1,700 bp. In some embodiments, the circular NA is about 1,800 bp. In some embodiments, the circular NA is about 1,900 bp. In some embodiments, the circular NA is about 2,000 bp. In some embodiments, the circular NA is about 2,200 bp. In some embodiments, the circular NA is about 2,400 bp. In some embodiments, the circular NA is about 2,600 bp. In some embodiments, the circular NA is about 2,800 bp. In some embodiments, the circular NA is about 3,000 bp. In some embodiments, the circular NA is about 3,300 bp. In some embodiments, the circular NA is about 3,600 bp. In some embodiments, the circular NA is about 3,900 bp. In some embodiments, the circular NA is about 4,000 bp. In some embodiments, the circular NA is about 4,400 bp. In some embodiments, the circular NA is about 4,800 bp. In some embodiments, the circular NA is about 5,000 bp. In some embodiments, the circular NA is about 5,500 bp. In some embodiments, the circular NA is about 10,000 bp. In some embodiments, the circular NA is about 11,000 bp. In some embodiments, the circular NA is about 12,000 bp. In some embodiments, the circular NA is about 13,000 bp. In some embodiments, the circular NA is about 14,000 bp. In some embodiments, the circular NA is about 15,000 bp. In some embodiments, the circular NA is about 16,000 bp. In some embodiments, the circular NA is about 17,000 bp. In some embodiments, the circular NA is about 18,000 bp. In some embodiments, the circular NA is about 19,000 bp. In some embodiments, the circular NA is about 20,000 bp. In some embodiments, the circular NA is about 22,000 bp. In some embodiments, the circular NA is about 24,000 bp. In some embodiments, the circular NA is about 25,000 bp.In some embodiments, the circular NA is about 27,500 bp. In some embodiments, the circular NA is about 30,000 bp. In some embodiments, the circular NA is about 32,000 bp. In some embodiments, the circular NA is about 34,000 bp. In some embodiments, the circular NA is about 36,000 bp. In some embodiments, the circular NA is about 38,000 bp. In some embodiments, the circular NA is about 40,000 bp. In some embodiments, the circular NA is about 44,000 bp. In some embodiments, the circular NA is about 48,000 bp. In some embodiments, the circular NA is about 50,000 bp. In some embodiments, the circular NA is about 55,000 bp. In some embodiments, the circular NA is about 60,000 bp. In some embodiments, the circular NA is about 65,000 bp. In some embodiments, the circular NA is about 70,000 bp. In some embodiments, the circular NA is about 75,000 bp. In some embodiments, the circular NA is about 80,000 bp. In some embodiments, the circular NA is about 85,000 bp. In some embodiments, the circular NA is about 90,000 bp. In some embodiments, the circular NA is about 95,000 bp. In some embodiments, the circular NA is about 100,000 bp. In some embodiments, the circular NA is about 110,000 bp. In some embodiments, the circular NA is about 120,000 bp. In some embodiments, the circular NA is about 125,000 bp. In some embodiments, the circular NA is about 130,000 bp. In some embodiments, the circular NA is about 135,000 bp. In some embodiments, the circular NA is about 140,000 bp. In some embodiments, the circular NA is about 145,000 bp. In some embodiments, the circular NA is about 150,000 bp. In some embodiments, the circular NA is about 160,000 bp. In some embodiments, the circular NA is about 170,000 bp. In some embodiments, the circular NA is about 175,000 bp. In some embodiments, the circular NA is about 180,000 bp. In some embodiments, the circular NA is about 190,000 bp. In some embodiments, the circular NA is about 200,000 bp. In some embodiments, the circular NA is about 210,000 bp. In some embodiments, the circular NA is about 220,000 bp. In some embodiments, the circular NA is about 225,000 bp.In some embodiments, the circular NA is about 230,000 bp. In some embodiments, the circular NA is about 240,000 bp. In some embodiments, the circular NA is about 250,000 bp.

[0072] Synthetic nucleic acid amplification

[0073] Typical protocols for amplifying NA synthesis fragments include using methods such as polymerase chain reaction (“PCR”) to amplify target DNA fragments. In vitro amplification by PCR has been successfully used in laboratories since the mid-1980s. Although PCR is rapid and affordable, it relies on rapid thermal cycling, which is impractical for large-scale applications. However, in order to continue its amplification, the PCR product can be ligated into a suitable vector and then transformed into a host cell such as a bacterial or yeast cell for in vivo amplification. After transformation, clones (single cell colonies derived from one cell) are identified that express the vector containing the desired target DNA fragment. Typically, such positive clones are identified by screening many colonies (at least dozens, possibly hundreds) to determine the presence of the vector containing the target DNA fragment. Screening is usually carried out by restriction mapping, and DNA sequencing can be used to verify the integrity of the vector and the presence of the complete target DNA fragment. This process is time-consuming, labor-intensive, requires the design and production of very specific DNA primer sequences, and provides multiple opportunities for mutagenesis to occur.

[0074] Once a clone containing the vector with the target DNA fragment is selected, the cells from that clone are amplified. As the cells of the selected clone proliferate, the vector is amplified. Then the amplified vector containing the target DNA fragment is recovered from the cells. When recovering the amplified DNA, there are usually problems associated with separating the amplified NA from the cells and unwanted cellular components. Although many methods have been designed for this purification over the years, they are still time-consuming and ineffective (i.e., cellular contaminants often remain with the isolated NA).

[0075] Rolling circle amplification

[0076] In nature, the replication of circular DNA (including plasmids and some viral genomes) often occurs by rolling circle amplification (“RCA”), whereby the circular DNA template is replicated into long linear concatemers of tandem repeats (e.g., amplicons). This process can also be carried out on circular RNA templates. An optimized in vitro RCA system is provided herein for large-scale NA production in a cell-free system, using a streamlined template, highly specific or random primers, and a polymerase.

[0077] The term RCA describes the ability of an RCA-type polymerase (also referred to herein as an RCA polymerase) to continuously advance around a circular NA template (DNA or RNA) while extending a hybridized primer. This results in the formation of linear single-stranded products with multiple repeats of the amplified NA. These linear single-stranded products serve as the basis for multiple hybridization, primer extension, and strand displacement events, leading to the formation of multiplexed NA products that also contain multiple repeats of the amplified DNA. Thus, in the tandem NA products, there are multiple copies of each amplified "single unit" NA molecule. RCA polymerases are particularly preferred for the methods disclosed herein. The products of the RCA-type strand displacement replication process typically require complex processing to release single units of NA.

[0078] To date, most RCA techniques have used Phi29 DNA polymerase, although other polymerases can also be used. This is because Phi29 polymerase is highly processive, which allows it to rapidly synthesize long concatemers of NA, and has strand displacement activity, which allows it to continuously synthesize new NA sequences while displacing any secondary primers it encounters. In addition, it can produce large amounts of NA in a relatively short period of time without thermal cycling. Moreover, Phi29 polymerase has an extremely low average error rate and can use RNA or DNA as a template.

[0079] The RCA reaction can utilize single-stranded NA templates or double-stranded NA templates. In some embodiments, a modified plasmid lacking the typical genetic sequences required for plasmid selection and replication in bacteria is used as a template. Any vector template can be used. In some embodiments, the template can be a circular expression cassette that contains at least one heterologous gene of interest flanked by genetic elements (e.g., promoters, poly-A tails, etc.) required for expression and processing of the expression product in the target cell. Although the methods disclosed herein can be used to amplify genes from conventional plasmids, streamlined templates that do not contain additional genetic sequences offer several benefits: they eliminate foreign sequences that may inadvertently silence the expression of the heterologous gene of interest; smaller constructs are more compact and can be taken up more efficiently by target cells, resulting in higher transfection efficiencies; and they are more cost-effective due to the lower production requirements for shorter expression cassettes, a statistical increase in the fidelity of the final product, and a reduced need for deep purification.

[0080] Although most RCA techniques use Phi29 DNA polymerase, it is contemplated that the disclosed methods are not limited to the use of Phi29 DNA polymerase. For example, U.S. Patent Nos. 6,576,448 and 6,235,502 disclose the use of bacterial DNA polymerase III in RCA. DNA polymerase III is reported to have clamp-like activity, providing a DNA synthesis rate of about 700 - 800 nucleotides per second, which can be optimized by adding helicase or stabilizing proteins. In addition, bacterial DNA polymerase I has also been used in RCA to amplify templates. DNA polymerase I mainly uses single-stranded templates, allowing small circular templates to form easily without steric hindrance, which is a problem often associated with the replication of very short double-stranded templates. U.S. Patent No. 5,614,365 discloses a modified DNA polymerase I that includes a sequence from T7 DNA polymerase to increase its efficiency up to 500-fold. This polymerase has a reduced ability to distinguish between deoxynucleotides and dideoxynucleotides. Other DNA polymerases useful in the methods disclosed herein include M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, VENT DNA polymerase, DEEP VENT DNA polymerase, KlenTaq DNA polymerase, the Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, reverse transcriptase, and their derivatives. Some embodiments of the methods disclosed herein use processive strand displacement polymerases, such as Phi29-like polymerases (such as those found in Phi29-like bacteriophages; see, e.g., Microbiol MolBiol Rev. 2001 Jun; 65(2):261 - 287), to efficiently amplify templates without thermal cycling. Preferred embodiments use Phi29 or Phi29-like polymerases, but other polymerases such as Pol I and Pol III, T7 DNA polymerase, and their derivatives can also be used.

[0081] To allow amplification according to the methods disclosed herein, it is preferred that the NA template is also contacted with one or more DNA or RNA primers. The primers can be non-specific (i.e., random sequences) or can be specific for one or more sequences contained in the NA template. Preferably, the primers are random sequences to allow non-specific initiation at any site on the NA template. This allows efficient amplification by multiple priming reactions from each template strand. Examples of random primers are hexamers, heptamers, octamers, nonamers, decamers or sequences of longer length, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The length of the random primers can be from 6 to 30, from 8 to 30 or from 12 to 30 nucleotides. Random primers are typically provided as a mixture of oligonucleotides that represent all potential combinations of the polymers in the NA template (e.g., hexamers, heptamers, octamers or nonamers, etc.).

[0082] In other embodiments, the NA primers are specific. As used herein, a specific NA primer is a primer having a sequence that is at least 80% complementary to the sequence in the template from which amplification is desired to begin. In this embodiment, a pair of primers can be used to specifically amplify the portion of the template that is internal to the two primer binding sites. The primers can be unlabeled or can contain one or more labels, such as a radionuclide or a fluorescent molecule. In some embodiments, the primers contain chemically modified nucleotides. The primer length / sequence can generally be selected based on considerations of the hybridization temperature, i.e., the temperature at which the primers are capable of binding to the template during the amplification step.

[0083] Contact of the NA template with the RCA polymerase and one or more primers occurs under conditions that facilitate hybridization of the primers to the template. The conditions include the presence of single-stranded NA that allows primer hybridization. In some embodiments, the double-stranded NA is denatured by any method known in the art (e.g., incubation at a temperature above 80°C) to provide single-stranded NA. The conditions also include the temperature and buffer that facilitate hybridization of the primers to the template. Suitable hybridization conditions can be selected according to the nature of the primers. Examples of preferred hybridization conditions used in the methods disclosed herein include buffer 30 mM Tris-HCl pH = 7.5, 20 mM KCl, 8 mM MgCl 2 . The hybridization can be carried out by gradually cooling to the desired reaction temperature after denaturation.

[0084] Once the NA template is contacted with a polymerase and one or more primers, it is incubated under conditions that promote amplification of the template. Preferably, the conditions promote amplification of the template by strand displacement replication of one strand to displace the replicated strand. The conditions include using any temperature that permits NA amplification, typically in the range of 20 to 90 °C. Preferred temperature ranges can be from about 20 to about 40 °C, or from about 25 to about 35 °C. Generally, a suitable temperature is selected based on the temperature at which a particular polymerase has optimal activity. This information is typically available and forms part of the general knowledge of a person skilled in the art. For example, the suitable temperature range for Phi29 DNA polymerase is from about 25 to about 35 °C, preferably about 30 °C. A person skilled in the art can generally determine the suitable temperature for effective amplification in the methods disclosed herein. For example, the process can be carried out over a range of temperatures, and the yield of amplified NA can be monitored to determine the optimal temperature range for a given polymerase.

[0085] Other conditions that promote template amplification include the presence of a polymerase and one or more primers. These conditions also include the presence of dNTPs (dATP, dTTP, dCTP, and dGTP) or NTPs (ATP, UTP, GTP, and CTP), a suitable buffer / pH, and other factors required for enzyme performance or stability. Suitable conditions include any conditions known in the art for providing polymerase activity.

[0086] For example, the pH can be in the range of 3 to 10, preferably 5 to 8 or about 7, such as about 7.5. The pH value can be maintained within this range by using one or more buffers. These buffers include, but are not limited to, MES, Bis-Tris, ADA, ACES, PIPES, MOBS, MOPS, MOPSO, Bis-Tris propane, BES, TES, HEPES, DIPSO, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, tris(hydroxymethyl)methylglycine (Tricine), Gly-Gly, Bicine, HE PBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, phosphates, citric acid-sodium hydrogen phosphate, citric acid-sodium citrate, sodium acetate-acetic acid, imidazole, and sodium carbonate-sodium bicarbonate. The reaction can also include salts of divalent metals, such as, but not limited to, magnesium salts (Mg 2+ ) and manganese salts (Mn 2+ ), including chlorides, acetates, and sulfates. Salts of monovalent metals, such as sodium (Na + ) and potassium (K + ) salts, such as potassium chloride, can also be included. Other salts that can be included are ammonium salts, particularly ammonium sulfate.

[0087] It may also include a detergent. Examples of suitable detergents include Triton TM X-100 (octoxynol), 20 (polyethylene glycol sorbitan monolaurate) and its derivatives. A stabilizer may also be included in the reaction. Any suitable stabilizer can be used; in particular, bovine serum albumin (BSA) and other stabilizing proteins are useful in the methods disclosed herein. The reaction conditions can also be improved by adding reagents that relax the NA coil and make it easier to denature the template. These reagents include, for example, dimethyl sulfoxide (DMSO), formamide, glycerol, and betaine.

[0088] The specific concentration of a particular reagent can be selected according to previous examples in the art and further optimized to meet specific requirements. As an example, a suitable reaction buffer used in RCA-based methods in the art contains 50 mM Tris HCl, pH = 7.5, 10 mM MgCl 2 、20 mM (NH 4 ) 2 SO 4 、5% glycerol, 0.2 mM BSA, and 1 mM dNTP. In the methods disclosed herein, a preferred reaction buffer for RCA amplification contains 35 mM Tris-HCl, 50 mM KCl, 14 mM MgCl 2 、10 mM (NH 4 ) 2 SO 4 、4 mM DTT, and 1 mM dNTP. This buffer is particularly suitable for Phi29 RCA polymerase.

[0089] Ligation reaction

[0090] The methods herein include generating circular closed NA from linear concatemer molecules. Ligases are a class of enzymes that catalyze the formation of a phosphodiester bond between the 3'-OH and 5'-phosphate groups of an NA strand, covalently binding two NA molecules together, or cyclizing a single NA molecule. In one aspect, under conditions that promote the ligation reaction, the linearized NA after digestion with a first endonuclease is contacted with at least one ligase. In some embodiments, the ligase used is T4 DNA ligase. Other ligases useful in the methods disclosed herein include, but are not limited to, T4 RNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, or Escherichia coli DNA ligase.

[0091] An important feature of the synthetic NA production process described herein is the volume of the ligation reaction. As the concentration of NA in the ligation reaction decreases, the reaction promotes the formation of monomeric circular NA (by intramolecular self-ligation) rather than the formation of high molecular weight polymers (by intermolecular ligation). One way to maintain a low concentration of linear NA in the ligation reaction is to add the template NA to the reaction in a staggered manner, which separates the total reaction NA (linear and circular NA) from the NA of the input reaction (only linear NA). When the linear NA in the reaction is cyclized, the concentration of linear NA decreases and more can be added while maintaining a low concentration. A method called stepwise (SBS) ligation has been described in the art to keep the linear template DNA low during the ligation reaction. The SBS method involves intermittently adding very small (<100bp) linear ssDNA molecules to the reaction buffer in 20-minute cycles over a total of 2 hours. However, when applied to large-sized (>250bp) dsDNA molecules, the SBS method has been shown to be only slightly better than conventional ligation (see Figure 5A ).

[0092] Accordingly, a new method has been developed and applied to the methods herein that allows for high-yield production of monomeric ligation products using linear dsDNA templates: the Substrate Continuous Addition Method (SCAM). SCAM involves adding linear NA to the ligation reaction in a slow, continuous manner. In some embodiments, a pump (e.g., a syringe pump or a peristaltic pump) is used for the addition of linear NA. In some embodiments, SCAM is carried out in a tube, such as a 50 mL conical tube. In some embodiments, SCAM is carried out in a larger container, such as a flask (e.g., an Erlenmeyer flask), such as a 125 mL flask.

[0093] In some embodiments, the first digestion product is added to the ligation reaction mixture at a rate of about 1% to about 20% of the final ligation reaction volume per hour over a period of 5 - 14 hours. In some embodiments, the addition is at a rate of about 2% to about 20% of the final ligation reaction volume per hour, or about 3% to about 20% of the final ligation reaction volume per hour, or about 4% to about 20% of the final ligation reaction volume per hour, or about 5% to about 20% of the final ligation reaction volume per hour, or about 6% to about 20% of the final ligation reaction volume per hour, or about 7% to about 20% of the final ligation reaction volume per hour, or about 8% to about 20% of the final ligation reaction volume per hour, or about 9% to about 20% of the final ligation reaction volume per hour, or about 10% to about 20% of the final ligation reaction volume per hour, or about 11% to about 20% of the final ligation reaction volume per hour, or about 12% to about 20% of the final ligation reaction volume per hour, or about 13% to about 20% of the final ligation reaction volume per hour, or about 14% to about 20% of the final ligation reaction volume per hour, or about 15% to about 20% of the final ligation reaction volume per hour, or about 16% to about 20% of the final ligation reaction volume per hour, or about 17% to about 20% of the final ligation reaction volume per hour, or about 18% to about 20% of the final ligation reaction volume per hour, or about 19% to about 20% of the final ligation reaction volume per hour, or about 1% to about 19% of the final ligation reaction volume per hour, or about 2% to about 19% of the final ligation reaction volume per hour, or about 3% to about 19% of the final ligation reaction volume per hour, or about 4% to about 19% of the final ligation reaction volume per hour, or about 5% to about 19% of the final ligation reaction volume per hour, or about 6% to about 19% of the final ligation reaction volume per hour, or about 7% to about 19% of the final ligation reaction volume per hour, or about 8% to about 19% of the final ligation reaction volume per hour, or about 9% to about 19% of the final ligation reaction volume per hour, or about 10% to about 19% of the final ligation reaction volume per hour, or about 11% to about 19% of the final ligation reaction volume per hour, or about 12% to about 19% of the final ligation reaction volume per hour, or about 13% to about 19% of the final ligation reaction volume per hour, or about 14% to about 19% of the final ligation reaction volume per hour, or about 15% to about 19% of the final ligation reaction volume per hour, or about 16% to about 19% of the final ligation reaction volume per hour, or about 17% to about 19% of the final ligation reaction volume per hour, or about 18% to about 19% of the final ligation reaction volume per hour, or about 1% to about 18% of the final ligation reaction volume per hour, or about 2% to about 18% of the final ligation reaction volume per hour, or about 3% to about 18% of the final ligation reaction volume per hour, or about 4% to about 18% of the final ligation reaction volume per hour, or about 5% to about 18% of the final ligation reaction volume per hour, or about 6% to about 18% of the final ligation reaction volume per hour, or about 7% to about 18% of the final ligation reaction volume per hour, or about 8% to about 18% of the final ligation reaction volume per hour, or about 9% to about 18% of the final ligation reaction volume per hour, or about 10% to about 18% of the final ligation reaction volume per hour, or about 11% to about 18% of the final ligation reaction volume per hour, or about 12% to about 18% of the final ligation reaction volume per hour, or about 13% to about 18% of the final ligation reaction volume per hour, or about 14% to about 18% of the final ligation reaction volume per hour, or about 15% to about 18% of the final ligation reaction volume per hour, or about 16% to about 18% of the final ligation reaction volume per hour, or about 17% to about 18% of the final ligation reaction volume per hour, or about 1% to about 17% of the final ligation reaction volume per hour, or about 2% to about 17% of the final ligation reaction volume per hour, or about 3% to about 17% of the final ligation reaction volume per hour, or about 4% to about 17% of the final ligation reaction volume per hour, or about 5% to about 17% of the final ligation reaction volume per hour, or about 6% to about 17% of the final ligation reaction volume per hour, or about 7% to about 17% of the final ligation reaction volume per hour, or about 8% to about 17% of the final ligation reaction volume per hour, or about 9% to about 17% of the final ligation reaction volume per hour, or about 10% to about 17% of the final ligation reaction volume per hour, or about 11% to about 17% of the final ligation reaction volume per hour, or about 12% to about 17% of the final ligation reaction volume per hour, or about 13% to about 17% of the final ligation reaction volume per hour, or about 14% to about 17% of the final ligation reaction volume per hour, or about 15% to about 17% of the final ligation reaction volume per hour, or about 16% to about 17% of the final ligation reaction volume per hour, or about 1% to about 16% of the final ligation reaction volume per hour, or about 2% to about 16% of the final ligation reaction volume per hour, or about 3% to about 16% of the final ligation reaction volume per hour, or about 4% to about 16% of the final ligation reaction volume per hour, or about 5% to about 16% of the final ligation reaction volume per hour, or about 6% to about 16% of the final ligation reaction volume per hour, or about 7% to about 16% of the final ligation reaction volume per hour, or about 8% to about 16% of the final ligation reaction volume per hour, or about 9% to about 16% of the final ligation reaction volume per hour, or about 10% to about 16% of the final ligation reaction volume per hour, or about 11% to about 16% of the final ligation reaction volume per hour, or about 12% to about 16% of the final ligation reaction volume per hour, or about 13% to about 16% of the final ligation reaction volume per hour, or about 14% to about 16% of the final ligation reaction volume per hour, or about 15% to about 16% of the final ligation reaction volume per hour, or about 1% to about 15% of the final ligation reaction volume per hour, or about 2% to about 15% of the final ligation reaction volume per hour, or about 3% to about 15% of the final ligation reaction volume per hour,or from about 4% to about 15%, or from about 5% to about 15%, or from about 6% to about 15%, or from about 7% to about 15%, or from about 8% to about 15%, or from about 9% to about 15%, or from about 10% to about 15%, or from about 11% to about 15%, or from about 12% to about 15%, or from about 13% to about 15%, or from about 14% to about 15%, or from about 1% to about 14%, or from about 2% to about 14%, or from about 3% to about 14%, or from about 4% to about 14%, or from about 5% to about 14%, or from about 6% to about 14%, or from about 7% to about 14%, or from about 8% to about 14%, or from about 9% to about 14%, or from about 10% to about 14%, or from about 11% to about 14%, or from about 12% to about 14%, or from about 13% to about 14%, or from about 1% to about 13%, or from about 2% to about 13%, or from about 3% to about 13%, or from about 4% to about 13%, or from about 4% to about 13%, or from about 5% to about 13%, or from about 6% to about 13%, or from about 7% to about 13%, or from about 8% to about 13%, or from about 9% to about 13%, or from about 10% to about 13%, or from about 11% to about 13%, or from about 12% to about 13%, or from about 1% to about 12%, or from about 2% to about 12%, or from about 3% to about 12%, or from about 4% to about 12%, or from about 5% to about 12%, or from about 6% to about 12%, or from about 7% to about 12%, or from about 8% to about 12%, or from about 9% to about 12%, or from about 10% to about 12%, or from about 11% to about 12%, or from about 1% to about 11%, or from about 2% to about 11%, or from about 3% to about 11%, or from about 4% to about 11%, or from about 5% to about 11%, or from about 6% to about 11%, or from about 7% to about 11%, or from about 8% to about 11%, or from about 9% to about 11%, or from about 10% to about 11%, or from about 1% to about 10%, or from about 2% to about 10%, or from about 3% to about 10%, or from about 4% to about 10%, or from about 5% to about 10%, or from about 6% to about 10%, or from about 7% to about 10%, or from about 8% to about 10%, or from about 9% to about 10%, or from about 1% to about 9% or from about 2% to about 9%, or from about 3% to about 9%, or from about 4% to about 9%, or from about 5% to about 9%, or from about 6% to about 9%, or from about 7% to about 9%, or from about 8% to about 9%, or from about 1% to about 8%, or from about 2% to about 8%, or from about 3% to about 8%, or from about 4% to about 8%, or from about 5% to about 8%, or from about 6% to about 8%, or from about 7% to about 8%, or from about 1% to about 7%, or from about 2% to about 7%, or from about 3% to about 7%, or from about 4% to about 7%, or from about 5% to about 7%, or from about 6% to about 7%, or from about 1% to about 6%, or from about 2% to about 6%, or from about 3% to about 6%, or from about 4% to about 6%, or from about 5% to about 6%, or from about 1% to about 5%, or from about 2% to about 5%, or from about 3% to about 5%, or from about 4% to about 5%, or from about 1% to about 4%, or from about 2% to about 4%, or from about 3% to about 4%, or from about 1% to about 3%, or from about 2% to about 3%Or from about 1% to about 2%.

[0094] In some embodiments, the first digestion product is added to the ligation reaction mixture over a period of 6 - 14 hours, or 7 - 14 hours, or 8 - 14 hours, or 9 - 14 hours, or 10 - 14 hours, or 11 - 14 hours, or 12 - 14 hours, or 13 - 14 hours, or 5 - 13 hours, or 6 - 13 hours, or 7 - 13 hours, or 8 - 13 hours, or 9 - 13 hours, or 10 - 13 hours, or 11 - 13 hours, or 12 - 13 hours, or 5 - 12 hours, or 6 - 12 hours, or 7 - 12 hours, or 8 - 12 hours, or 9 - 12 hours, or 10 - 12 hours, or 11 - 12 hours, or 5 - 11 hours, or 6 - 11 hours, or 7 - 11 hours, or 8 - 11 hours, or 9 - 11 hours or 10 - 11 hours, or 5 - 10 hours, or 6 - 10 hours, or 7 - 10 hours, or 8 - 10 hours, or 9 - 10 hours, or 5 - 9 hours, or 6 - 9 hours, or 7 - 9 hours, or 8 - 9 hours, or 5 - 8 hours, or 6 - 8 hours, or 7 - 8 hours, or 5 - 7 hours, or 5 - 6 hours. In some embodiments, the first digestion product is added to the ligation reaction mixture over a period of about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours or about 14 hours or longer. Preferably, the first digestion product is added to the ligation reaction mixture at a rate of about 3% to about 5% of the final ligation reaction volume per hour over a 10 - 12 hour period.

[0095] Endonucleases, topoisomerases and exonucleases, as well as nucleic acid purification

[0096] In some embodiments, the methods disclosed herein include a processing step of using an endonuclease to cleave a NA molecule, resulting in the production of multiple nucleotide units. Endonucleases are a class of enzymes that cleave phosphodiester bonds within a polynucleotide chain, and they can facilitate the cleavage of NA molecules as further described herein. Different families of endonucleases have different requirements for the initiation of cleavage. For example, DNase I family endonucleases cleave DNA nonspecifically, meaning they will cleave DNA at almost any double-stranded position. Optionally, restriction endonucleases contain domains that recognize very specific nucleotide sequences and thus cleave only at those specific sites. Restriction endonucleases or other sequence-specific endonucleases are preferably used in the methods disclosed herein. Types of endonucleases useful in the methods disclosed herein include, but are not limited to, type I restriction endonucleases, type II restriction endonucleases, type IIs restriction endonucleases, type III restriction endonucleases, endonuclease III, endonuclease IV, endonuclease V, endonuclease VIII, T7 endonuclease I, T4 endonuclease V, T4 endonuclease VII, deoxyribonuclease I, deoxyribonuclease II, deoxyribonuclease III, IV, and RNA endonucleases, including engineered RNA endonucleases with customized sequence specificity. Other sequence-specific endonucleases that can be used include RNA-guided endonucleases, such as any CRISPR / Cas endonuclease (and similar endonucleases, e.g., Cas9, Cpf1, etc.).

[0097] In some embodiments, to ensure that the endonuclease no longer cleaves the NA molecule after the digestion reaction is complete, methods for inactivating the endonuclease are used. A common method for inactivating an enzyme is to heat the reaction solution containing the enzyme to a temperature that promotes protein denaturation, e.g., >65 °C and hold for >15 minutes. Such a high temperature will disrupt important protein domains and alter the shape of the enzyme so that its substrate cannot fit with the active site, thereby inactivating it. Another method is to change the pH of the reaction solution containing the enzyme to a pH that alters the hydrogen bond and salt bridge interactions in the protein tertiary structure, also inactivating it. In some embodiments, the methods herein include inactivating the endonuclease after the endonuclease reaction is complete. In some embodiments, the endonuclease is inactivated by heat inactivation, e.g., heating the reaction solution to >65 °C and holding for >15 minutes. In some examples, the pH of the reaction solution is changed by adding an acid or a base to inactivate the endonuclease. In some embodiments, hydrochloric acid (HCl) is used to lower the pH of the reaction solution, and sodium hydroxide (NaOH) is used to increase the pH of the reaction solution after inactivation. Other acids and bases that can be used include, but are not limited to, acetic acid (C 2 H 4 O 2 ), boric acid (H 3BO 3 ) Citric acid (C 6 H 8 O 7 ) Nitric acid (HNO 3 ) Sulfuric acid (H 2 SO 4 ) Calcium hydroxide (Ca(OH) 2 ) Ammonia (NH 3 ) Sodium bicarbonate (NaHCO 3 ) or sodium carbonate (Na 2 CO 3 ).

[0098] The structure of the closed-end NA molecule, including circular NA molecules, such as the ligation products described herein, promotes the occurrence of NA supercoiling. Supercoiling is a property of closed-end NA caused by the tight topological coupling between the supercoiled and double-helical structures of NA, resulting in a structurally compact molecule. Due to its inherent physical properties, supercoiling typically occurs in closed-end NA molecules. When in a circular conformation, the primary coiled structure of double-helical NA promotes further coiling into a supercoiled structure. In some embodiments, the methods herein produce supercoiled products by ligating monomer insertion sequences.

[0099] In some embodiments, the circular ligation product is at least partially supercoiled, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% supercoiled.

[0100] In some embodiments, the methods herein include generating further supercoiled NA from open - looped NA by enzymatic reactions. It is not uncommon that cellular processes such as replication, transcription, recombination, and rearrangement result in topological changes of NA, leading to problems of NA distortion, which can be resolved by the action of topoisomerases. In vitro, topoisomerases are useful because they have the ability to alter the topological state of DNA and RNA. DNA topoisomerases, for example, introduce transient single - strand (type I) or double - strand (type II) breaks in the phosphodiester backbone of DNA to allow positive or negative supercoiling (more or less tightly wound). By contacting NA with topoisomerases in vitro, it is possible to synthetically determine the supercoiled state of NA. In some embodiments, the topoisomerase used is DNA gyrase. In some embodiments, the topoisomerase used is Escherichia coli DNA gyrase. Other topoisomerases useful in the methods disclosed herein include, but are not limited to, Cre recombinase, Staphylococcus aureus DNA gyrase, DNA topoisomerase 2 - α, or DNA topoisomerase 2 - β.

[0101] In some embodiments, the methods herein include using exonucleases to remove linear and open - looped NA from the supercoiled product. Exonucleases are a class of enzymes that digest NA strands with open 3’ or 5’ ends, ultimately resulting in the elimination of these strands. In the present disclosure, exonucleases can be used to further purify the amplified NA product. In some embodiments, the exonuclease used is T5 exonuclease. Other exonucleases useful in the methods disclosed herein include, but are not limited to, exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VIII, exonuclease T, λ exonuclease, or T7 exonuclease.

[0102] In some embodiments, the methods herein include further purifying the amplified NA product using NA purification techniques known in the art. These methods can include, but are not limited to, isopropanol precipitation, methanol precipitation, ethanol precipitation, ion - exchange chromatography including anion - exchange chromatography, solid - phase purification, electrophoresis, affinity chromatography, reverse - phase chromatography, or size - exclusion chromatography. As known in the art, certain chromatographic methods can be performed as column chromatography or batch chromatography. Combinations of various purification techniques can also be used.

[0103] Definitions

[0104] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, and reference is made to the disclosure text, which provides a general guide to many of the terms used in this application for those skilled in the art. If there is any conflict between the definitions listed herein and those in the reference publications, the definitions provided herein shall prevail.

[0105] As used herein, "nucleic acid" (NA) or "polynucleotide" refers to an organic molecule comprising two or more covalently bonded nucleotides. As used herein, "nucleotide" refers to an organic molecule that comprises: 1) a nucleoside, which comprises a sugar covalently bonded to a nitrogenous base (nucleobase); and 2) a phosphate group, which is covalently bonded to the sugar of the nucleoside. Nucleotides in a polynucleotide are typically linked by phosphodiester bonds, wherein the 3′ carbon of the sugar of the first nucleotide is linked to the 5′ carbon of the sugar of the second nucleic acid via a bridging phosphate group. Typically, the bridging phosphate comprises two non-bridging oxygen atoms and two bridging oxygen atoms, the non-bridging oxygen atoms being bonded only to the phosphorus atom of the phosphate, each bridging oxygen atom linking the phosphorus atom to the 3′ carbon of the first nucleotide or the 5′ carbon of the second nucleotide. In a nucleic acid sequence that describes the nucleotide sequence in a nucleic acid, if the 3′ carbon of the first nucleotide is linked to the 5′ carbon of the second nucleotide, the first nucleotide is said to be located 5′ (upstream) of the second nucleotide. Similarly, if the 5′ carbon of the second nucleotide is linked to the 3′ carbon of the first nucleotide, the second nucleotide is said to be located 3′ (downstream) of the first nucleotide. Nucleic acid sequences are typically read in the 5′→3′ order, starting with the 5′ nucleotide and ending with the 3′ nucleotide.

[0106] As used herein, the term "circular NA" or "circular NA vector" refers to a nucleic acid molecule in circular form. The circular NA can be DNA or RNA. The circular NA vector can be monomeric, dimeric, trimeric, tetrameric, pentameric, hexameric, etc. Preferably, the circular NA vector is monomeric. In other preferred embodiments, the circular NA vector is a monomeric supercoiled circular DNA molecule. In some embodiments, the NA vector is open circular. In some embodiments, the DNA vector is double-stranded circular. This circular form is generally capable of being amplified into tandem linear NA by rolling circle amplification. The term "circular NA vector" may be used interchangeably herein with the terms "NA vector", "circular NA molecule", and "covalently closed circular NA vector". Those skilled in the art will understand that, as described herein, such circular vectors can include covalently closed vectors with supercoiling and complex DNA topologies.

[0107] In some embodiments, the NA is linear NA. Linear NA can be DNA or RNA. Linear NA is an NA having a 5′-terminal nucleotide and a 3′-terminal nucleotide. The 5′-terminal nucleotide of linear NA is covalently bound to only one adjacent nucleotide of the NA, and the adjacent nucleotide appears 3′ of the 5′-terminal nucleotide in the NA nucleic acid sequence. The 3′-terminal nucleotide of linear NA is covalently bound to only one adjacent nucleotide of the NA, and the adjacent nucleotide appears 5′ of the 3′-terminal nucleotide in the NA nucleic acid sequence. In a nucleic acid sequence containing each nucleotide of linear NA in 5′-to-3′ order, the 5′-terminal nucleotide is the first nucleotide in the sequence, and the 3′-terminal nucleotide is the last nucleotide in the sequence. In some embodiments, the linear NA is self-ligating to produce circular NA.

[0108] RNA molecules that can be translated are referred to as messenger RNA, or mRNA. DNA or RNA sequences encode proteins via codons. A codon refers to a group of three nucleotides in a nucleic acid (such as DNA or RNA) sequence. An anticodon refers to a group of three nucleotides within a nucleic acid, such as transfer RNA (tRNA), which are complementary to a codon such that the codon of the first nucleic acid binds to the anticodon of the second nucleic acid via hydrogen bonds between the codon and anticodon bases. For example, the codon 5′-AUG-3′ on mRNA has the corresponding anticodon 3′-UAC-5′ on tRNA. During translation, a tRNA having an anticodon complementary to the codon to be translated binds to the codon on mRNA, typically to deliver the amino acid corresponding to the codon to be translated or to facilitate the termination of translation and the release of the translated polypeptide from the ribosome.

[0109] As used herein, "modified nucleotide" refers to a nucleotide whose structure is not the canonical structure of an adenosine nucleotide, cytidine nucleotide, guanosine nucleotide, or uridine nucleotide. The canonical structure of a molecule refers to the structure typically known in the art for the molecule named. The canonical structure of an adenosine nucleotide containing an adenine base, ribose, and one or more phosphate groups is shown below in the form of adenosine monophosphate:

[0110]

[0111] The canonical structure of AMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which the oxygen atoms of the phosphate and / or the 3′ oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0112] The canonical structure of a cytidine nucleotide containing a cytosine base, ribose, and one or more phosphate groups is shown below in the form of cytidine monophosphate:

[0113] The canonical structure of CMP also refers to a structure in which one or more hydroxyl groups of phosphoric acid and / or one or more hydroxyl groups of sugar are deprotonated, and a structure in which the oxygen atom of phosphoric acid and / or the 3'-oxygen atom of sugar binds to an adjacent nucleotide in a nucleic acid sequence.

[0114] The canonical structure of a guanine nucleotide containing a guanine base, ribose, and one or more phosphate groups is shown below in the form of guanosine monophosphate:

[0115] The canonical structure of GMP also refers to a structure in which one or more hydroxyl groups of phosphoric acid and / or one or more hydroxyl groups of sugar are deprotonated, and a structure in which the oxygen atom of phosphoric acid and / or the 3'-oxygen atom of sugar binds to an adjacent nucleotide in a nucleic acid sequence.

[0116] The canonical structure of a uracil nucleotide containing a uracil base, ribose, and one or more phosphate groups is shown below in the form of uridine monophosphate:

[0117] The canonical structure of UMP also refers to a structure in which one or more hydroxyl groups of phosphoric acid and / or one or more hydroxyl groups of sugar are deprotonated, and a structure in which the oxygen atom of phosphoric acid and / or the 3'-oxygen atom of sugar binds to an adjacent nucleotide in a nucleic acid sequence.

[0118] Due to one or more modifications in the sugar, nitrogenous base, or phosphate of a nucleotide, the structure of a modified nucleotide may be different from that of a typical nucleotide. In some embodiments, a modified nucleotide contains a modified nucleoside that is not in the canonical structure of adenosine, cytidine, guanosine, or uridine.

[0119] An example of the canonical structure of adenosine (a type of adenosine nucleoside) is as follows:

[0120] The canonical structure of adenosine also refers to a structure in which one or more hydroxyl groups of phosphoric acid and / or one or more hydroxyl groups of sugar are deprotonated, a structure in which the 5'-carbon in the nucleic acid sequence binds to the 5'-phosphate, and a structure in which the 3'-oxygen atom in the nucleic acid sequence binds to the 5'-phosphate of an adjacent nucleotide.

[0121] An example of the canonical structure of cytidine (a type of cytidine nucleoside) is as follows:

[0122] The canonical structure of cytidine also refers to a structure in which one or more hydroxyl groups of phosphoric acid and / or one or more hydroxyl groups of sugar are deprotonated, a structure in which the 5'-carbon in the nucleic acid sequence binds to the 5'-phosphate, and a structure in which the 3'-oxygen atom in the nucleic acid sequence binds to the 5'-phosphate of an adjacent nucleotide.

[0123] An example of the canonical structure of guanosine (a guanine nucleoside) is as follows:

[0124] The canonical structure of guanosine also refers to a structure in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, a structure in which the 5′ carbon in the nucleic acid sequence is bound to the 5′ phosphate, and a structure in which the 3′ oxygen atom in the nucleic acid sequence is bound to the 5′ phosphate of an adjacent nucleotide.

[0125] An example of the canonical structure of uridine (a uracil nucleoside) is as follows:

[0126] The canonical structure of uridine also refers to a structure in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, a structure in which the 5′ carbon in the nucleic acid sequence is bound to the 5′ phosphate, and a structure in which the 3′ oxygen atom in the nucleic acid sequence is bound to the 5′ phosphate of an adjacent nucleotide.

[0127] As used herein, "poly-A tail" refers to a nucleic acid sequence containing adenosine nucleotides that is attached to the 3′ end of a nucleic acid (such as RNA). The poly-A tail or poly-A region can consist of 25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100% adenosine nucleotides. The adenosine nucleotides contained in the poly-A tail can be canonical adenosine nucleotides or modified (non-canonical) adenosine nucleotides.

[0128] As used herein, "ligase" refers to an enzyme capable of forming a covalent bond between two nucleotides, and the process of "ligating" refers to forming a covalent bond between two nucleotides. As used herein, "concatemer" refers to a NA molecule containing multiple copies of the same or substantially the same NA sequence (such as a subunit) that are typically tandemly linked.

[0129] As used herein, the term "isolated" refers to being artificially produced. In some embodiments, with respect to an NA vector, the term "isolated" refers to an NA vector that is: (i) amplified in vitro (e.g., in a cell-free environment), e.g., by rolling circle amplification or polymerase chain reaction (PCR); (ii) recombinantly produced by molecular cloning; (iii) purified, such as by restriction endonuclease cleavage and separation by gel electrophoresis, or column chromatography; or (iv) synthesized, e.g., by chemical synthesis. An isolated NA vector is a vector that is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector is considered isolated if the 5' and 3' restriction endonuclease sites in the vector are known, or if the polymerase chain reaction (PCR) primer sequences of the vector have been published, but an NA sequence that exists in its native state in the natural host of the NA sequence is not considered isolated. An isolated NA vector can be substantially purified, but this is not required.

[0130] As used herein, a "cell-free method" of producing an NA vector refers to a method that does not rely on the inclusion of any NA within a host cell (such as a bacterial (e.g., Escherichia coli) host cell) to facilitate any step of the method. For example, a cell-free method occurs in a suitable solution (e.g., a buffer solution) within one or more synthetic containers (e.g., glass or plastic tubes or other containers), where enzymes and other reagents can be added to facilitate NA amplification, modification, and isolation.

[0131] As used herein, a "vector" refers to an NA molecule capable of carrying a heterologous gene into a target cell, where the heterologous gene can then be replicated, processed, and / or expressed in the target cell.

[0132] Translation is the process of guiding protein production using an RNA coding sequence. The first step of translation is initiation, in which the ribosome binds to the mRNA and the first transfer RNA (tRNA) carrying the first amino acid binds to the first codon or start codon. The next stage of translation, elongation, consists of three steps. First, the second tRNA with an anticodon binds to the mRNA, which is complementary to the start (START) codon or the second codon and carries the second amino acid. Second, the carbon atom of the terminal non-side chain carboxylic acid portion of the first amino acid reacts with the nitrogen of the terminal non-side chain amino portion of the second amino acid carried, forming a peptide bond between the two amino acids, the second amino acid binds to the second tRNA, and the first amino acid binds to the second amino acid but not to the first tRNA. Third, the first tRNA dissociates from the mRNA, and the ribosome moves along the mRNA such that the position of the first tRNA bound to the ribosome is now occupied by the second tRNA, and the position previously occupied by the second tRNA is now empty for an additional tRNA carrying an additional amino acid to bind to the mRNA. These three steps: 1) binding of the tRNA carrying the amino acid, 2) formation of the peptide bond, which adds the additional amino acid to the growing polypeptide, and 3) movement of the ribosome along the mRNA, continue until the ribosome reaches the stop (STOP) codon, which results in the termination of translation. Typically, the tRNA that binds to the stop codon does not carry an amino acid, so the binding of the tRNA that does not carry an amino acid during the elongation step causes the bond between the polypeptide and the tRNA carrying the last amino acid in the polypeptide to break, thus releasing the polypeptide from the ribosome. Optionally, if no tRNA binds to the stop codon, the ribosome can dissociate from the mRNA and release the polypeptide.

[0133] As used herein, "target cell" refers to any cell that expresses or is intended to express a target gene. A vector can be introduced into target cells present in a subject (in situ) or target cells in culture by a variety of methods including electroporation. In some embodiments, the target cell is a post-mitotic cell. Target cells include vertebrate and invertebrate cells (and cell lines of animal origin). Representative examples of vertebrate cells include mammalian cells such as human, rodent (e.g., rat and mouse), and ungulate (e.g., bovine, goat, sheep, and pig). Alternatively, the target cell can be a stem cell (e.g., a pluripotent cell (i.e., a cell whose progeny can differentiate into several restricted cell types such as hematopoietic stem cells or other stem cells)) or a totipotent cell (i.e., a cell whose progeny can become any cell type in an organism, such as embryonic stem cells and somatic stem cells such as hematopoietic cells). In other embodiments, the target cells include oocytes, eggs, embryonic cells, fertilized eggs, sperm cells, and somatic (non-stem) mature cells from a variety of organs or tissues such as liver cells, nerve cells, muscle cells, and blood cells (e.g., lymphocytes).

[0134] "Host cell" refers to any cell that contains a NA vector of interest. As described in the present disclosure, a host cell can be used as a recipient for a NA vector. The term includes the progeny of the original cell that has been transfected. Thus, "host cell" as used herein can refer to a cell that has been transfected with a heterologous gene (e.g., by a NA vector as described herein), and any progeny thereof that carry the NA vector of interest. It should be understood that the progeny of a single parental cell may not be identical to the original parent in morphology or genomic or total NA complement due to natural, accidental, or intentional mutations.

[0135] The term "heterologous gene" refers to a gene that is not naturally present in the host or target cell in which it is expressed. For example, a heterologous gene can be a mammalian gene, such as a therapeutic gene (e.g., a gene encoding a therapeutic protein such as a therapeutic replacement protein, an antigen-binding protein, etc.), such as a mammalian gene encoding a therapeutic protein. In some embodiments, the heterologous gene encodes a protein or a portion thereof that is defective or absent in the target cell and / or subject (e.g., a therapeutic replacement protein). In some embodiments, the heterologous gene contains one or more exons encoding a protein that is defective or absent in the target cell and / or subject. In some embodiments, the heterologous gene includes therapeutic NA such as therapeutic RNA (e.g., microRNA, siRNA, shRNA, or guide RNA compatible with the Cas nuclease system) or therapeutic DNA (e.g., antisense oligonucleotides, aptamers, or ribozymes).

[0136] The term "promoter" refers to a sequence that regulates the transcription of a heterologous gene operably linked to the promoter. The promoter provides a sequence that is sufficient to direct the transcription and / or recognition site for RNA polymerase and other transcription factors required for efficient transcription, and can direct cell-specific expression. In addition to the sequence sufficient to direct transcription, the promoter sequence can also include sequences of other regulatory elements involved in regulating transcription (e.g., enhancers, Kozak sequences, and introns). Examples of promoters known in the art and useful for the vectors described herein include the CMV promoter, the hEF1a promoter, the CBA promoter, the smCBA promoter, inducible promoters such as TRE and TRE3G, and those derived from immunoglobulin genes, SV40, or other tissue-specific genes. Standard techniques for generating functional promoters by mixing and matching known regulatory elements are known in the art. A "truncated promoter" can also be generated from a promoter fragment or by mixing and matching fragments of known regulatory elements; for example, the smCBA promoter is a truncated form of the CBA promoter.

[0137] As used herein, an "open reading frame" ("ORF"), such as an ORF encoding a protein, refers to a nucleic acid sequence that contains a coding sequence that, when translated, results in the production of a protein. The nucleic acid sequence can be an RNA sequence, in which case translation of the RNA sequence produces a polypeptide having the amino acid sequence of the protein. The nucleic acid sequence can be a DNA sequence, in which case a protein is produced when RNA polymerase transcribes an RNA molecule containing an RNA sequence complementary to the DNA sequence, and translation of the RNA sequence produces a polypeptide having the amino acid sequence of the protein. An ORF typically begins with a start codon, such as AUG in an RNA sequence (ATG in a DNA sequence), and ends with a stop codon, such as UAG, UAA, or UGA in an RNA sequence (TAG, TAA, or TGA in a DNA sequence), and the number of bases between the G of the start codon and the T or U of the stop codon is a multiple of 3 (e.g., 3, 6, 9, 12, etc.).

[0138] In some embodiments of the synthetic circular NA provided herein, the circular NA contains a 5′ untranslated region (5′UTR) and a 3′ untranslated region (3′UTR). The 5′ and 3′UTRs are sequences within the RNA that do not encode the amino acids of the protein encoded by the RNA and are thus not part of the open reading frame. The 5′UTR is located 5′ (upstream) of the open reading frame. The 3′UTR is located 3′ (downstream) of the open reading frame. In some embodiments, the 3′UTR contains one or more nucleotides that are located 3′ of the open reading frame of the RNA and 5′ (upstream) of the poly-A region.

[0139] In some aspects, the present disclosure provides compositions comprising any of the synthetic cyclic NAs, delivery agents, or cells provided herein. In some embodiments, the composition further comprises one or more additional reagents, such as nucleotides, nucleic acids, amino acids, peptides, proteins, small molecules, aptamers, lipids, or carbohydrates. In some embodiments, the additional reagent has a therapeutic effect when administered to a subject. In some embodiments, the additional reagent is a reagent for modulating the expression and / or activity of one or more gene products (e.g., proteins) in a subject. In some embodiments, the additional reagent is a nucleic acid for reducing the expression and / or activity of one or more gene products (e.g., proteins), such as short hairpin RNA (shRNA), small interfering RNA (siRNA), or antisense oligonucleotide (ASO). In some embodiments, the additional reagent is an inhibitor for reducing the activity of one or more gene products (e.g., proteins). In some embodiments, the reagent is a small molecule inhibitor. In some embodiments, the additional reagent is a reagent for enhancing the immune response of a subject. In some embodiments, the additional reagent is an antigen, such as a nucleic acid antigen, a protein antigen, or a phospholipid antigen. In some embodiments, the additional reagent is an adjuvant, such as aluminum hydroxide or potassium aluminum sulfate (alum), monophosphoryl lipid A (MPL), water-in-oil emulsion (e.g., squalene emulsion), cytosine-phosphate-guanine (CpG) oligodeoxynucleotide, or other adjuvants known in the art. See, e.g., Di Pasquale, A et al. Vaccines. 2015. 3(2):320-343. In some embodiments, the composition is a pharmaceutical composition comprising any one of the synthetic cyclic NAs, delivery agents, or cells provided herein, and a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients, carriers, buffers, stabilizers, isotonic agents, preservatives, or antioxidants, or other substances well known to those skilled in the art. These materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance may depend on the route of administration, such as parenteral, intramuscular, intradermal, sublingual, oral, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal administration.

[0140] In some aspects, the present disclosure provides methods of administering any of the synthetic circular NAs, delivery agents, cells, compositions, or pharmaceutical compositions provided herein to a subject. In some embodiments, the subject is a human. In some embodiments, the administration is parenteral, intramuscular, intradermal, sublingual, oral, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal administration. In some embodiments, the composition is stored at below 50°C, below 40°C, below 30°C, below 20°C, below 10°C, below 0°C, below -10°C, below -20°C, below -30°C, below -40°C, below -50°C, below -60°C, below -70°C, or below -80°C, such that the nucleic acid is relatively stable over time. In some embodiments, the synthetic circular NA is introduced into the cells of a subject by in vivo electroporation. In vivo electroporation is the process of using electrical pulses to introduce nucleic acids or other molecules into the cells of a subject, which facilitates the passage of nucleic acids or other molecules across the cell membrane and / or cell wall. See, e.g., Somiari et al. Molecular Therapy., 2000. 2(3):178-187. The synthetic circular NA to be delivered is administered to the subject, such as by injection, and electrical pulses are applied to the injection site, whereby the nucleic acid is electrofacilitated into the cells at the administration site. In some embodiments, the synthetic circular NA is delivered to and taken up by the cells of a subject (e.g., locally at the administration site or cells of the entire subject) by a delivery agent that binds (e.g., conjugates) to the synthetic circular NA. In some embodiments, the synthetic circular NA is administered together with other components such as buffers and / or excipients to increase the electroporation efficiency.

[0141] The terms "a" and "an" mean "one or more". For example, "a gene" is understood to represent one or more such genes. Thus, the terms "a" and "an", "one or more", "at least one" are used interchangeably herein.

[0142] Unless otherwise indicated, the term "about" as used herein refers to a value that varies within ±10% of a reference value.

[0143] The phrase "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that may be present conjointly in some cases and separately in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so joined. Other elements may optionally be present in addition to the elements specifically identified by the "and / or" clause, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, in one embodiment, when used in conjunction with open-ended language such as "comprising", a reference to "A and / or B" can refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so forth.

[0144] As used in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one, but also more than one, of a plurality or series of elements, and optionally including additional unlisted items. Only terms that expressly state the contrary, such as "only one" or "exactly one", or when used in a claim "consisting of", refer to exactly one element of a plurality or series of elements. In general, the term "or" as used herein represents an exclusive alternative (i.e., "one or the other, but not both") only when preceded by an exclusive term such as "either", "one", "only one" or "exactly one". When used in a claim, "consisting essentially of" shall have the ordinary meaning as used in the field of patent law.

[0145] As used in the specification and claims, with respect to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each specifically listed element in the list of elements, and not excluding any combinations of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to in the phrase "at least one", whether or not related to those specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") can, in one embodiment, refer to at least one, optionally including more than one A, with no B present (and optionally including elements other than B); in another embodiment, refer to at least one, optionally including more than one B, with no A present (and optionally including elements other than A); in yet another embodiment, refer to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0146] In view of the specification and practice of the invention disclosed herein, other embodiments of the invention will be apparent to those skilled in the art. The specification and examples are considered to be exemplary only, and the true scope and spirit of the invention are indicated by the appended claims. In case of any conflict in definitions between various sources or references, the definitions provided herein shall prevail.

[0147] Examples

[0148] Example 1: Development of a cell - free method for generating synthetic circular nucleic acids using rolling - circle amplification

[0149] Figure 1 A schematic diagram of the method disclosed herein is described, in which a starting synthetic circular NA template is amplified by the steps shown. Steps 1-4 can be supplemented with steps 5-7, in which the amplified NA solution is contacted with a topoisomerase to supercoil it, contacted with an exonuclease to remove linear NA, and further purified to obtain a purer final product. All reaction steps use a common buffer and no purification is required between steps.

[0150] Figures 2A - 2B A rolling circle amplification (RCA) process using Example NA and polymerase is described. As Figure 2AAs shown, circular DNA molecules (labeled plasmids) can be used as NA templates. Incubating 5 μg / mL plasmid with 50 μM random DNA primers, 2 mM dNTP molecules, and 200 U / mL Phi29 DNA polymerase in a buffer solution results in the amplification of long single-stranded concatemers. The DNA primers then bind to the concatemers, and the Phi29 polymerase generates complementary strands, producing a double-stranded linear DNA product containing multiple copies of the starting plasmid sequence. Figure 2B The results of RCA of the plasmid for 90 minutes (lane 2) and 16 hours (lane 3) are shown. Lane 1 shows a DNA ladder for size reference. Lanes 2 and 3 illustrate the presence of long, double-stranded concatemers that migrate slowly in the gel and remain in the loading well, with a greater amount of concatemers observed after 16 hours compared to 90 minutes of RCA.

[0151] Figures 3A - 3D A schematic diagram depicting the amplification of an NA template using the method provided herein. Examples of starting NA templates are shown in Figure 3A wherein the circular template contains a backbone sequence, an insert sequence, cleavage sites (RE1) at the 5' and 3' ends of the insert sequence, and an endonuclease cleavage site (RE2) within the backbone that does not have the same recognition sequence as RE1. In this example, the cleavage sites at the 5' and 3' ends of the insert sequence are the same cleavage site, but this is not necessary. Performing RCA on the template generates a linear concatemer (RCA amplification product) as shown in Figure 3B The horizontal lines depict the RE1 and RE2 cleavage sites in the linear concatemer. The RCA amplification product is then contacted with an endonuclease that cleaves at RE1, producing a Figure 3C shown linear product (first digestion product) that contains separate insert and backbone sequences. The insert sequence is flanked by RE1 cleavage sites and does not contain RE2 cleavage sites. The first digestion product is incubated with a ligase to promote self-ligation, and examples of possible products are shown in Figure 3D (ligation products). For example, in some embodiments, the ligation product generated by the method described herein contains a single copy of the insert sequence (ligation product 1), while in other embodiments, it contains more than one copy of the insert sequence (ligation product 4). In some embodiments, the ligation product contains a single copy of the backbone (ligation product 2), and in some embodiments, it contains more than one copy of the backbone (ligation product 5). In some embodiments, the ligation product contains at least one insert sequence and at least one backbone (ligation product 3).

[0152] Having as in Figure 3AThe DNA plasmid of the shown design is subjected to RCA and a first digestion with the first restriction enzyme EcoRI (RE1), followed by ligation. The DNA template is slowly added to a container containing T4 DNA ligase, ATP, universal buffer, and water using the SCAM method described herein. Slowly adding the DNA over 12 hours (substrate continuous addition method, i.e., SCAM) results in the production of the major monomeric circular construct (see Figure 5). The products of the first digestion and SCAM ligation are subjected to DNA agarose gel electrophoresis (lanes 2 and 3, respectively) (Figure 3E). Lanes 1 and 2 show linear and supercoiled DNA ladders, respectively, for size reference. Lane 2 shows the linear insert sequence and backbone products after digestion with EcoRI (RE1) (higher and lower bands, respectively); lane 3 shows the DNA species present after self-ligation ( Figure 3D the possibilities are shown in).

[0153] Figures 4A - 4C A DNA gel showing the inactivation of the endonuclease at a specific pH is shown. To determine the pH at which EcoRI is inactivated, multiple reaction solutions are prepared with all reagents except DNA (i.e., the reaction solution contains buffer [New England BioLabs, proprietary composition], EcoRI, and water). The pH of each solution is changed using 12% HCl to include pH values from 7.8, 7.0, 6.0, 5.0, 4.0, 3.0, or 2.0. After 5 minutes, the pH of all solutions is restored to 7.8 using 3M NaOH. After 5 minutes, a circular DNA plasmid template is added to each reaction solution and incubated at 37 °C for 2 hours. Figure 4A The reaction products after inactivating EcoRI using different pH values are shown. pH values from 7.8 - 5.0 do not inactivate EcoRI; under these pH conditions, EcoRI retains its endonuclease activity, resulting in the production of linearized DNA (lanes 2 - 5). Lanes 6 - 8 show that pH values from 4.0 - 2.0 result in significant inactivation of EcoRI, inhibiting its endonuclease activity and retaining the circular DNA template. Lane 1 shows a linear DNA ladder for size reference. Figure 4B Data from a similar experimental setup is shown, but the inactivation of the HindIII endonuclease is tested. Similar to Figure 4A the same, pH values ≤ 4.0 result in significant inactivation of the HindIII endonuclease. Lane 1 shows a DNA ladder for size reference. The sample in lane 8 undergoes a pH change (lowered to pH = 4.0 with HCl; restored to pH = 7.8 with NaOH), but does not contain the HindIII endonuclease. Figure 4CShows the experimental results of contacting supercoiled DNA (SC, lane 2) with HindIII inactivated by three different methods: heat inactivation at 80 °C for 20 minutes (HI, lane 3), pH inactivation (pH = 4.0, 5 minutes; pH, lane 4), or combined heat and pH inactivation (HIpH, lane 5). The solution was then added to a ligation reaction containing T4 DNA ligase, incubated overnight at room temperature, and the products were then subjected to gel electrophoresis. Compared to the SC control (lane 2), the levels of linearized plasmid were similar in the samples with inactivated endonuclease (lanes 3-5), demonstrating that pH inactivation does not significantly affect the ligation reaction efficiency. Lane 1 shows a linear DNA ladder used for size reference.

[0154] Figures 5A - 5B Shows the results of ligation reactions using the stepwise method (SBS) or the substrate continuous addition method (SCAM). To first test the efficiency of SBS for conventional ligation reactions, each reaction was carried out in parallel with the same linear DNA substrate ( Figure 5A ). For the conventional ligation reaction, all ligation reagents (T4 DNA ligase, T4 DNA ligase buffer [New England BioLabs, proprietary composition], and DNA) were added to the reaction simultaneously and allowed to incubate at room temperature for 2 hours (CTRL). In the SBS samples, all ligation reagents and 20% of the final DNA volume were added at the start of the reaction. Starting 20 minutes later, 20% of the final DNA volume was added at 5 time points at 20-minute intervals, and after adding the last 20% of the DNA, it was incubated for 20 minutes, with a total reaction time of 2 hours. The samples were loaded onto a DNA gel in triplicate with increasing amounts of DNA, and a linear and a supercoiled DNA ladder were loaded simultaneously as size references (lanes 1 and 2, respectively). Comparison between the circular monomer bands in the SBS and conventional ligation lanes revealed a slight increase in circular monomers (50%-70%) when using the SBS method (see lanes 4, 6, and 8 compared to lanes 3, 5, and 7). A second experiment was conducted to test the ligation efficiency using SCAM compared to the conventional ligation reaction ( Figure 5B ). Similar to Figure 5A , a conventional ligation reaction was carried out in which all ligation reagents were added to the reaction simultaneously and allowed to incubate at 25 °C for 20 hours (CTRL, lane 3). In the SCAM samples, all ligation reagents were first mixed to produce an initial volume of 10 mL. Then, at 25 °C, 10 mL of the DNA substrate was continuously added at a rate of 0.6 mL / hour over 16 hours, with a final volume of 20 mL. After adding the last batch of substrate, the reaction continued for 4 hours. The samples were loaded onto a gel, and a linear and a supercoiled DNA ladder were loaded for size reference ( Figure 5B)。Comparison between the circular monomer bands in SCAM and traditional ligation lanes revealed a significant increase (158%) in the production of circular monomers when using SCAM, demonstrating that SCAM ligation is significantly more efficient in generating circular monomer products from dsDNA templates compared to SBS and traditional ligation methods (see comparison of lane 4 with lane 3). Note that Figure 5A and Figure 5B the DNA constructs used in the experiments described in Figure 5A have a large enough difference in the sizes of the insert and backbone sequences to be separated on a gel; Figure 5B the insert and backbone sequences in

[0155] are of similar size and thus appear as a single band on the gel. The ligation products from Fig. 3E contain the desired amplified circular monomer insert sequence (ligation product 1), and also contain sequences containing the vector backbone ( Figure 6A , ligation products 2, 3, and 5). Figure 6B shows a schematic illustration of linearizing these unwanted backbone-containing products. By contacting the ligation products with an endonuclease that cuts at RE2, any circular products containing the vector backbone are cleaved, specifically linearizing the unwanted NA sequences ( Figure 6B , second digestion products 2, 3, and 5). Figure 6A and 6B The numbers in Figure 6C show the products generated from each corresponding digested circular plasmid.

[0156] Figure 7A depicts a schematic illustration of the process of supercoiling circular NA. A topoisomerase used to change the topological state of NA can contact relaxed NA (as shown for the plasmid in Figure 7A ) to facilitate supercoiling, generating a more compact DNA molecule (supercoiled product). The second digestion products from Figure 6C are contacted with Escherichia coli DNA gyrase for 2 hours or overnight. Figure 7B shows a DNA gel that was run to compare the sizes of the second digestion products before (lane 2) and after (lane 3) supercoiling for 2 hours or after supercoiling overnight (lane 4). After supercoiling, the second digestion products migrate through the gel at the same rate as the linearized backbone (XhoI digestion), with the disappearance of the ladder-like appearance in lane 2. Lane 1 shows a DNA ladder for size reference.

[0157] Figure 8A The process of removing unwanted linear DNA from the second digestion product is described. Although the supercoiled product contains the desired circular insert sequence ( Figure 8A , products 1 and 4), it also contains the products with linear backbone remaining from the second digestion ( Figure 8A , products 2, 3 and 5). To remove these unwanted products, the Figure 7B supercoiled product is contacted with an exonuclease, which digests linear and open circular NA. Figure 8B DNA gel electrophoresis is shown to compare the sizes of the supercoiled product before (lane 2) and after (lane 3) digestion with T5 exonuclease. The linear monomers, polymers and materials captured in the well of lane 2 are not present in lane 3, which mainly contains supercoiled circular inserts. Lane 1 shows the DNA ladder for size reference.

[0158] from Figure 8B the exonuclease product is further purified by anion exchange chromatography followed by isopropanol precipitation. Figure 9 A DNA gel is shown that was run to demonstrate the purity of the supercoiled circular insert sequence obtained after performing Figure 1 steps 1 - 7 (see lane 3). Linear and supercoiled DNA ladders are shown in lanes 1 and 2 respectively.

[0159] Equivalents

[0160] Although multiple inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily conceive of various other devices and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each such variation and / or modification is considered to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend on the specific application for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to determine using only routine experimentation, many equivalents to the specific inventive embodiments described herein. Accordingly, it should be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, the embodiments of the present invention may be practiced in a manner different from that specifically described and claimed. The inventive embodiments of the present disclosure are directed to each and every separate feature, system, article, material, kit, and / or method described herein. Moreover, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0161] All references, patents, and patent applications disclosed herein are incorporated herein by reference, and in some cases, they may include the entire document.

[0162] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method need not be limited to the order in which the steps or acts of the method are recited.

[0163] In the claims as well as the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," etc., are to be understood as open-ended, i.e., meaning including but not limited to. As stated in Section 2111.03 of the United States Patent and Trademark Office's Manual of Patent Examining Procedure, only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively. It should be understood that embodiments described herein using open-ended transitional phrases (e.g., "comprising") should also be understood in alternative embodiments as being "consisting of" and "consisting essentially of" the features described by the open-ended transitional phrase. For example, if the present disclosure describes "a composition comprising A and B," the present disclosure also contemplates alternative embodiments of "a composition consisting of A and B" and "a composition consisting essentially of A and B."

Claims

1. A method for amplifying and synthesizing circular nucleic acids, which comprises: a) providing a circular nucleic acid template, which comprises (i) a backbone, wherein the backbone comprises one or more internal endonuclease cleavage sites, and (ii) an insert sequence, wherein the insert sequence comprises endonuclease cleavage sites at its 5' and 3' termini; b) amplifying the circular nucleic acid template by rolling circle amplification to produce an amplification product; c) contacting the amplification product with a first endonuclease under digestion conditions to produce a first digestion product; d) adding the first digestion product to a ligation reaction mixture comprising a ligase, wherein the first digestion product is added to the ligation reaction mixture at a rate of about 1% to about 20% of the final ligation reaction volume per hour over a period of 5 - 14 hours; and e) incubating the ligation reaction mixture to produce a circular ligation product, wherein the circular ligation product is at least partially supercoiled.

2. The method according to claim 1, wherein the first digestion product is added to the ligation reaction mixture at a rate of about 3% to about 5% of the final ligation reaction volume per hour over a period of 10 - 12 hours.

3. The method according to claim 1 or 2, wherein the circular ligation product is contacted with a second endonuclease under digestion conditions to produce a second digestion product, wherein the first endonuclease and the second endonuclease do not have the same recognition site.

4. The method according to any one of claims 1 - 3, which further comprises contacting the circular ligation product or the second digestion product with a topoisomerase under conditions that promote supercoiling to produce a supercoiled product.

5. The method according to any one of claims 1 - 4, which further comprises contacting the ligation product, the second digestion product, or the supercoiled product with an exonuclease that digests single - stranded nucleic acids and open - circular nucleic acids to produce a final reaction product comprising synthetic circular nucleic acids substantially free of linear nucleic acids.

6. The method according to any one of claims 1 - 5, which further comprises purifying the circular ligation product, the second digestion product, the supercoiled product, or the final reaction product.

7. The method according to claim 6, wherein the final reaction product is purified by chromatography, such as ion - exchange chromatography, affinity chromatography, reverse - phase chromatography, or size - exclusion chromatography; isopropanol precipitation, methanol precipitation, ethanol precipitation, solid - phase purification, electrophoresis, or a combination thereof.

8. The method according to claim 7, wherein the ion - exchange chromatography is anion - exchange chromatography.

9. The method according to any one of claims 1 - 8, which further comprises inactivating the first and / or second endonuclease by lowering the pH of the solution to a pH of 3 - 5 after step b) of claim 1 and / or claim 4, respectively.

10. The method according to any one of claims 1 - 9, wherein the nucleic acid template comprises: a. a nucleic acid fragment to be amplified; b. two recognition sites for a first endonuclease, which are located at the 5' and 3' termini of the nucleic acid fragment to be amplified, respectively, and c. a recognition site for a second endonuclease in the vector backbone.

11. The method according to claim 10, wherein the nucleic acid template further comprises a vector backbone.

12. The method according to any one of claims 1-11, wherein the rolling circle amplification is carried out using Phi29 DNA polymerase, bacterial DNA polymerase III, bacterial DNA polymerase I, modified DNA polymerase I, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, VENT DNA polymerase, DEEP VENT DNA polymerase, KlenTaq DNA polymerase, the Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, or reverse transcriptase.

13. The method according to any one of claims 1-12, wherein the rolling circle amplification is carried out using Phi29 DNA polymerase.

14. The method according to any one of claims 1-13, wherein the ligase is T4 DNA ligase, T4 RNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, or Escherichia coli DNA ligase.

15. The method according to claim 14, wherein the ligase is T4 DNA ligase.

16. The method according to any one of claims 4-15, wherein the topoisomerase is DNA gyrase.

17. The method according to claim 16, wherein the DNA gyrase is Escherichia coli DNA gyrase, Staphylococcus aureus DNA gyrase, DNA topoisomerase 2-α, or DNA topoisomerase 2-β.

18. The method according to claim 17, wherein the DNA gyrase is Escherichia coli DNA gyrase.

19. The method according to any one of claims 5-18, wherein the exonuclease is T5 exonuclease, exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VIII, exonuclease T, λ exonuclease, or T7 exonuclease.

20. The method according to claim 19, wherein the exonuclease is T5 exonuclease.

21. The method according to any one of claims 1-20, wherein the first endonuclease or the second endonuclease is a type I restriction endonuclease, a type II restriction endonuclease, a type IIs restriction endonuclease, a type III restriction endonuclease, endonuclease III, endonuclease IV, endonuclease V, endonuclease VIII, T7 endonuclease I, T4 endonuclease V, T4 endonuclease VII, deoxyribonuclease I, deoxyribonuclease II, deoxyribonuclease III, deoxyribonuclease IV, an RNA endonuclease, including an engineered RNA endonuclease with customized sequence specificity, or an RNA-guided endonuclease, such as a CRISPR / Cas endonuclease.

22. A synthetic circular nucleic acid produced by the method according to any one of claims 1-21.

23. A composition comprising the synthetic circular nucleic acid according to claim 22 and a pharmaceutically acceptable carrier.

24. A cell comprising the synthetic circular nucleic acid according to claim 22 or the composition according to claim 23.

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