Cell-free method for producing synthetic circular nucleic acids

Abstract

The present disclosure relates to a method for cell-free circular nucleic acid amplification using rolling circle amplification.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 398,354, filed August 16, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to a cell-free circular nucleic acid amplification method. [Background technology]

[0003] Since their development, synthetic nucleic acid (NA) molecules have become increasingly useful in biomedical research, medicine, and therapy. The use of these molecules has been applied in many fields, enabling, for example, the ability to combine unique sequences from different organisms to generate constructs that offer specific advantages and utility. For example, certain sequences found in bacteria or viruses can be useful for enhancing the expression of mammalian genes. By generating synthetic NA molecules containing such bacterial or viral sequences and mammalian genes on the same construct, gene expression levels can be enhanced to levels otherwise not normally achievable in mammalian cells, even to therapeutic doses. Furthermore, the ability to synthesize NA molecules allows for the generation of molecules with unique sequences, such as specific genetic variants, polymorphisms, or mutations, with great precision.

[0004] Current methods for producing useful amounts of synthetic NA molecules, for example for laboratory use or as therapeutic agents, require the use of non-mammalian cell lines. However, while these methods allow for the production of sufficient amounts of synthetic NA molecules, the resulting NA molecules often contain impurities resulting from the amplified cell lines. In addition, fidelity and expression are difficult to control, and these methods require expensive and specialized equipment. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure provides a cost-effective method for highly amplifying NAs in a synthetic cell-free environment, allowing for strict control of the amplification and processing reactions and eliminating concerns associated with cell-mediated NA amplification. [Means for solving the problem]

[0006] In some aspects, provided herein are methods for amplifying synthetic circular nucleic acids. The method includes providing a circular nucleic acid template comprising (i) a backbone comprising one or more internal endonuclease cleavage sites and (ii) an insert sequence comprising endonuclease cleavage sites on the 5'-end and the 3'-end, or providing a circular nucleic acid template comprising (i) an insert sequence flanked by first endonuclease cleavage sites and (ii) a backbone comprising one or more second internal endonuclease cleavage sites. The method further includes amplifying the circular nucleic acid template to produce an amplification product, contacting the amplification product with a first endonuclease under digestion conditions to thereby produce a first digestion product, adding the first digestion product to a ligation reaction mixture comprising a ligase enzyme, and incubating the ligation reaction mixture to thereby produce a circular ligation product, wherein the circular ligation product is at least partially supercoiled.

[0007] In some embodiments, the backbone comprises more than one internal endonuclease cleavage site.

[0008] In some embodiments, the backbone comprises two or more different internal endonuclease cleavage sites.

[0009] In some embodiments, the backbone comprises between 1 and 5 internal endonuclease cleavage sites.

[0010] In some embodiments, the endonuclease cleavage sites flanking the insert sequence are the same endonuclease cleavage site.

[0011] In some embodiments, the endonuclease cleavage sites flanking the insert sequence are different from each other.

[0012] 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 for 5 to 14 hours.

[0013] 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 for 5 to 14 hours.

[0014] 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 for 10 to 12 hours.

[0015] 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 for 10 to 12 hours.

[0016] In some embodiments, the circular ligation product is contacted with a second endonuclease under conditions for digestion, thereby producing a second digestion product, wherein the first and second endonucleases do not have the same recognition site.

[0017] 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, thereby producing a supercoiled product.

[0018] In some embodiments, the method further comprises contacting the ligation product, second digestion product, or supercoiled product with an exonuclease that digests single-stranded nucleic acids and open-circular nucleic acids, thereby producing a final reaction product comprising synthetic circular nucleic acids that are substantially free of linear nucleic acids.

[0019] 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, for example, 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.

[0020] In some embodiments, the method further comprises, after digestion with the first endonuclease and / or digestion with the second endonuclease, inactivating the first and / or second endonuclease by lowering the pH of the solution to a pH between 3 and 5.

[0021] In some embodiments, the nucleic acid template comprises a nucleic acid fragment to be amplified, two recognition sites for a first endonuclease at each of the 5' and 3' ends of the nucleic acid fragment to be amplified, and a recognition site for a second endonuclease in a vector backbone, hi some embodiments, the nucleic acid template further comprises a vector backbone.

[0022] In some embodiments, rolling circle amplification is performed using Phi29 DNA polymerase. In some embodiments, rolling circle amplification is performed using bacterial DNA polymerase III. In some embodiments, rolling circle amplification is performed using bacterial DNA polymerase I. In some embodiments, rolling circle amplification is performed using modified DNA polymerase I. In some embodiments, rolling circle amplification is performed using M2 DNA polymerase. In some embodiments, rolling circle amplification is performed using B103 DNA polymerase. In some embodiments, rolling circle amplification is performed using GA-1 DNA polymerase. In some embodiments, rolling circle amplification is performed using phi-PRD1 polymerase. In some embodiments, rolling circle amplification is performed using VENT DNA polymerase. In some embodiments, rolling circle amplification is performed using DEEP VENT DNA polymerase. In some embodiments, rolling circle amplification is performed using KlenTaq DNA polymerase. In some embodiments, rolling circle amplification is performed using the Klenow fragment of DNA polymerase I. In some embodiments, rolling circle amplification is performed using DNA polymerase III. In some embodiments, rolling circle amplification is performed using T3 DNA polymerase. In some embodiments, rolling circle amplification is performed using T4 DNA polymerase. In some embodiments, rolling circle amplification is performed using T5 DNA polymerase. In some embodiments, rolling circle amplification is performed using T7 DNA polymerase. In some embodiments, rolling circle amplification is performed using Bst polymerase. In some embodiments, rolling circle amplification is performed using rBST DNA polymerase. In some embodiments, rolling circle amplification is performed using N29 DNA polymerase. In some embodiments, rolling circle amplification is performed using TopoTaq DNA polymerase.In some embodiments, rolling circle amplification is performed using T7 RNA polymerase. In some embodiments, rolling circle amplification is performed using SP6 RNA polymerase. In some embodiments, rolling circle amplification is performed using T3 RNA polymerase. In some embodiments, rolling circle amplification is performed using reverse transcriptase.

[0023] 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.

[0024] In some embodiments, the topoisomerase is DNA gyrase. In some embodiments, the DNA gyrase is E. 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-alpha. In some embodiments, the DNA gyrase is DNA topoisomerase 2-beta.

[0025] 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 lambda exonuclease. In some embodiments, the exonuclease is T7 exonuclease.

[0026] 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 DNase I. In some embodiments, the first endonuclease or the second endonuclease is DNase II. In some embodiments, the first endonuclease or the second endonuclease is DNase III. In some embodiments, the first endonuclease or the second endonuclease is DNase 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-directed 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.

[0027] Another aspect provides synthetic circular nucleic acids produced by the methods provided herein.

[0028] In some embodiments, provided herein are cells comprising the 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.

[0029] In some embodiments, provided herein is a delivery agent comprising a synthetic circular nucleic acid 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 microparticle, a polymer nanoparticle or microparticle, a protein nanoparticle or microparticle, or a solid nanoparticle or microparticle.

[0030] In some embodiments, provided herein are compositions comprising a synthetic circular nucleic acid produced by the methods provided herein, wherein the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition further comprises an additional agent. In some embodiments, the additional agent has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is a nucleotide, nucleic acid, amino acid, peptide, protein, small molecule, aptamer, lipid, or carbohydrate. In some embodiments, the composition is for use in the prevention or treatment of a disease or disorder in a subject in need thereof.

[0031] In some embodiments, provided herein are methods that include introducing into a cell a synthetic circular nucleic acid produced by the methods provided herein. In some embodiments, the method includes introducing the synthetic circular nucleic acid into a subject. In some embodiments, the subject is a human. In some embodiments, the method includes preventing or treating a disease in a subject in need thereof. These and other aspects of the disclosure are further described below.

[0032] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference 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 in no way limit the scope of the present disclosure. [Brief explanation of the drawings]

[0033] [Figure 1] 1 shows a step-by-step overview of the NA amplification method described herein, 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 by NA supercoiling, exonuclease digestion, and purification by methods such as column chromatography or isopropanol precipitation. [Figure 2] Figure 2A demonstrates the rolling circle amplification (RCA) step of the methods provided herein. Figure 2A shows an outline of the RCA method, the first step of the disclosed method. RCA is performed to amplify a circular DNA template containing both the desired expression cassette insert and the vector backbone, resulting in long linear concatemers with multiple copies of the insert. Figure 2B shows the results of RCA performed on the plasmid for 90 minutes (lane 2) and 16 hours (lane 3). Lane 1 shows a DNA ladder for size reference. The numbers on the left of the gel indicate the length of each corresponding ladder band in base pairs (bp). [Figure 3]This figure shows the process for the first endonuclease digestion (FIG. 3C) and ligation (FIG. 3D) of the RCA amplification product (FIG. 3B) generated from the nucleic acid template (FIG. 3A). Through these steps, in some embodiments, a circular NA molecule containing only the expression cassette insert, the desired final product, can be generated. 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. [Figure 4-1] This figure shows the effect of varying the pH of a common reaction buffer on the activity of endonuclease enzymes. 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 on the left of the gel indicate the length of each corresponding ladder band in base pairs (bp). In Figure 4B, lane 1 shows a linear DNA ladder, and lanes 2-7 show pH values ​​of 7.8, 7.0, 6.0, 5.0, 4.0, and 3.0, respectively. Lane 8 is a control in which no HindIII enzyme was added to the reaction. The numbers on the left of the gel indicate the length of each corresponding ladder band in base pairs (bp). Figure 4C shows the results of an experiment in which supercoiled DNA (SC) was inactivated by three different methods: heat inactivation (HI), pH inactivation (pH), or a combination of heat and pH (HIpH) before contacting it with HindIII. This solution was then poured into a ligation reaction containing T4 DNA ligase, and after overnight incubation at room temperature, the product was electrophoresed on a gel. Lane 1 shows the DNA ladder, lane 2 shows the control untreated SC DNA, and lanes 3-5 show the HI, pH, and HIpH samples, respectively. Numbers on the left of the gel indicate the length of each corresponding ladder band in kilobases (kb). Numbers corresponding to the experimental bands indicate the intensity reading of each band. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5]This figure demonstrates the beneficial effect of utilizing sequential substrate addition (SCAM) to generate greater amounts of circular monomer during a ligation reaction. In Figure 5A, lanes 1 and 2 show linear and supercoiled DNA ladders, respectively. The numbers on the left and right of the gel indicate the lengths of the corresponding ladder bands in base pairs (bp) for the linear ladder (left) and the supercoiled ladder (right). Lanes 3, 5, and 7 show products from a conventional ligation reaction loaded with increasing amounts (200 ng, 400 ng, and 800 ng, respectively). Lanes 4, 6, and 8 show products from an SBS ligation reaction loaded with 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 on the left and right of the gel indicate the lengths of the corresponding ladder bands in kilobases (kb) for the linear ladder (left) and the supercoiled ladder (right). Lane 3 shows the ligation product produced by conventional ligation reaction (CTRL). Lane 4 shows the ligation product produced by SCAM ligation (SCAM). Numbers corresponding to experimental bands indicate the intensity reading of each band. [Figure 6-1] Figures 6A and 6B demonstrate the ligation step of the methods provided herein. Figure 6A shows five non-limiting different possible ligation products that can be produced by the methods provided herein. Figure 6B shows the products of a second endonuclease digestion performed to remove the ligation product containing the vector backbone. Figure 6C shows a DNA gel run to compare the sizes of the ligation products before (lane 2; ligation) and after (lane 3; XhoI digestion) the second digestion. Lane 1 shows a linear DNA ladder for size reference. The numbers to the left of the gel indicate the length of each corresponding ladder band in base pairs (bp). [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7]Figure 7A demonstrates the supercoiling step of the methods provided herein. Figure 7A demonstrates how topoisomerase enzymes act to supercoil NAs, resulting in small NA molecules that migrate quickly through agarose gels due to increased density after supercoiling. In Figure 7B, lane 2 shows the vector digested with XhoI (XhoI digestion), lane 3 shows the results of supercoiling the digested vector with DNA gyrase for 2 hours (2 hr), and lane 4 shows the results of supercoiling the digested vector with DNA gyrase overnight (O / N). Lane 1 shows a linear DNA ladder. The numbers on the left side of the gel indicate the length of each corresponding ladder band in base pairs (bp). [Figure 8] Figures 8A and 8B demonstrate the exonuclease step of the methods provided herein. Figure 8A demonstrates the results of contacting the products from the second endonuclease digestion with an exonuclease enzyme, which acts to remove linear and open-circular NAs. This step allows for further purification of the amplified NA product. In Figure 8B, lane 1 shows the linear DNA ladder, lane 2 shows the results of supercoiling the ligation reaction product with DNA gyrase, and lane 3 shows the results of contacting the supercoiled product with exonuclease to remove unwanted NAs (T5). The numbers on the left side of the gel indicate the corresponding ladder bands (1-14) and correspond to the lengths of the DNA ladder bands shown in lane 1 of Figure 7B. [Figure 9]

[0023] Figure 1 demonstrates the purity of the final NA product produced using the methods 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 on the left of the gel indicate the length of each corresponding ladder band in base pairs (bp). DETAILED DESCRIPTION OF THE INVENTION

[0034] Traditional cell-based methods for amplifying nucleic acids (NAs) in large quantities are costly and often error-prone. For example, the use of bacteria to amplify NAs requires growing large quantities of bacteria in expensive fermenters and maintaining sterile conditions to prevent contamination of the bacterial culture. Furthermore, bacteria must be lysed to release the amplified NAs, and the NAs must be washed and purified from other bacterial components, such as endotoxins, that are toxic to mammals. Typical NA purification procedures from bacteria and other cell sources include the use of organic compounds, mutagens, and toxic compounds, including phenol, ethidium bromide, and cesium chloride, as well as enzymes, such as lysozyme, proteinase K, and RNase A. All of these compounds pose potential health hazards if injected as contaminants into NA vaccines or other therapeutic NA agents.

[0035] In addition to cost issues, the use of bacteria can often pose challenges to the fidelity of amplification methods. The complex biochemical environment of bacterial cells makes it 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 may also cause problems with the production of the desired NA molecule.

[0036] In contrast, cell-free (synthetic) NA amplification can result in significant cost savings due to streamlined production and simplified purification. Additionally, it eliminates impurities typically associated with conventional methods such as those described herein. In particular, cell-free enzymatic methods for NA amplification do not require the use of host cells, effectively eliminating the problems associated with cell-based NA amplification. Provided herein are methods for amplifying circular NA molecules in a synthetic manner, enabling the production of highly pure amplification products in a cost-effective and time-efficient manner.

[0037] Provided herein are methods for amplifying synthetic circular nucleic acid (NA) molecules (e.g., circular NA vectors described herein). In particular, the methods provided herein include in vitro synthesis of NA (e.g., in the absence of cells (i.e., cell-free)), which provides a purer composition of the resulting NA molecule compared to NA derived from bacteria or yeast, allowing for faster and more efficient NA synthesis.

[0038] In some embodiments, amplification of NA vectors using the methods disclosed herein begins with preparing a sample containing circular NAs. In some embodiments, circular NAs are produced by self-ligation of linear NAs. This can be done by any method known in the art, for example, by contacting linear NAs with a ligase enzyme under conditions suitable for self-ligation. In some embodiments, circular NAs are DNA. In some embodiments, circular NAs are RNA. In some embodiments, circular NAs include 1) an expression cassette with a heterologous gene (i.e., insert), and 2) a backbone (i.e., scaffold) containing a nucleotide sequence that provides isolation, expression, and / or amplification properties. In some embodiments, circular NAs are 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 NAs are specifically degraded, for example, using an exonuclease, to purify circular NAs.

[0039] 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 (5') of one or more heterologous genes. Additionally or alternatively, the circular NA vector can comprise 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 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).

[0040] In some embodiments, the poly-A tail comprises between 50 and 100, between 100 and 150, between 150 and 200, between 200 and 300, between 300 and 400, or between 400 and 500 nucleotides. In some embodiments, between 25 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100%, 70 and 100%, 80 and 100%, 90 and 100%, 95 and 100%, 96 and 100%, 97 and 100%, 98 and 100%, or 99 and 100% of the nucleotides in the poly-A tail are adenosine nucleotides.

[0041] 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 of the same gene. As used herein, "the same gene" refers to NA sequences encoding proteins with 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 with 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., via dimerization, e.g., the heavy and light chains of an antibody or fragment thereof), or do not interact. In some embodiments, the heterologous gene comprises one or more trans-splicing molecules or portions thereof (e.g., binding domains).

[0042] In some embodiments, the NA vector containing the expression cassette is amplified in vitro by rolling circle amplification (RCA) by incubating the NA in a cell-free preparation 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., dNTPs or NTPs, 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 contains one or more nucleotide analogs, e.g., unnatural nucleotides.

[0043] In some embodiments, the nucleotide analogue contains a modified phosphate, which results in a modified internucleotide linkage when incorporated into the nascent NA. The modified phosphate used in the present invention can be, but is not limited to, phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate and guanidinopropyl phosphoramidate. In some embodiments, more than one modified phosphate is used.

[0044] In some embodiments, the nucleotide analog comprises a modified sugar. Modified sugars for use in the present invention include 2'-deoxyfluoro (2FA), L-adenosine (LA), 2'-deoxyadenosine (dA), locked nucleic acid (LNA), 2'-methoxy (2OMe), 2'-methoxyethoxy (2MOE), 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'-deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, The modified sugar may be, but is not limited to, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 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.

[0045] In some embodiments, the nucleotide analog comprises a modified nucleobase. Modified nucleobases for use in the present invention include inosine, xanthine, aliaminouracil, aliaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenosine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl) propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, ara adenine, ara cytosine , araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil,Cyanine 7-aminoallyluracil, dabsyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OB EA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-carboxamido-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-Methyl-thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio- The modified nucleobase may be, but is not limited to, N6-threonylcarbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, more than one modified nucleobase is used.

[0046] The polymerase amplifies the NA molecule by rolling circle amplification, thereby generating linear concatemers containing multiple copies of the NA molecule. Suitable polymerases include thermophilic polymerases, polymerases characterized by high processivity due to GC-rich sequences, and polymerases with NA displacement ability.

[0047] The resulting concatemers are digested using an endonuclease (e.g., a restriction endonuclease) to cleave the concatemerized NA molecules and generate linear NA molecules of unit length containing the heterologous gene, if present in the NA molecule subjected to the amplification method. Self-ligation of the linear NA molecules (e.g., by adding a ligase enzyme) optionally results in a circular synthetic NA molecule containing the complete heterologous gene. Because the NA replication and amplification described herein can be performed using a polymerase under cell-free conditions, the synthetic NA molecule can be isolated from the bacterial components of the plasmid into which it was cloned, thus removing bacterial signatures, such as bacterial CpG motifs and / or dam or dcm methylation, from the isolated vector.

[0048] In one aspect, the present invention provides a cell-free method for amplifying an isolated NA vector by (i) preparing a sample containing a circular NA vector containing an expression cassette, wherein the expression cassette contains (a) a heterologous gene containing a first endonuclease cleavage site at the 5'-end and 3'-end 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 thereby generate linear concatemers; (iii) using a first endonuclease to digest the linear concatemers to separate the expression cassette from the backbone; and (iv) incubating the expression cassette under conditions that allow self-ligation, thereby producing a circular ligation product containing the expression cassette. In some embodiments, the method further comprises using a second endonuclease selected to cleave the circular NA product containing the vector backbone to digest the ligation product, thereby producing a second digestion product. In some embodiments, the method further comprises contacting the ligation product or the second digestion product with a topoisomerase enzyme to supercoil the remaining circular NA product, i.e., the circular NA product lacking the vector backbone. In some embodiments, the supercoiled NA can be a polymeric supercoiled NA. In some embodiments, the supercoiled NA can be a 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, the open relaxed circular NA is separated from the supercoiled NA in the purification step.

[0049] Embodiments of the methods disclosed herein combine several technologies for the purpose of affordable production of large quantities of NAs for therapeutic, diagnostic, and research applications. Due to the cell-free nature of the methods disclosed herein, there are no sources of endotoxin contamination other than those minimally contained in the reagents used. Additional advantages include the ability to produce large quantities of product, such as fermentation, in small laboratory flasks; the need for only a minimal number of reagents; the ability to produce large quantities of product in a relatively short period of time; and streamlined purification procedures.

[0050] synthetic nucleic acid molecule In one aspect, the methods disclosed herein provide an isolated circular NA (e.g., a circular NA vector), 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) a recombination site. For example, in some embodiments, the circular NA lacks an origin of replication, a drug resistance gene, and a recombination site. In some embodiments, the circular NA comprises one or more heterologous genes. In some embodiments, the one or more heterologous genes comprise an open reading frame (ORF). In some embodiments, the one or more heterologous genes encode proteins. In some embodiments, the protein is a monomer (e.g., a monomeric protein having a secondary structure, a tertiary structure, and / or a 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 may be a multicistronic or multi-transcription unit sequence.

[0051] In some embodiments, the heterologous gene comprises a spacer. As used herein, a spacer refers to a nucleotide sequence placed between coding sequences in a polycistronic gene locus or polycistronic mRNA to facilitate translation or processing of one or more coding sequences into one or more separate proteins. Non-limiting examples of a spacer are nucleotide sequences encoding an internal ribosome entry site (IRES), a self-cleaving peptide coding sequence, and an endogenous protease cleavage site. In some embodiments, the spacer is an IRES. As used herein, an IRES refers to a DNA sequence that, when transcribed into RNA, allows translation initiation from an internal region of an RNA (e.g., an mRNA). Translation in eukaryotes typically begins at the 5' cap of an mRNA, so only one translation event occurs for each mRNA. However, an IRES can initiate translation independently of the 5' cap and act as an additional ribosome recruitment site, thereby resulting in the co-expression of one or more proteins from a single mRNA.

[0052] 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 in some members of the picornavirus family. The 2A self-cleaving peptide can undergo self-cleavage to generate a mature protein through a translational effect known as "stop-go" or "stop-carry" (Wang et al. (2015), Nature Scientific Reports 5:16237). The term "self-cleaving" is a misnomer, as these peptides are thought to function by causing the ribosome to skip peptide bond synthesis at the C-terminus of the 2A element, resulting in a separation between the end of the 2A sequence and the next downstream peptide. The "cleavage" occurs between the glycine and proline residues found at the C-terminus, meaning that the upstream cistron has a few additional residues at its end, while the downstream cistron begins with a proline.

[0053] In some embodiments, the spacer encodes a cleavage site for a protease that is endogenous to the host cell. Non-limiting examples of proteases are trypsin, elastase, matrix metalloproteinases (MMPs), and pepsin.

[0054] In some embodiments, the protein is a therapeutic protein. As used herein, a "therapeutic protein" refers to a protein that, when expressed in a subject, e.g., a human subject, having or at risk of developing a disease or disorder, prevents, reduces, or alleviates one or more signs or symptoms of the disease. A therapeutic protein can be, for example, an enzyme, a clotting 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 coagulation factor, an enzyme-activating 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 gene that is mutated in a subject. As a non-limiting example, a mutation in a gene encoding such a protein can cause a decrease in the level of the protein expressed in one or more cells of the subject. Thus, expression of a therapeutic protein can compensate for a mutation in a gene encoding such a protein in a subject. In some embodiments, the enzyme is an epigenetic regulator. In some embodiments, the epigenetic regulator is a histone methyltransferase, histone demethylase, histone acetylase, DNA methyltransferase, or 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. Antibodies include fragments capable of binding to the antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', dis-scFv, single-domain antibodies (sdAb), (Fab')2 (including chemically linked (Fab')2), NANOBODIES®, chimeric antibodies, and humanized antibodies.

[0055] In another aspect, the methods disclosed herein provide an NA vector comprising one or more non-coding heterologous genes. In some embodiments, the non-coding heterologous gene is a therapeutic NA. As used herein, a therapeutic nucleic acid (NA) is a nucleic acid or related compound that alters 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, an siRNA, an shRNA, an miRNA, a gRNA, or a CRISPRi molecule.

[0056] In some embodiments, a heterologous gene comprises a reporter sequence in addition to a sequence encoding a protein or therapeutic nucleic acid. In some embodiments, reporter genes are used, for example, to confirm the expression of heterologous genes in specific cells and tissues. Reporter sequences that can be provided in heterologous genes 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 others known in the art. When associated with regulatory elements that drive their expression, reporter sequences provide a signal that is detectable by conventional means, including enzymatic assays, radioactive assays, colorimetric assays, fluorometric or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry. For example, if the reporter sequence is a LacZ gene, the presence of LacZ expression product in the presence of appropriate reagents generates a detectable signal. If the reporter sequence expresses green fluorescent protein or luciferase, the presence of the green fluorescent protein or luciferase (with appropriate reagents) will produce a detectable signal that can be measured visually by the production of color or light in a luminometer.

[0057] In another aspect, provided herein is an NA vector comprising a promoter sequence upstream (5') of one or more heterologous genes. In some embodiments, the NA vector comprises a polyadenylation site downstream of one or more heterologous genes. In some embodiments of any of the above aspects, the one or more heterologous genes comprise a trans-splicing molecule or a portion thereof (e.g., a binding domain). In another aspect, provided herein is an isolated circular NA vector comprising one or more therapeutic NAs. Such isolated circular NA vectors lack an origin of replication and / or a drug resistance gene and lack a recombination site. In some embodiments, the NA vector comprises a terminal repeat sequence. In some embodiments, the terminal repeat sequence is at least 10 base pairs (bp) in length. In some embodiments, the NA vector lacks bacterial plasmid DNA. In some embodiments, the NA vector comprises one or more unmethylated GATC sequences, one or more unmethylated CCAGG sequences, and / or one or more CCTGG sequences. Additionally or alternatively, the NA vector (a) lacks an immunogenic bacterial signature; (b) lacks an RNA polymerase pause site; and / or (c) is substantially devoid of CpG islands.

[0058] In some embodiments, the NA vectors used herein (e.g., circular NA vectors) contain conventional control elements operably linked to a heterologous gene in a manner that allows transcription, translation, and / or expression in target cells. 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 native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized. A promoter region is operably linked to a heterologous gene if it can effect transcription of that gene and the resulting transcript can be translated into the desired protein. Promoters useful as part of the NA vectors described herein include constitutive promoters and inducible promoters.

[0059] In another aspect, provided herein is an isolated linear NA molecule, optionally produced by a cell-free method, comprising multiple identical concatemers, each of which 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) a replication origin (e.g., a bacterial replication origin) and / or a drug resistance gene; and (b) a recombination site. For example, in some embodiments, the isolated linear NA molecule lacks a replication origin, a drug resistance gene, and a recombination site. 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 insert.

[0060] In some embodiments, the circular NAs provided herein are greater than 250 base pairs. In some embodiments, the circular NAs range from about 250 base pairs to a maximum of about 250,000 base pairs (i.e., 250 bp to 250,000 bp). In some embodiments, the circular NAs are between 250 and 1000 bp. In some embodiments, the circular NAs are between 1000 and 10,000 bp. In some embodiments, the circular NAs are between 10,000 and 25,000 bp. In some embodiments, the circular NAs are between 25,000 bp and 50,000 bp. In some embodiments, the circular NAs are between 50,000 bp and 75,000 bp. In some embodiments, the circular NAs are between 75,000 bp and 100,000 bp. In some embodiments, the circular NAs are between 100,000 bp and 125,000 bp. In some embodiments, the circular NA is between 125,000 bp and 150,000 bp. In some embodiments, the circular NA is between 150,000 bp and 175,000 bp. In some embodiments, the circular NA is between 175,000 bp and 200,000 bp. In some embodiments, the circular NA is between 200,000 bp and 225,000 bp. In some embodiments, the circular NA is between 225,000 bp and 250,000 bp.

[0061] In some embodiments, the cyclic NAs are about 500 bp. In some embodiments, the cyclic NAs are about 1,000 bp. In some embodiments, the cyclic NAs are about 1,100 bp. In some embodiments, the cyclic NAs are about 1,200 bp. In some embodiments, the cyclic NAs are about 1,300 bp. In some embodiments, the cyclic NAs are about 1,400 bp. In some embodiments, the cyclic NAs are about 1,500 bp. In some embodiments, the cyclic NAs are about 1,600 bp. In some embodiments, the cyclic NAs are about 1,700 bp. In some embodiments, the cyclic NAs are about 1,800 bp. In some embodiments, the cyclic NAs are about 1,900 bp. In some embodiments, the cyclic NAs are about 2,000 bp. In some embodiments, the cyclic NAs are about 2,200 bp. In some embodiments, the cyclic NAs are about 2,400 bp. In some embodiments, the cyclic NAs are about 2,600 bp. In some embodiments, the cyclic NAs are about 2,800 bp. In some embodiments, the cyclic NAs are about 3,000 bp. In some embodiments, the cyclic NAs are about 3,300 bp. In some embodiments, the cyclic NAs are about 3,600 bp. In some embodiments, the cyclic NAs are about 3,900 bp. In some embodiments, the cyclic NAs are about 4,000 bp. In some embodiments, the cyclic NAs are about 4,400 bp. In some embodiments, the cyclic NAs are about 4,800 bp. In some embodiments, the cyclic NAs are about 5,000 bp. In some embodiments, the cyclic NAs are about 5,500 bp. In some embodiments, the cyclic NAs are about 10,000 bp. In some embodiments, the cyclic NAs are about 11,000 bp. In some embodiments, the cyclic NAs are about 12,000 bp. In some embodiments, the cyclic NAs are about 13,000 bp. In some embodiments, the cyclic NAs are about 14,000 bp. In some embodiments, the cyclic NAs are about 15,000 bp. In some embodiments, the cyclic NAs are about 16,000 bp. In some embodiments, the cyclic NAs are 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 cyclic NAs are about 22,000 bp. In some embodiments, the cyclic NAs are about 24,000 bp. In some embodiments, the cyclic NAs are about 25,000 bp. In some embodiments, the cyclic NAs are about 27,500 bp. In some embodiments, the cyclic NAs are about 30,000 bp. In some embodiments, the cyclic NAs are about 32,000 bp. In some embodiments, the cyclic NAs are about 34,000 bp. In some embodiments, the cyclic NAs are about 36,000 bp. In some embodiments, the cyclic NAs are about 38,000 bp. In some embodiments, the cyclic NAs are about 40,000 bp. In some embodiments, the cyclic NAs are about 44,000 bp. In some embodiments, the cyclic NAs are about 48,000 bp. In some embodiments, the cyclic NAs are about 50,000 bp. In some embodiments, the cyclic NAs are about 55,000 bp. In some embodiments, the cyclic NAs are about 60,000 bp. In some embodiments, the cyclic NAs are about 65,000 bp. In some embodiments, the circular NAs are about 70,000 bp. In some embodiments, the circular NAs are about 75,000 bp. In some embodiments, the circular NAs are about 80,000 bp. In some embodiments, the circular NAs are about 85,000 bp. In some embodiments, the circular NAs are about 90,000 bp. In some embodiments, the circular NAs are about 95,000 bp. In some embodiments, the circular NAs are about 100,000 bp. In some embodiments, the circular NAs are about 110,000 bp. In some embodiments, the circular NAs are about 120,000 bp. In some embodiments, the circular NAs are about 125,000 bp. In some embodiments, the circular NAs are about 130,000 bp. In some embodiments, the circular NAs are about 135,000 bp. In some embodiments, the circular NAs are about 140,000 bp. In some embodiments, the circular NAs are about 145,000 bp. In some embodiments, the circular NAs are 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 NAs are about 190,000 bp. In some embodiments, the circular NAs are about 200,000 bp. In some embodiments, the circular NAs are about 210,000 bp. In some embodiments, the circular NAs are about 220,000 bp. In some embodiments, the circular NAs are about 225,000 bp. In some embodiments, the circular NAs are about 230,000 bp. In some embodiments, the circular NAs are about 240,000 bp. In some embodiments, the circular NAs are about 250,000 bp.

[0062] Synthetic Nucleic Acid Amplification A typical protocol for amplifying synthetic fragments of NA involves amplifying the target DNA fragment using a method such as the polymerase chain reaction ("PCR"). In vitro amplification by PCR has been used successfully in laboratories since the mid-1980s, and while PCR is rapid and affordable, it relies on rapid thermal cycling, making it impractical for large-scale applications. Nevertheless, to continue the amplification, the PCR product can then be ligated into an appropriate vector, which is then transformed into host cells, such as bacteria or yeast cells, for in vivo amplification. After transformation, clones (individual colonies of cells derived from a single cell) expressing the vector containing the desired target DNA fragment are identified. Typically, such positive clones are identified by screening many colonies (at least tens, and sometimes hundreds) for the presence of the vector carrying the target DNA fragment. Screening is typically performed by restriction mapping, and DNA sequencing can be used to confirm the integrity of the vector and the presence of the complete target DNA fragment. This method is time-consuming and labor-intensive, requires the design and preparation of highly specific DNA primer sequences, and provides many opportunities for mutagenesis to occur.

[0063] Once a clone containing a vector carrying the target DNA fragment is selected, cells derived from this clone are grown. As the cells of the selected clone increase, the vector is amplified. The amplified vector containing the target DNA fragment is then recovered from the cells. When recovering the amplified DNA, there is often a problem associated with separating the amplified NA from cells and unwanted cellular components. Many methods have been devised over the years for such purification, but they remain time-consuming and ineffective (i.e., cellular contaminants often remain in the isolated NA).

[0064] Rolling Circle Amplification In nature, replication of circular DNA, including plasmids and some viral genomes, frequently occurs by rolling circle amplification ("RCA"), whereby circular DNA templates are replicated into long linear concatemers of tandem repeats (e.g., amplicons). This method can also be performed on circular RNA templates. Provided herein is an optimized in vitro RCA system for use in a cell-free system for large-scale NA production, using streamlined templates, highly specific or random primers, and polymerases.

[0065] The term RCA refers to the ability of an RCA-type polymerase (also referred to herein as RCA polymerase) to continuously proceed around a circular NA template (DNA or RNA) while extending a hybridized primer. This results in the formation of linear single-stranded products containing multiple repeats of the amplified NA. These linear single-stranded products serve as the basis for multiple hybridization, primer extension, and strand displacement events, resulting in the formation of concatemeric NA products that again contain multiple repeats of the amplified DNA. Thus, in concatemeric NA products, multiple copies of each amplified "single unit" NA molecule are present. RCA polymerase is particularly preferred for use in the methods disclosed herein. Products of RCA-type strand displacement replication methods traditionally require complex processing to release single units of NA.

[0066] Most RCA techniques to date have utilized Phi29 DNA polymerase, but other polymerases can also be used. This is because Phi29 polymerase is highly processive, allowing it to rapidly synthesize long concatemers of NAs, and its strand-displacing activity allows it to continuously synthesize new NA sequences while displacing any secondary primers it encounters. Additionally, it can produce large amounts of NAs in a relatively short time without the need for thermal cycling. Furthermore, Phi29 polymerase has an extremely low average error rate and can use either RNA or DNA as a template.

[0067] The RCA reaction can utilize either a single-stranded or double-stranded NA template. In some embodiments, a modified plasmid lacking typical gene sequences required for plasmid selection and replication in bacteria is used as the template. Any vector template can be used. In some embodiments, the template can be a circular expression cassette containing at least one heterologous gene of interest flanked by genetic elements (e.g., promoter, poly-A tail, etc.) necessary for expression and processing of the expression product in the target cell. While the methods disclosed herein can be used to amplify genes from conventional plasmids, streamlined templates without extraneous gene sequences offer several advantages. They eliminate extraneous sequences that may inadvertently silence expression of the heterologous gene of interest. Smaller constructs are more compact and can be more efficiently taken up by target cells, resulting in higher transfection efficiencies. They are also more cost-effective due to the lower production requirements of shorter expression cassettes, a statistical increase in the fidelity of the final product, and reduced need for extensive purification.

[0068] While 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 reportedly possesses clamp-like activity, providing a DNA synthesis rate of approximately 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 to amplify templates in RCA. DNA polymerase I primarily utilizes single-stranded templates and can readily form small circular templates without the steric hindrance that often poses problems when replicating extremely short double-stranded templates. U.S. Patent No. 5,614,365 discloses a modified DNA polymerase I containing sequences derived from T7 DNA polymerase to increase efficiency by up to 500-fold. This polymerase exhibits a reduced ability to discriminate 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, 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 derivatives thereof.Some embodiments of the methods disclosed herein use processive strand-displacing polymerases, such as Phi29-like polymerases (e.g., those found in Phi29-like phages; see, e.g., Microbiol Mol Biol Rev. 2001 Jun;65(2):261-287), to efficiently amplify templates without the need for thermal cycling. Preferred embodiments use Phi29 or Phi29-like polymerases, although other polymerases, such as Pol I and Pol III, T7 DNA polymerase, and their derivatives, can also be used.

[0069] To enable amplification according to the methods disclosed herein, the NA template is preferably also contacted with one or more DNA or RNA primers. The primers may be nonspecific (i.e., random in sequence) or specific to one or more sequences contained within the NA template. The primers are preferably random in sequence to allow nonspecific initiation at any site on the NA template. This allows for highly efficient amplification through multiple initiation reactions from each template strand. Examples of random primers are hexamers, heptamers, octamers, nonamers, decamers, or longer sequences, 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. Random primers can be 6-30, 8-30, or 12-30 nucleotides in length. Random primers are typically provided as a mixture of oligonucleotides representing all possible combinations of polymers in the NA template (eg, hexamers, heptamers, octamers, or nonamers).

[0070] In other embodiments, the NA primer is specific. As used herein, a specific NA primer is one that has a sequence that is at least 80% complementary to a sequence in the template from which amplification is desired to begin. In this embodiment, a pair of primers can be used to specifically amplify a portion of the template located within the two primer binding sites. The primers may be unlabeled or may contain one or more labels, such as radionuclides and fluorescent molecules. In some embodiments, the primers contain chemically modified nucleotides. The length / sequence of the primer can typically be selected based on consideration of the hybridization temperature, i.e., so that it can bind to the template at the temperature used in the amplification step.

[0071] The NA template is contacted with the RCA polymerase and one or more primers under conditions that promote hybridization of the primer with the template. These conditions include the presence of single-stranded NAs that allow hybridization of the primer. In some embodiments, single-stranded NAs are provided by denaturing double-stranded NAs by any method known in the art (e.g., incubation at a temperature above 80°C). These conditions also include a temperature and buffer that promote hybridization of the primer with the template. Appropriate hybridization conditions can be selected depending on the characteristics of the primer. An example of a preferred hybridization condition used in the method disclosed herein includes a buffer of 30 mM Tris-HCl pH=7.5, 20 mM KCl, and 8 mM MgCl. Hybridization can be performed after denaturation by gradually cooling to the desired reaction temperature.

[0072] Once the NA template has been contacted with the polymerase and one or more primers, there is then an incubation step under conditions that promote amplification of the template. Preferably, these conditions promote amplification of the template by displacement of the replicated strand by strand displacement replication of another strand. These conditions include the use of any temperature that allows for NA amplification, generally in the range of 20 to 90°C. Preferred temperature ranges can be about 20 to about 40°C or about 25 to about 35°C. Typically, the appropriate temperature is selected based on the temperature at which a particular polymerase has optimal activity. This information is generally available and forms part of the general knowledge of those skilled in the art. For example, the appropriate temperature range for Phi29 DNA polymerase is about 25 to about 35°C, preferably about 30°C. Those skilled in the art will be able to routinely identify the appropriate temperature for efficient amplification in the methods disclosed herein. For example, the method can be performed over a range of temperatures, and the yield of amplified NAs can be monitored to identify the optimal temperature range for a given polymerase.

[0073] Other conditions that promote amplification of a template include the presence of a polymerase and one or more primers. Conditions also include the presence of dNTPs (dATP, dTTP, dCTP, and dGTP) or NTPs (ATP, UTP, GTP, and CTP), an appropriate buffer / pH, and other factors required for enzyme performance or stability. Suitable conditions include any conditions used to provide activity of a polymerase enzyme known in the art.

[0074] For example, the pH may be within the range of 3 to 10, preferably 5 to 8 or about 7, e.g., about 7.5. The pH may be maintained within this range by the use of one or more buffers. Such 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, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, phosphate, citrate-sodium hydrogen phosphate, citric acid-sodium citrate, sodium acetate-acetic acid, imidazole, and sodium carbonate-sodium bicarbonate. This reaction also may be carried out with salts of divalent metals, such as magnesium (Mg), including, but not limited to, chloride, acetate, and sulfate. 2+ ) and manganese (Mn 2+ ) salts of monovalent metals, such as sodium (Na + ) and potassium (K + ) salts, such as potassium chloride. Other salts that may be included are ammonium salts, especially ammonium sulfate.

[0075] A detergent may also be included. Examples of suitable detergents include Triton™ X-100 (octoxynol), TWEEN® 20 (polyethylene glycol sorbitan monolaurate), and derivatives of either of these. 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. Reaction conditions can also be improved by adding agents that reduce NA coiling and make template denaturation easier. Such agents include, for example, dimethyl sulfoxide (DMSO), formamide, glycerol, and betaine.

[0076] The specific concentrations of specific reagents can be selected based on prior art and further optimized to suit 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, 20 mM (NH)SO, 5% glycerol, 0.2 mM BSA, and 1 mM dNTPs. A preferred reaction buffer used in RCA amplification in the method disclosed herein contains 35 mM Tris-HCl, 50 mM KCl, 14 mM MgCl, 10 mM (NH)SO, 4 mM DTT, and 1 mM dNTPs. This buffer is particularly suitable for use with Phi29 RCA polymerase.

[0077] Ligation Reaction The method herein involves generating closed circular NAs from linear concatemer molecules. Ligases are a class of enzymes that catalyze the formation of a phosphodiester bond between the 3'-OH group and the 5'-phosphate group of an NA chain, covalently linking two NA molecules together or circularizing a single NA molecule. In one aspect, the linear NAs after the first endonuclease digestion are contacted with at least one ligase enzyme under conditions that promote the ligation reaction. In some embodiments, the ligase enzyme used is T4 DNA ligase. Other ligase enzymes 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 E. coli DNA ligase.

[0078] A key feature of the synthetic NA production method described herein is the volume of the ligation reaction. As the concentration of NAs in the ligation reaction decreases, the reaction favors the formation of monomeric circular NAs (by intramolecular self-ligation) as opposed to high-molecular-weight polymers (by intermolecular ligation). A method for maintaining a low concentration of linear NAs in the ligation reaction is to stagger the addition of template NAs to the reaction, thereby separating the total reaction NAs (linear and circular) from the NAs input to the reaction (linear NAs only). As linear NAs in the reaction circularize, their concentration decreases, allowing for the addition of more linear NAs while maintaining a low concentration. A method known as step-by-step (SBS) ligation has been described in the art to maintain a low concentration of linear template DNA during the ligation reaction. The SBS method involves intermittently adding very small (<100 bp) linear ssDNA molecules to the reaction buffer in 20-minute increments over a total period of 2 hours. However, when applied to dsDNA molecules of substantial size (>250 bp), the SBS method proved to be slightly superior to conventional ligation (see Figure 5A).

[0079] As a result, a novel method that enables high production of monomeric ligation products using linear dsDNA templates, called sequential substrate addition (SCAM), has been developed and applied to the method herein. SCAM involves the slow, continuous addition of linear NAs to a ligation reaction. In some embodiments, the addition of linear NAs is performed using a pump (e.g., a syringe pump or a peristaltic pump). In some embodiments, SCAM is performed in a tube, such as a 50 mL conical tube. In some embodiments, SCAM is performed in a larger vessel, such as a flask (e.g., an Erlenmeyer flask), for example, a 125 mL flask.

[0080] 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 for 5 to 14 hours, or about 2% to about 20%, or about 3% to about 20%, or about 4% to about 20%, or about 5% to about 20%, or about 6% to about 20%, or about 7% to about 20%, or about 8% to about 20%, or about 9% to about 20%, or about 10% to about 20%, or about 11% to about 20%, or about 12% to about 20%, or about 13% to about 20%, or about 14% to about 20%, or about 15% to about 20%, or about 16% to about 20%, or about 17% to about 20%, or about 18% to about 20% of the final ligation reaction volume per hour. or about 19% to about 20%, or about 1% to about 19%, or about 2% to about 19%, or about 3% to about 19%, or about 4% to about 19%, or about 5% to about 19%, or about 6% to about 19%, or about 7% to about 19%, or about 8% to about 19%, or about 9% to about 19%, or about 10% to about 19%, or about 11% to about 19%, or about 12% to about 19%, or about 13% to about 19%, or about 14% to about 19%, or about 15% to about 19%, or about 16% to about 19%, or about 17% to about 19%, or about 18% to about 19%, or about 1% to about 18 %, or about 2% to about 18%, or about 3% to about 18%, or about 4% to about 18%, or about 5% to about 18%, or about 6% to about 18%, or about 7% to about 18%, or about 8% to about 18%, or about 9% to about 18%, or about 10% to about 18%, or about 11% to about 18%, or about 12% to about 18%, or about 13% to about 18%, or about 14% to about 18%, or about 15% to about 18%, or about 16% to about 18%, or about 17% to about 18%, or about 1% to about 17%, or about 2% to about 17%, or about 3% to about 17%, or about 4% to about 17% %, or about 5% to about 17%, or about 6% to about 17%, or about 7% to about 17%, or about 8% to about 17%, or about 9% to about 17%, or about 10% to about 17%, or about 11% to about 17%, or about 12% to about 17%, or about 13% to about 17%, or about 14% to about 17%, or about 15% to about 17%, or about 16% to about 17%, or about 1% to about 16%, or about 2% to about 16%, or about 3% to about 16%, or about 4% to about 16%, or about 5% to about 16%, or about 6% to about 16%, or about 7% to about 16%, or about 8% to about 16%,or about 9% to about 16%, or about 10% to about 16%, or about 11% to about 16%, or about 12% to about 16%, or about 13% to about 16%, or about 14% to about 16%, or about 15% to about 16%, or about 1% to about 15%, or about 2% to about 15%, or about 3% to about 15%, or about 4% to about 15%, or about 5% to about 15%, or about 6% to about 15%, or about 7% to about 15%, or about 8% to about 15%, or about 9% to about 15%, or about 10% to about 15%, or about 11% to about 15%, or about 12% to about 15%, or about 13% to about 15%, or about 14% to about 15%, or about 1% to about 14%, or about 2% to about 14%, or about 3% to about 14%, or about 4% to about 14%, or about 5% to about 14%, or about 6% to about 14%, or about 7% to about 14%, or about 8% to about 14%, or about 9% to about 14%, or about 10% to about 14%, or about 11% to about 14%, or about 12% to about 14%, or about 13% to about 14%, or about 1% to about 13%, or about 2% to about 14%, or about 3% to about 14%, or about 4% to about 13%, or about 4% to about 13%, or about 5% to about 13%, or about 6% to about 13%, or about 7% to about 13%, or about 8% to about 13%, or about 9% to about 13% , or about 10% to about 13%, or about 11% to about 13%, or about 12% to about 13%, or about 1% to about 12%, or about 2% to about 12%, or about 3% to about 12%, or about 4% to about 12%, or about 5% to about 12%, or about 6% to about 12%, or about 7% to about 12%, or about 8% to about 12%, or about 9% to about 12%, or about 10% to about 12%, or about 11% to about 12%, or about 1% to about 11%, or about 2% to about 11%, or about 3% to about 11%, or about 4% to about 11%, or about 5% to about 11%, or about 6% to about 11%, or about 7% to about 11%, or about 8% to about 11%, or is about 9% to about 11%, or about 10% to about 11%, or about 1% to about 10%, or about 2% to about 10%, or about 3% to about 10%, or about 4% to about 10%, or about 5% to about 10%, or about 6% to about 10%, or about 7% to about 10%, or about 8% to about 10%, or about 9% to about 10%, or about 1% to about 9%, or about 2% to about 9%, or about 3% to about 9%, or about 4% to about 9%, or about 5% to about 9%, or about 6% to about 9%, or about 7% to about 9%, or about 8% to about 9%, or about 1% to about 8%, or about 2% to about 8%, or about 3% to about 8%, or about 4% to about 8%, or about 5% to about 8%,Or about 6% to about 8%, or about 7% to about 8%, or about 1% to about 7%, or about 2% to about 7%, or about 3% to about 7%, or about 4% to about 7%, or about 5% to about 7%, or about 6% to about 7%, or about 1% to about 6%, or about 2% to about 6%, or about 3% to about 6%, or about 4% to about 6%, or about 5% to about 6%, or about 1% to about 5%, or about 2% to about 5%, or about 3% to about 5%, or about 4% to about 5%, or about 1% to about 4%, or about 2% to about 4%, or about 3% to about 4%, or about 1% to about 3%, or about 2% to about 3%, or about 1% to about 2%.

[0081] In some embodiments, the first digestion product is spun for 6 to 14 hours, or 7 to 14 hours, or 8 to 14 hours, or 9 to 14 hours, or 10 to 14 hours, or 11 to 14 hours, or 12 to 14 hours, or 13 to 14 hours, or 5 to 13 hours, or 6 to 13 hours, or 7 to 13 hours, or 8 to 13 hours, or 9 to 13 hours, or 10 to 13 hours, or 11 to 13 hours, or 12 to 13 hours, or 5 to 12 hours, or 6 to 12 hours, or 7 to 12 hours, or 8 to 12 hours, or 9 to 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 for 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 more. 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 for 10 to 12 hours.

[0082] Endonuclease, topoisomerase and exonuclease enzymes, and nucleic acid purification In some embodiments, the methods disclosed herein include a processing step that utilizes an endonuclease enzyme to separate NA molecules, resulting in the production of multiple nucleotide units. Endonucleases are a class of enzymes that cleave phosphodiester bonds within polynucleotide chains, which can facilitate the separation of NA molecules as described further herein. Different families of endonucleases have different requirements for initiating cleavage. For example, endonucleases of the DNase I family cleave DNA nonspecifically, meaning that they essentially cleave DNA at any double-stranded site. Alternatively, restriction endonucleases contain domains that recognize very specific nucleotide sequences and therefore cleave only at those specific sites. Restriction endonucleases or other sequence-specific endonucleases are preferred for use in the methods disclosed herein. The categories of endonuclease enzymes useful in the methods disclosed herein include 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, DNase I, DNase II, DNase III, DNase IV, and RNA endonucleases, including engineered RNA endonucleases with customized sequence specificity.Other sequence-specific endonucleases that can be used include, but are not limited to, RNA-directed endonucleases, such as CRISPR / Cas endonucleases (and similar endonucleases, such as Cas9, Cpf1, etc.).

[0083] In some embodiments, a method for inactivating the endonuclease enzyme is used to ensure that the endonuclease enzyme does not continue to cleave NA molecules after the digestion reaction is complete. A common method for enzyme inactivation is to heat the reaction solution containing the enzyme to a temperature that promotes protein denaturation, for example, 65°C or higher, for 15 minutes or more. Such high temperatures can damage important protein domains and change the shape of the enzyme, making it impossible for the substrate to fit into the active site, thereby inactivating the enzyme. Another method is to change the pH of the reaction solution containing the enzyme to a pH that changes the hydrogen bond and salt bridge interactions in the tertiary structure of the protein, similarly inactivating the enzyme. In some embodiments, the methods herein include inactivating the endonuclease enzyme after the endonuclease reaction is complete. In some embodiments, the endonuclease enzyme is inactivated by heat inactivation, for example, by heating the reaction solution to 65°C or higher for 15 minutes or more. In some embodiments, the endonuclease enzyme is inactivated by changing the pH of the reaction solution by adding an acid or base. 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 (C2H4O2), boric acid (H3BO3), citric acid (C6H8O7), nitric acid (HNO3), sulfuric acid (H2SO4), calcium hydroxide (Ca(OH)2), ammonia (NH3), sodium bicarbonate (NaHCO3), or sodium carbonate (Na2CO3).

[0084] The structure of a circular NA molecule, such as a closed-end NA molecule containing the ligation product described herein, promotes the formation of supercoils in the NA. Supercoil formation is a characteristic of closed-end NA caused by the tight topological bond between the coiled and double-helical structures of the NA, resulting in a structurally compact molecule. Due to their inherent physical properties, supercoil formation often occurs in closed-end NA molecules. When in a circular conformation, the primary coil structure of the double-helical NA promotes further coiling into a supercoiled structure. In some embodiments, the method herein generates a supercoiled product by ligating a monomer insertion sequence.

[0085] 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 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% supercoiled.

[0086] In some embodiments, the methods herein involve generating further supercoiled NA from open-circular NA through an enzymatic reaction. Cellular processes, such as replication, transcription, recombination, and rearrangement, often result in changes in the topology of NA, causing problems with NA torsion, which can be addressed by the action of topoisomerase enzymes. In vitro, topoisomerase enzymes are useful because they have the ability to change the topological state of DNA and RNA. For example, DNA topoisomerase introduces temporary single-strand (type I) or double-strand (type II) breaks in the phosphate backbone of DNA, allowing for positive or negative supercoiling (tighter or weaker coiling). By contacting NA with a topoisomerase enzyme in vitro, it is possible to synthetically determine the supercoil state of NA. In some embodiments, the topoisomerase used is DNA gyrase. In some embodiments, the topoisomerase used is E. coli DNA gyrase. Other topoisomerase enzymes useful in the methods disclosed herein include, but are not limited to, Cre recombinase, Staphylococcus aureus DNA gyrase, DNA topoisomerase 2-alpha, or DNA topoisomerase 2-beta.

[0087] In some embodiments, the methods herein involve using an exonuclease enzyme to remove linear and open-circular NA from supercoiled products. Exonucleases are a class of enzymes that act by digesting NA strands with open 3' or 5' ends, ultimately resulting in the removal of these strands. In the present disclosure, exonuclease enzymes can be used to further purify the amplified NA product. In some embodiments, the exonuclease used is T5 exonuclease. Other exonuclease enzymes 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, lambda exonuclease, or T7 exonuclease.

[0088] In some embodiments, the methods herein include further purifying the amplified NA product using NA purification techniques known in the art. These methods may 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.

[0089] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to the published literature, which provides general guidance to those skilled in the art for many of the terms used in this application. In all cases where there is a conflict between a definition set forth herein and a definition in a referenced publication, the definition provided herein shall control.

[0090] As used herein, "nucleic acid" (NA) or "polynucleotide" refers to an organic molecule comprising two or more covalently linked nucleotides. As used herein, "nucleotide" refers to an organic molecule comprising: 1) a nucleoside comprising a sugar covalently linked to a nitrogenous base (nucleobase); and 2) a phosphate group covalently linked to the sugar of the nucleoside. The nucleotides in a polynucleotide are typically linked by phosphodiester bonds, with the 3' carbon of the sugar of a first nucleotide linked to the 5' carbon of the sugar of a second nucleic acid by a bridging phosphate group. Typically, the bridging phosphate contains two non-bridging oxygen atoms bonded only to the phosphorus atom of the phosphate, and two bridging oxygen atoms, each linking the phosphorus atom to either the 3' carbon of the first nucleotide or the 5' carbon of the second nucleotide. In a nucleic acid sequence describing the order of nucleotides in a nucleic acid, a first nucleotide is said to be 5' (upstream) of a second nucleotide if the 3' carbon of the first nucleotide is linked to the 5' carbon 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 3' (downstream) of the first nucleotide. Nucleic acid sequences are typically read in 5'→3' order, beginning with the 5' nucleotide and ending with the 3' nucleotide.

[0091] As used herein, the term "circular NA" or "circular NA vector" refers to a nucleic acid molecule in a circular form. Circular NA may be DNA or RNA. Circular NA vectors may be monomeric, dimeric, trimeric, tetrameric, pentameric, hexamer, etc. Preferably, the circular NA vector is monomeric. In another preferred embodiment, 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. Such circular forms can typically be amplified into concatemeric linear NAs by rolling circle amplification. As used herein, the term "circular NA vector" is used interchangeably with the terms "NA vector," "circular NA molecule," and "covalently closed circular NA vector." Those skilled in the art will understand that such circular vectors may include covalently closed vectors with supercoiling and complex DNA topology, as described herein.

[0092] In some embodiments, the NA is a linear NA. Linear NA can be DNA or RNA. Linear NA is an NA with 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 is located on the 3' side of the 5'-terminal nucleotide in the nucleic acid sequence of the NA. The 3'-terminal nucleotide of linear NA is covalently bound to only one adjacent nucleotide of the NA, and the adjacent nucleotide is located on the 5' side of the 3'-terminal nucleotide in the nucleic acid sequence of the NA. In a nucleic acid sequence containing all nucleotides of linear NA in the order of 5' to 3', 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, linear NA is self-ligated to produce a circular NA.

[0093] RNA molecules that can be translated are called 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 a DNA or RNA sequence. An anticodon refers to a group of three nucleotides in a nucleic acid, such as a transfer RNA (tRNA), that is complementary to the codon, thereby associating the codon of a first nucleic acid with the anticodon of a second nucleic acid through hydrogen bonding between the bases of the codon and the anticodon. For example, the codon 5'-AUG-3' on an mRNA corresponds to the anticodon 3'-UAC-5' on a tRNA. During translation, a tRNA with an anticodon complementary to the codon to be translated associates with the codon on the mRNA to generally deliver the amino acid corresponding to the codon to be translated or to facilitate the termination of translation and release of the translated polypeptide from the ribosome.

[0094] As used herein, "modified nucleotide" refers to a nucleotide having a structure that is not the standard structure of an adenosine, cytidine, guanine, or uracil nucleotide. The standard structure of a molecule refers to the structure generally known in the art to be the structure referred to by the molecular name. The standard structure of an adenosine nucleotide, containing an adenine base, a ribose sugar, and one or more phosphate groups, is shown below in the form of adenosine monophosphate: [ka] .

[0095] The canonical structure of AMP 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, and in which the oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0096] The standard structure of a cytosine nucleotide, containing a cytosine base, a ribose sugar, and one or more phosphate groups, is shown below in the form of cytidine monophosphate: [ka] The canonical structure of a CMP 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, and in which the oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are attached to adjacent nucleotides in a nucleic acid sequence.

[0097] The standard structure of a guanine nucleotide, containing a guanine base, a ribose sugar, and one or more phosphate groups, is shown below in the form of guanosine monophosphate: [ka] The canonical structure of GMP 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, and in which the oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are attached to adjacent nucleotides in a nucleic acid sequence.

[0098] The standard structure of a uracil nucleotide, containing a uracil base, a ribose sugar, and one or more phosphate groups, is shown below in the form of uridine monophosphate: [ka] The canonical structure of a UMP 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, and in which the oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to adjacent nucleotides in a nucleic acid sequence.

[0099] The structure of a modified nucleotide can differ from that of a standard nucleotide by modification of one or more of the nucleotide's sugar, nitrogenous base, or phosphate. In some embodiments, a modified nucleotide comprises a modified nucleoside that is not of the standard structure of an adenine nucleoside, a cytosine nucleoside, a guanine nucleoside, or a uracil nucleoside.

[0100] An example of the standard structure of the adenine nucleoside adenosine is reproduced below: [ka] The canonical structure of adenosine 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, the 5' carbon is bound to the 5' phosphate in a nucleic acid sequence, and the 3' oxygen atom is bound to the 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0101] An example of the canonical structure of the cytosine nucleoside cytidine is reproduced below: [ka] The canonical structure of cytidine 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, the 5' carbon is bound to the 5' phosphate in a nucleic acid sequence, and the 3' oxygen atom is bound to the 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0102] An example of the canonical structure of the guanine nucleoside guanosine is reproduced below: [ka] 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, the 5' carbon is attached to the 5' phosphate in a nucleic acid sequence, and the 3' oxygen atom is attached to the 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0103] An example of the canonical structure of uridine, the uracil nucleoside, is reproduced below: [ka] The standard 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, the 5' carbon is bound to the 5' phosphate in a nucleic acid sequence, and the 3' oxygen atom is bound to the 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0104] As used herein, a "poly-A tail" refers to a nucleic acid sequence comprising adenosine nucleotides attached to the 3' end of a nucleic acid, e.g., RNA. A poly-A tail or poly-A region can be composed 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 standard adenosine nucleotides or modified (non-standard) adenosine nucleotides.

[0105] As used herein, "ligase" refers to an enzyme capable of forming a covalent bond between two nucleotides, and the method of "ligation" refers to the formation of a covalent bond between two nucleotides. As used herein, "concatamer" refers to an NA molecule containing multiple copies of the same or substantially identical NA sequence (e.g., subunits), typically linked in series.

[0106] As used herein, the term "isolated" means artificially produced. In some embodiments, with respect to an NA vector, the term "isolated" refers to (i) an NA vector amplified in vitro (e.g., in a cell-free environment), for example, by rolling circle amplification or polymerase chain reaction (PCR); (ii) an NA vector recombinantly produced by molecular cloning; (iii) an NA vector purified, for example, by endonuclease cleavage and gel electrophoretic fractionation or column chromatography; or (iv) an NA vector synthesized, for example, by chemical synthesis. An isolated NA vector is one that can be easily manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector for which the 5' and 3' endonuclease sites are known or the polymerase chain reaction (PCR) primer sequences are disclosed is considered isolated, but an NA sequence present natively in its natural host is not. An isolated NA vector may be substantially purified, but may not be purified.

[0107] As used herein, a "cell-free method" for producing an NA vector refers to a method that does not rely on the containment of any NA within a host cell, such as a bacterial (e.g., E. coli) host cell, to facilitate any step of the method. For example, a cell-free method may be carried out in one or more synthetic vessels (e.g., glass or plastic tubes or other vessels) in a suitable solution (e.g., buffer), to which enzymes and other agents can be added to facilitate the amplification, modification, and isolation of the NA.

[0108] As used herein, "vector" refers to a NA molecule capable of carrying a heterologous gene in a target cell, where the heterologous gene can then be replicated, processed and / or expressed within the target cell.

[0109] Translation is the process of using an RNA coding sequence to direct the production of a protein. The first step in translation is initiation, in which a ribosome associates with an mRNA, and a first transfer RNA (tRNA) carrying a first amino acid associates with the first or START codon. The next stage of translation, elongation, involves three steps. First, a second tRNA carrying a second amino acid, with an anticodon complementary to the codon following the START or second codon, associates with the mRNA. Second, the carbon atom of the terminal non-side chain carboxylic acid moiety of the first amino acid reacts with the nitrogen of the terminal non-side chain amino moiety of the second amino acid, 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 proceeds along the mRNA, so that the position where the first tRNA was associated with the ribosome is now occupied by the second tRNA, and the position previously occupied by the second tRNA is now free for a further tRNA carrying an additional amino acid to associate with the mRNA. These three steps—1) association of the tRNA carrying an amino acid, 2) peptide bond formation that adds the additional amino acid to the growing polypeptide, and 3) progression of the ribosome along the mRNA—continue until the ribosome reaches a STOP codon, resulting in the end of translation. Generally, the tRNA associated with the STOP codon does not carry an amino acid, so association of a tRNA that does not carry an amino acid during the elongation step results in cleavage of the bond between the polypeptide and the tRNA carrying the final amino acid in the polypeptide, thereby releasing the polypeptide from the ribosome. Alternatively, if a tRNA is not associated with the STOP codon, the ribosome may dissociate from the mRNA and release the polypeptide.

[0110] 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 a target cell present in a subject (in situ) or in culture by various methods, including electroporation. In some embodiments, the target cell is a post-mitotic cell. Target cells include animal cells (and cell lines derived from animals), both vertebrate and invertebrate. Representative examples of vertebrate cells include mammalian cells, such as human, rodent (e.g., rat and mouse), and ungulate (e.g., cow, 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, e.g., hematopoietic stem cells or other stem cells)), or a totipotent cell (i.e., a cell whose progeny can become any cell type of an organism, e.g., embryonic stem cells and somatic stem cells, e.g., hematopoietic cells). In yet other embodiments, target cells include oocytes, eggs, embryonic cells, zygotes, sperm cells, and somatic (non-stem) mature cells from various organs or tissues, such as liver cells, nerve cells, muscle cells, and blood cells (e.g., lymphocytes).

[0111] "Host cell" refers to any cell that harbors a desired NA vector. Host cells can be used as recipients of the NA vectors described in this disclosure. This term includes the progeny of the original transfected cell. Thus, as used herein, "host cell" can refer to either a cell that has been transfected with a heterologous gene (e.g., by an NA vector described herein) and its progeny that harbor the desired NA vector. It is understood that the progeny of a single parent cell may not be completely identical in morphology or genomic or total NA complement to the original parent due to natural, accidental, or deliberate mutation.

[0112] The term "heterologous gene" refers to a gene that does not naturally occur 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, e.g., a therapeutic replacement protein, an antigen-binding protein, etc.), e.g., a mammalian gene encoding a therapeutic protein. In some embodiments, a heterologous gene encodes a protein or portion thereof (e.g., a therapeutic replacement protein) that is missing or absent in a target cell and / or subject. In some embodiments, a heterologous gene comprises one or more exons that encode a protein that is missing or absent in a target cell and / or subject. In some embodiments, a heterologous gene comprises a therapeutic NA, such as a therapeutic RNA (e.g., a microRNA, siRNA, shRNA, or guide RNA compatible with a Cas nuclease system) or a therapeutic DNA (e.g., an antisense oligonucleotide, aptamer, or ribozyme).

[0113] The term "promoter" refers to a sequence that regulates transcription of a heterologous gene operably linked to it. A promoter can provide sufficient sequences to direct transcription and / or recognition sites for RNA polymerase and other transcription factors necessary for efficient transcription, directing cell-specific expression. In addition to sequences sufficient to direct transcription, a promoter sequence can also include sequences for other regulatory elements involved in modulating transcription (e.g., enhancers, Kozak sequences, and introns). Examples of promoters known in the art and useful in the vectors described herein include the CMV promoter, hEF1a promoter, CBA promoter, smCBA promoter, inducible promoters such as TRE and TRE3G, and promoters derived from immunoglobulin genes, SV40, or other tissue-specific genes. Standard techniques for creating functional promoters by mixing and matching known regulatory elements are known in the art. "Truncated promoters" can also be generated from promoter fragments or by mixing and matching fragments of known regulatory elements. For example, the smCBA promoter is a truncated version of the CBA promoter.

[0114] As used herein, an "open reading frame" ("ORF"), e.g., an ORF encoding a protein, refers to a nucleic acid sequence comprising a coding sequence that results in the production of a protein when the ORF is translated. The nucleic acid sequence may be an RNA sequence, where translation of the RNA sequence produces a polypeptide having the amino acid sequence of the protein. The nucleic acid sequence may be a DNA sequence, where a protein is produced when an RNA polymerase uses the DNA sequence to transcribe an RNA molecule comprising 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, e.g., AUG in the RNA sequence (ATG in the DNA sequence), and ends with a STOP codon, e.g., UAG, UAA, or UGA in the RNA sequence (TAG, TAA, or TGA in the DNA sequence), where the number of bases between the start codon G and the STOP codon T or U is a multiple of three (e.g., 3, 6, 9, 12, etc.).

[0115] In some embodiments of the synthetic circular NAs provided herein, the circular NAs comprise 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 therefore are not part of the open reading frame. The 5'UTR is 5' (upstream) of the open reading frame. The 3'UTR is 3' (downstream) of the open reading frame. In some embodiments, the 3'UTR is 3' of the open reading frame and comprises one or more nucleotides that are 5' (upstream) of the poly-A region of the RNA.

[0116] 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 compositions further comprise one or more additional agents, such as nucleotides, nucleic acids, amino acids, peptides, proteins, small molecules, aptamers, lipids, or carbohydrates. In some embodiments, the additional agents have a therapeutic effect when administered to a subject. In some embodiments, the additional agents are agents for use in modulating the expression and / or activity of one or more gene products (e.g., proteins) in a subject. In some embodiments, the additional agents are nucleic acids for use in 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 oligonucleotides (ASO). In some embodiments, the additional agents are inhibitors for reducing the activity of one or more gene products (e.g., proteins). In some embodiments, the agents are small molecule inhibitors. In some embodiments, the additional agents are agents for enhancing an immune response in a subject. In some embodiments, the additional agents are antigens, such as nucleic acid antigens, protein antigens, or phospholipid antigens. In some embodiments, the additional agent is an adjuvant, such as aluminum hydroxide or aluminum potassium sulfate (alum), monophosphoryl lipid A (MPL), an oil-in-water emulsion (e.g., a squalene emulsion), cytosine phosphoguanine (CpG) oligodeoxynucleotide, or another adjuvant known in the art. See, for example, 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, tonicity agents, preservatives, or antioxidants, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.The precise nature of the carrier or other material may depend on the route of administration, for example parenteral, intramuscular, intradermal, sublingual, buccal, intraocular, intranasal, subcutaneous, intrathecal, intratumor, oral, vaginal or rectal.

[0117] In some aspects, the present disclosure provides a method for administering any of the synthetic cyclic NAs, delivery agents, cells, compositions, or pharmaceutical compositions provided herein to a subject. In some embodiments, the subject is a human. In some embodiments, administration is parenteral, intramuscular, intradermal, sublingual, oral, intraocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal. 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 so that the nucleic acid is relatively stable over time. In some embodiments, the synthetic cyclic NAs are introduced into the subject's cells by in vivo electroporation. In vivo electroporation is a method for introducing nucleic acids or other molecules into the subject's cells using an electric pulse that promotes the passage of nucleic acids or other molecules through the cell membrane and / or cell wall. See, for example, Somiari et al., Molecular Therapy., 2000. 2(3):178-187. The synthetic cyclic NA to be delivered is administered to a subject, for example, by injection, and an electric pulse is applied to the injection site, whereby the electricity promotes the entry of the nucleic acid into cells at the administration site. In some embodiments, the synthetic cyclic NA is delivered and taken up into cells of a subject (e.g., local cells at the administration site or the entire subject) via a delivery agent associated (e.g., conjugated) with the synthetic cyclic NA. In some embodiments, the synthetic cyclic NA is administered with other elements that enhance electroporation efficiency, such as buffers and / or excipients.

[0118] The terms "a" and "an" mean "one or more." For example, "a gene" is understood to refer to one or more such genes. Thus, "a" and "an," "one or more (or an)," and "at least one (or an)" are used interchangeably herein.

[0119] As used herein, the term "about" refers to a value within a ±10% variation from the reference value, unless otherwise specified.

[0120] As used in the specification and claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present 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 conjoined. Other elements, whether related or unrelated to the elements specifically identified, may optionally be present other than the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language, e.g., "comprising," can refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements), etc.

[0121] As used in this specification and the 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" shall be construed as being inclusive, i.e., the inclusion of at least one, but more than one, of a number or list of elements, and possibly further including items not listed. Only terms clearly indicating the contrary, e.g., "only one of" or "exactly one of," or when used in the claims, "consisting of" shall refer to the inclusion of exactly one element of a number or list of elements. In general, as used herein, the term "or" shall be construed as indicating exclusive alternatives (i.e., "one or the other, but not both") only when followed by terms of exclusivity, e.g., "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0122] As used in this specification and claims, the phrase "at least one" in reference to a list of one or more elements 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 and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the possibility that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements specifically identified, may optionally be present. 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 refer to, in one embodiment, at least one A, optionally including more than one, with no B (and optionally including elements other than B); in another embodiment, at least one B, optionally including more than one, with no A (and optionally including elements other than A); in yet another embodiment, at least one A, optionally including more than one, and at least one B, optionally including more than one (and optionally including other elements); etc.

[0123] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein, and it is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims. In the event of any conflict in definitions among various sources or references, the definitions provided herein shall control.

[0124] [Example] [Example 1] Development of a cell-free method for producing synthetic circular nucleic acids using rolling circle amplification Figure 1 shows a schematic diagram of the method disclosed herein, 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 enzyme to supercoil it, and with an exonuclease to remove linear NAs, followed by further purification for a purer final product. All reaction steps utilize a common buffer, and no purification is required between steps.

[0125] Figures 2A-2B show the method of rolling circle amplification (RCA) using example NAs and polymerases. As shown in Figure 2A, circular DNA molecules (labeled plasmids) can be used as NA templates. 5 μg / mL of plasmid was incubated with 50 μM random DNA primers, 2 mM dNTPs, and 200 U / mL Phi29 DNA polymerase in buffer to amplify long single-stranded concatemers. The DNA primers then bound to the concatemers, and Phi29 polymerase produced the complementary strand, resulting in double-stranded linear DNA products containing multiple copies of the starting plasmid sequence. Figure 2B shows the results of RCA performed on the plasmid for 90 minutes (lane 2) and 16 hours (lane 3). Lane 1 shows a DNA ladder for size reference. Lanes 2 and 3 indicate the presence of long double-stranded concatemers that migrated slowly in the gel and anchored in the loading well; greater amounts of concatemers were observed after 16 hours compared to 90 minutes of RCA.

[0126] Figures 3A-3D show a schematic diagram of amplifying a NA template using the methods provided herein. An example of a starting NA template is shown in Figure 3A, where 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 share 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 need not be the case. Performing RCA on the template yields linear concatemers as shown in Figure 3B (RCA amplification product). The horizontal lines indicate the cleavage sites of RE1 and RE2 in the linear concatemers. The RCA amplification product is then contacted with an endonuclease that cleaves at RE1, yielding a linear product shown in Figure 3C (first digestion product), which contains a separate insert sequence and backbone sequence. The insert sequence is flanked by RE1 cleavage sites and does not contain an RE2 cleavage site. The first digestion product is incubated with a ligase enzyme to promote self-ligation, examples of which are shown in Figure 3D (ligation products). For example, in some embodiments, the ligation product produced by the methods described herein contains a single copy of the insert (ligation product 1), while in other embodiments, it contains more than one copy of the insert (ligation product 4). In some embodiments, the ligation product contains a single copy of the backbone (ligation product 2), while 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 and at least one backbone (ligation product 3).

[0127] A DNA plasmid with the design shown in Figure 3A underwent a first digestion with RCA and the first restriction enzyme EcoRI (RE1) before undergoing ligation. The DNA template was slowly added to a vessel containing T4 DNA ligase, ATP, a common buffer, and water using the SCAM method described herein. Slow addition of DNA over a 12-hour period (sequential substrate addition, or SCAM) produced primarily monomeric circular constructs (see Figure 5). The products of the first digestion and SCAM ligation were run on a DNA agarose gel (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 and backbone product (higher and lower bands, respectively) after digestion with EcoRI (RE1), and lane 3 shows the DNA species present after self-ligation (the potential shown in Figure 3D).

[0128] Figures 4A-4C show DNA gels demonstrating the inactivation of endonuclease enzymes at specific pHs. To determine the pH at which EcoRI was inactivated, multiple reaction solutions were prepared using all reagents except DNA (i.e., the reaction solutions contained CutSmart® Buffer [New England BioLabs, proprietary composition], EcoRI, and water). The pH values ​​of each solution were varied using 12% HCl to include pH values ​​of 7.8, 7.0, 6.0, 5.0, 4.0, 3.0, or 2.0. After 5 minutes, the pH of all solutions was returned to 7.8 using 3 M NaOH. After 5 minutes, circular DNA plasmid template was added to each reaction solution, which was then incubated at 37°C for 2 hours. Figure 4A shows the reaction products after inactivating EcoRI using different pH values. pH values ​​between 7.8 and 5.0 did not inactivate EcoRI; under these pH conditions, EcoRI retained its endonuclease activity and did not result in the production of linear DNA (lanes 2–5). Lanes 6–8 show that pH values ​​between 4.0 and 2.0 caused significant inactivation of EcoRI, inhibiting its endonuclease activity and preserving the circular DNA template. Lane 1 shows a linear DNA ladder for size reference. Figure 4B shows data from a similar experimental setup, but testing the inactivation of HindIII endonuclease. Similar to Figure 4A, pH values ​​below 4.0 caused significant inactivation of HindIII endonuclease. Lane 1 shows a DNA ladder for size reference. The sample in lane 8 underwent a pH change (lowering to pH 4.0 using HCl and returning to pH 7.8 using NaOH) but did not contain HindIII endonuclease. Figure 4C shows the results of an experiment in which supercoiled DNA (SC, lane 2) was 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 a combination of heat and pH (HI pH, lane 5), and then contacted with HindIII. This solution was then added to a ligation reaction containing T4 DNA ligase, and after overnight incubation at room temperature, the products were run on a gel.Compared to the SC control (lane 2), the levels of linearized plasmid were similar in the endonuclease-inactivated samples (lanes 3-5), demonstrating that pH inactivation does not significantly affect the ligation reaction efficiency. Lane 1 shows a linear DNA ladder for size reference.

[0129] Figures 5A-5B show the results of ligation reactions performed using the step-by-step (SBS) method or sequential substrate addition (SCAM) method. First, to test the efficiency of SBS versus conventional ligation reactions, each reaction was performed in parallel using the same linear DNA substrate (Figure 5A). For conventional ligation reactions, all ligation reagents (T4 DNA ligase, T4 DNA ligase buffer [New England BioLabs, proprietary composition], and DNA) were added to the reaction simultaneously and incubated at room temperature for 2 hours (CTRL). For SBS samples, all ligation reagents and 20% of the final DNA volume were added at the beginning of the reaction. Starting 20 minutes later, 20% of the final DNA volume was added at five time points, 20 minutes apart, for a total reaction time of 2 hours, with a 20-minute incubation period after the addition of the final 20% of DNA. Samples were loaded in triplicate on a DNA gel with increasing amounts of DNA, along with a linear DNA ladder and a supercoiled DNA ladder for size reference (lanes 1 and 2, respectively). Comparison of the circular monomer bands in the SBS and conventional ligation lanes revealed a slight increase (50%–70%) in the circular monomer when using the SBS method (see lanes 4, 6, and 8 compared to lanes 3, 5, and 7). A second experiment was performed to test the ligation efficiency using SCAM versus conventional ligation reactions (Figure 5B). Similar to Figure 5A, conventional ligation reactions were performed in which all ligation reagents were added simultaneously to the reaction and incubated at 25 °C for 20 h (CTRL, lane 3). For the SCAM sample, all ligation reagents were first combined to produce an initial volume of 10 mL. Then, 10 mL of DNA substrate was added continuously at a rate of 0.6 mL / h over 16 h at 25 °C to produce a final volume of 20 mL. After the addition of the last substrate, the reaction was allowed to continue for 4 h. The samples were loaded onto a gel along with a linear DNA ladder and a supercoiled DNA ladder for size reference (Figure 5B).Comparison of the circular monomer bands in the SCAM and conventional ligation lanes revealed a significant increase (158%) in circular monomer production when SCAM was used, demonstrating that SCAM ligation is significantly more effective at producing circular monomer product from dsDNA templates compared to SBS and conventional ligation methods (see lane 4 compared to lane 3). Note that the DNA constructs used in the experiments shown in Figures 5A and 5B were different. The sizes of the insert and backbone sequences in Figure 5A were different enough to be separated on the gel. The sizes of the insert and backbone sequences in Figure 5B were similar and therefore appear as a single band on the gel.

[0130] The ligation product from Figure 3E contained the desired amplified circular monomer insert sequence (ligation product 1), but it also contained sequences constituting the vector backbone (Figure 6A, ligation products 2, 3, and 5). Figure 6B shows a schematic diagram of linearizing these unwanted backbone-containing products. Contacting the ligation product with an endonuclease that cleaves at RE2 cleaves any circular product containing the vector backbone and specifically linearizes the unwanted NA sequence (Figure 6B, second digestion products 2, 3, and 5). The numbers in Figures 6A and 6B indicate the products generated from each corresponding digested circular plasmid. Figure 6C shows a DNA gel run comparing the sizes of the ligation products before (lane 2) and after (lane 3) the second digestion with XhoI (i.e., RE2), revealing a size shift only in the DNA product containing the longer vector backbone (the lower band in lane 2). Lane 1 shows a linear DNA ladder for size reference.

[0131] Figure 7A shows a schematic diagram of how circular NAs are supercoiled. When topoisomerase enzymes, which act by altering the topological state of NAs, come into contact with relaxed NAs (shown as a plasmid in Figure 7A), they promote supercoiling, resulting in smaller DNA molecules (supercoiled products). The secondary digestion products from Figure 6C were contacted with E. coli DNA gyrase for 2 hours or overnight. Figure 7B shows a DNA gel run to compare the size of the secondary digestion products before supercoiling (lane 2) and after supercoiling for 2 hours (lane 3) or overnight (lane 4). After supercoiling, the secondary digestion products migrated through the gel at the same rate as the linear backbone (XhoI digestion), and the ladder-like appearance present in lane 2 was lost. Lane 1 shows a DNA ladder for size reference.

[0132] Figure 8A shows a method for removing undesired linear DNA from the second digestion product. While the supercoiled product contains the desired circular insert sequence (Figure 8A, products 1 and 4), it also contains products containing the linear backbone remaining from the second digestion (Figure 8A, products 2, 3, and 5). To remove these undesired products, the supercoiled product from Figure 7B was contacted with an exonuclease enzyme that digests linear and open-circular DNA. Figure 8B shows a DNA gel run comparing the sizes of the supercoiled products before (lane 2) and after (lane 3) digestion with T5 exonuclease. The linear monomer, multimer, and material captured in the wells of lane 2 are absent in lane 3, which contains primarily supercoiled circular inserts. Lane 1 shows a DNA ladder for size reference.

[0133] The exonuclease product from Figure 8B was further purified by anion exchange chromatography followed by isopropanol precipitation. Figure 9 shows a DNA gel run to demonstrate the purity of the supercoiled circular insert obtained after performing steps 1-7 of Figure 1 (see lane 3). The linear and supercoiled DNA ladders are shown in lanes 1 and 2, respectively.

[0134] equivalent While several inventive embodiments have been described and illustrated herein, those skilled in the art can readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or achieving one or more of the advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0135] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and may in some cases include the entire specification.

[0136] Also, unless expressly indicated to the contrary, it should be understood that in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0137] As in the above specification, in the claims, all transitional phrases, such as "comprising," "including," "holding," "having," "containing," "involving," "keeping," "consisting of," and the like, are understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as defined in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures. It should be understood that embodiments described herein using an open-ended transitional phrase (e.g., "comprising") are also contemplated, in alternative embodiments, as "consisting of" and "consisting essentially of" the feature described by the open-ended transitional phrase. For example, if the disclosure describes "a composition comprising A and B," the disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."

Claims

1. 1. A method for amplifying a synthetic circular nucleic acid, comprising: a) providing a circular nucleic acid template comprising (i) a backbone comprising one or more internal endonuclease cleavage sites, and (ii) an insert sequence comprising endonuclease cleavage sites at the 5' and 3' ends; b) amplifying the circular nucleic acid template by rolling circle amplification, thereby producing an amplification product; c) contacting the amplification product with a first endonuclease under conditions for digestion, thereby producing a first digestion product; d) adding the first digestion product to a ligation reaction mixture containing a ligase enzyme, 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 for 5 to 14 hours; and e) incubating the ligation reaction mixture, thereby producing a circular ligation product, wherein the circular ligation product is at least partially supercoiled. A method comprising:

2. 10. The method of 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 for 10 to 12 hours.

3. 3. The method of claim 1 or 2, wherein the circular ligation product is contacted with a second endonuclease under conditions for digestion, thereby producing a second digestion product, and wherein the first and second endonucleases do not have the same recognition site.

4. 4. The method of any one of claims 1 to 3, further comprising contacting the circular ligation product or the second digestion product with a topoisomerase under conditions that promote supercoiling, thereby producing a supercoiled product.

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

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

7. 7. The method of 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. 8. The method of claim 7, wherein the ion exchange chromatography is anion exchange chromatography.

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

10. The nucleic acid template is a. the nucleic acid fragment to be amplified; b. two recognition sites for a first endonuclease at the 5' and 3' ends of the nucleic acid fragment to be amplified, respectively; and c. A recognition site for a second endonuclease in the vector backbone 10. The method of any one of claims 1 to 9, comprising:

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

12. 12. The method of claim 1, wherein the rolling circle amplification is performed 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, 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. 13. The method of any one of claims 1 to 12, wherein rolling circle amplification is performed using Phi29 DNA polymerase.

14. 14. The method of claim 1, wherein the ligase is T4 DNA ligase, T4 RNA ligase, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase, or E. coli DNA ligase.

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

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

17. 17. The method of claim 16, wherein the DNA gyrase is Escherichia coli DNA gyrase, Staphylococcus aureus DNA gyrase, DNA topoisomerase 2-alpha, or DNA topoisomerase 2-beta.

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

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

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

21. 21. The method of any one of claims 1 to 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, DNase I, DNase II, DNase III, DNase IV, an RNA endonuclease or an RNA-directed endonuclease, including an engineered RNA endonuclease with customized sequence specificity, such as a CRISPR / Cas endonuclease.

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

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

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

Images