Formation of DNA constructs

Abstract

Provided herein are methods for generating circular nucleic acid molecules comprising providing a nucleic acid template, amplifying the nucleic acid template, and generating an amplification product comprising circular nucleic acids.

Description

FORMATION OF DNA CONSTRUCTSCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 620,031, filed on January 11, 2024, which is incorporated by reference in its entirety.BACKGROUND

[0002] The creation of high-quality large nucleic acid constructs is necessary for many therapeutic applications such as gene therapy and nucleic acid vaccines. Generating high percentages of nucleic acid monomers when creating nucleic acid constructs is difficult due to the continuous replicating nature of methods such as rolling circle amplification. Thus, there is a need for improved methods to create high-quality large nucleic acid monomeric constructs.INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF SUMMARY

[0004] Provided herein are methods for generating circular nucleic acids, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises XerC / D, ArgR, and PepA binding sites; b) amplifying the nucleic acid template by providing an amplification enzyme and primers for rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR) amplification to form a tandem repeat nucleic acid sequence comprising at least two recombination sites; and c) generating an amplification product comprising a circular nucleic acid; wherein the method comprises incubating the tandem repeat nucleic acid sequence with: i) a recombination enzyme comprising XerC / D recombinase polypeptides, mutant Cre, or a resolvase; and ii) accessory factors or cognate resolvases that promote monomeric resolution. In some embodiments, the accessory factors that promote monomeric resolution comprise ArgR and Pep A. In some embodiments, the accessory factors comprise a recombinase. In some embodiments, the nucleic acid template further comprises an expression cassette. In some embodiments, the resolvase is CinH, ParA, beta, Tn3, or gamma delta. In some embodiments, the amplification enzyme is phi29 DNA polymerase. In some embodiments, the primers for RCA comprise random hexamers or longer, sequence specific primers that anneal at greater than 30°C. In some embodiments, the primers for PCR amplification produce a linear product flanked byrecombination sites and are compatible with the accessory factors and the recombination enzyme. In some embodiments, the method further comprises sequencing the amplification product. In some embodiments, the sequencing comprises single molecule sequencing. In some embodiments, the sequencing comprises nanopore sequencing. In some embodiments, the method further comprises employing the ampl ification product in cell-based processes such transformation of bacteri a or yeast or transfection of mammalian cells. In some embodiments, the method further comprises adding an endonuclease and / or an exonuclease to remove a residual nicked dsDNA, a residual single stranded DNA, branched DNA, or a residual linear dsDNA from the amplification product. In some embodiments, the amplification product comprises at least 85% monomeric circular nucleic acids. In some embodiments, the amplification product comprises at least 95% monomeric circular nucleic acids. In some embodiments, the method further comprises processing the amplification product to generate a nucleic acid vaccine. In some embodiments, the method further comprises generating a large DNA construct. In some embodiments, the large DNA construct does not comprise extraneous sequences. In some embodiments, the method further comprises generating a gene. In some embodiments, the method further comprises generating a gene cluster. In some embodiments, the method further comprises generating a chromosome. In some embodiments, the method further comprises generating a genome. In some embodiments, the method occurs in vitro. In some embodiments, the method further comprises transfecting a cell free-produced DNA into cells. In some embodiments, the cells are mammalian cells. In some embodiments, the in vitro method is a transcription-translation (TX-TL) method. In some embodiments, the amplification product is used to generate an RNA. In some embodiments, the RNA is used to generate a protein. In some embodiments, the protein is a therapeutic protein. In some embodiments, the RNA is used as an RNA therapeutic. In some embodiments, the RNA therapeutic is formulated as a nanoparticle. In some embodiments, the amplification product is used as a DNA therapeutic. In some embodiments, the DNA therapeutic is formulated as a nanoparticle. In some embodiments, the method occurs in vivo. In some embodiments, the method occurs ex vivo. In some embodiments, the method further comprises barcoding the circular nucleic acid.

[0005] Provided herein are methods for generating an RNA vaccine, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises XerC / D, ArgR, and PepA binding sites; b) amplifying the nucleic acid template by providing an amplification enzyme and primers for rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR) amplification to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; and c) generating an RNA vaccine, wherein the method comprises incubating the tandem repeat nucleic acid sequence with: i) a recombination enzymecomprising XerCZD recombinase polypeptides, mutant Cre, or a resolvase; and ii) accessory factors or cognate resolvases that promote monomeric resolution.

[0006] Provided herein are methods for generating circular nucleic acids, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises a recombination site; b) amplifying the nucleic acid template by providing an amplification enzyme and primers for rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR) amplification to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; and c) generating an amplification product comprising a circular nucleic acid; wherein the method comprises incubating the tandem repeat nucleic acid sequence with: i) a recombination enzyme comprising a recombinase polypeptide; and ii) an accessory factor or cognate resolvase that promotes monomeric resolution. In some embodiments, the recombination site comprises a loxP binding site. In some embodiments, the recombinase polypeptide is a Cre recombinase polypeptide. In some embodiments, the recombinase polypeptide is a resolvase polypeptide. In some embodiments, the resolvase polypeptide is Cin l, ParA, beta, Tn3, or gamma delta. In some embodiments, the recombination site comprises a XerC / D binding site. In some embodiments, the recombinase polypeptide comprises a XerC polypeptide. In some embodiments, the method further comprises a second recombination enzyme comprising a XerD polypeptide. In some embodiments, the accessory factor that promotes monomeric resolution comprises ArgR. In some embodiments, the accessory factor that promotes monomeric resolution comprises PepA. In some embodiments, the accessory factor that promotes monomeric resolution comprises Sso7d. In some embodiments, the method further comprises a second accessory factor that promotes monomeric resolution. In some embodiments, the accessory factor that promotes monomeric resolution comprises ArgR and wherein the second accessoiy factor that promotes monomeric resolution is PepA. In some embodiments, the nucleic acid template further comprises an expression cassette. In some embodiments, the amplification enzyme is phi29 DNA polymerase. In some embodiments, the primers for RCA comprise random hexamers or longer, sequence specific primers that anneal at greater than 30°C. In some embodiments, the primers for PCR amplification produce a linear product flanked by recombination sites and are compatible with the accessory factors and the recombination enzyme. In some embodiments, the method further comprises sequencing the amplification product. In some embodiments, the sequencing comprises single molecule sequencing. In some embodiments, the sequencing comprises nanopore sequencing. In some embodiments, the method further comprises employing the amplification product in cell -based processes such transformation of bacteria or yeast or transfection of mammalian cells. In some embodiments, the method further comprises adding an endonucleaseand / or an exonuclease to remove a residual nicked dsDNA, a residual single stranded DNA, branched DNA, or a residual linear dsDNA from the amplification product. In some embodiments the amplification product comprises at least 85% monomeric circular nucleic acids. In some embodiments, the amplification product comprises at least 95% monomeric circular nucleic acids. In some embodiments, the method further comprises processing the amplification product to generate a nucleic acid vaccine. In some embodiments, the method further comprises generating a large DNA construct. In some embodiments, the large DNA construct does not comprise extraneous sequences. In some embodiments, the method further comprises generating a gene. In some embodiments, the method further comprises generating a gene cluster. In some embodiments, the method further comprises generating a chromosome. In some embodiments, the method further comprises generating a genome. In some embodiments, the method occurs in vitro. In some embodiments, the method further comprises transfecting a cell free-produced DNA into cells. In some embodiments, the cells are mammalian cells. In some embodiments, the in vitro method is a transcription-translation (TX-TL) system. In some embodiments, the amplification product is used to generate an RNA. In some embodiments, the RNA is used to generate a protein. In some embodiments, the protein is a therapeutic protein. In some embodiments, the RNA is used as an RNA therapeutic. In some embodiments, the RNA therapeutic is formulated as a nanoparticle. In some embodiments, the amplification product is used as a DNA therapeutic. In some embodiments, the DNA therapeutic is formulated as a nanoparticle. In some embodiments, the method occurs in vivo. In some embodiments, the method occurs ex vivo. In some embodiments, the method further comprises barcoding the circular nucleic acid.

[0007] Provided herein are methods for generating an RNA vaccine, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises a recombination site; b) amplifying the nucleic acid template by providing an amplification enzyme and primers for either rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR) amplification to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; and c) generating an RNA vaccine, wherein the method comprises incubating the tandem repeat nucleic acid sequence with: i) a recombination enzyme comprising a recombinase polypeptide; and ii) an accessory factor or cognate resolvase that promotes monomeric resolution.

[0008] Provided herein are methods for generating circular nucleic acids, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises a recombination site; b) amplifying the nucleic acid template to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; c) generating an amplification product; d) providing arecombinase; and e) generating a circularized product comprising at least 85% monomeric circular nucleic acids. In some embodiments, the amplification product comprises linear concatemers. In some embodiments, the method does not comprise use of accessory factors. In some embodiments, the method further comprises use of an accessory factor or a cognate resolvase that promotes monomeric resolution. In some embodiments, the accessory factor that promotes monomeric resolution comprises PepA. In some embodiments, the accessory factor that promotes monomeric resolution comprises Sso7d. In some embodiments, the method further comprises a second accessory factor that promotes monomeric resolution. In some embodiments, the accessory factor that promotes monomeric resolution comprises ArgR, and wherein the second accessory factor that promotes monomeric resolution is PepA. In some embodiments, the recombinase is beta, a beta recombinase peptide, Tn3, a Tn3 recombinase peptide, gamma delta, or a gamma delta recombinase peptide. In some embodiments, the recombinase comprises a serine recombinase. In some embodiments, the serine recombinase is a small serine recombinase. In some embodiments, the small serine recombinase is Cinll or a Cinll polypeptide. In some embodiments, the small serine recombinase is ParA or a ParA polypeptide. In some embodiments, the recombinase comprises a Cre polypeptide. In some embodiments, the recombinase comprises a XerC polypeptide. In some embodiments, the method further comprises a second recombinase comprising a XerD polypeptide. In some embodiments, the nucleic acid template further comprises an expression cassette. In some embodiments, the recombination site comprises an RS1 / RS2 binding site. In some embodiments, the recombination site comprises an MRS binding site. In some embodiments, the recombination site comprises a res binding site. In some embodiments, the amplification comprises rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR). In some embodiments, the method further comprises providing an amplification enzyme. In some embodiments, the amplification enzyme is phi29 DNA polymerase. In some embodiments, the method further comprises sequencing the amplification product or the circularized product. In some embodiments, the sequencing comprises single molecule sequencing. In some embodiments, the sequencing comprises nanopore sequencing. In some embodiments, the method further comprises employing the circularized product in cell-based processes such transformation of bacteria or yeast or transfection of mammalian cells. In some embodiments, the method further comprises adding an endonuclease and / or an exonuclease to remove a residual nicked dsDNA, a residual single stranded DNA, branched DNA, or a residual linear dsDNA from the amplification product or the circularized product. In some embodiments, the circularized product comprises at least 90% monomeric circular nucleic acids. In some embodiments, the circularized product comprises at least 95% monomeric circular nucleic acids. In some embodiments, the methodfurther comprises processing the circularized product to generate a nucleic acid vaccine. In some embodiments, the method further comprises generating a large DNA construct. In some embodiments, the large DNA construct does not comprise extraneous sequences. In some embodiments, the method further comprises generating a gene. In some embodiments, the method further comprises generating a gene cluster. In some embodiments, the method further comprises generating a chromosome. In some embodiments, the method further comprises generating a genome. In some embodiments, the method occurs in vitro. In some embodiments, the method further comprises transfecting the cell free-produced DNA into cells. In some embodiments, the cells are mammalian cells. In some embodiments, the in vitro method is a transcription-translation (TX-TL) method. In some embodiments, the circularized product is used to generate an RNA. In some embodiments, the RNA is used to generate a protein. In some embodiments, the protein is a therapeutic protein. In some embodiments, the RNA is used as an RNA therapeutic. In some embodiments, the RNA therapeutic is formulated as a nanoparticle. In some embodiments, the amplification product is used as a DNA therapeutic. In some embodiments, the DNA therapeutic is formulated as a nanoparticle. In some embodiments, the method occurs in vivo. In some embodiments, the method occurs ex vivo. In some embodiments, the method further comprises barcoding the circularized product.

[0009] Provided herein are methods for generating RNA vaccines, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises a recombination site; b) amplifying the nucleic acid template to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; c) generating an amplification product; d) providing a recombinase; and e) generating an RNA vaccine.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1A depicts an exemplary ColEl plasmid with a ColEl origin site (ColEl cer).

[0011] FIG. IB diagrams an exemplary target site for the XerC / D system, ColEl cer, showing the XerC, XerD, ArgR, and PepA binding sites.

[0012] FIG. 2 shows a DNA gel for CinH recombination in vitro of an MDA / RCA product. A 2.3 monomeric plasmid was produced.

[0013] FIG. 3 shows a plasmid map of SEQ ID NO: 25 with direct repeats of RS2 sites.

[0014] FIG. 4 shows a DNA gel for CinH recombination in vitro. A plasmid with direct repeats of RS2 sites was treated with MBP-CinH at various concentrations (3 nM, 9 nM, and 30 nM) followed by digestion with BaeGl restriction enzyme. The 3 nM MBP-CinH produced 1071 and 1529 base pair (bp) bands, as expected for recombination.DETAILED DESCRIPTION

[0015] The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques, which are within the skill of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

[0016] Cell-free nucleic acid amplification techniques (e.g., isothermal amplification techniques) are used to create high-quality nucleic acids. Isothermal amplification methods typically produce concatemers comprising linear tandem repeat units of input circular nucleic acid template sequences. These tandem repeat sequences can be useful for routine molecular biology experiments such as cloning and sequencing. However, they are seldom used in nucleic acid-based therapeutics because the transformation or transfection efficiencies of these concatemers are often lower than transformation or transfection efficiencies of monomeric sequences. Generation of increased proportions of monomeric sequences using isothermal amplification techniques (e.g., rolling circle amplification or multiple displacement amplification) allows for enhanced use of nucleic acids in subsequent reactions.

[0017] Definitions

[0018] Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when usedin this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0020] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0021] Nucleic Acids

[0022] A nucleic acid is a biopolymer that encodes information in a sequence. A nucleic acid can encompass single-, double-, or triple-stranded nucleic acids, or combinations thereof. A nucleic acid can be but is not limited to a deoxyribonucleic acid (DNA), a single stranded DNA (ssDNA), a double stranded DNA (dsDNA), a ribonucleic acid (RNA), a messenger RNA (mRNA), a transfer RNA, (tRNA), a ribosomal RNA (rRNA), a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a micro RNA, (miRNA), a small nucleolar RNA (snRNA), a long noncoding RNA (IncRNA), a threose nucleic acid (TNA), a glycol nucleic acid (GNA), a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a cyclohexenyl nucleic acids (CeNA), or a chimera or combinations thereof. Nucleic acids can encode functional genes.Alternatively, nucleic acids may not encode functional genes. Nucleic acids can be in a linear conformation. Alternatively, nucleic acids can be in a circular conformation (e.g., a plasmid, a minicircle, etc.). Nucleic acids can be natural nucleic acids. Alternatively or in addition to, nucleic acids can be engineered nucleic acids. Alternatively or in addition to, nucleic acids can be modified nucleic acids. Nucleic acids may also comprise modified nucleotide bases.

[0023] Nucleic acid sequences, also called oligonucleotides or polynucleotides can comprise exon regions, intron regions, intrinsically disordered regions (IDRs), 5’ untranslated regions (UTRs), 3’ UTRs, 5’ caps, poly-adenylation tails, or a combination thereof. Nucleic acid sequences can comprise genes. Alternatively or in addition to, nucleic acid sequences can comprise expression cassettes.

[0024] In some embodiments, nucleic acids can comprise recombination sites. Recombination sites are portions of nucleic acids where the nucleic acid strand can be broken or cut and then joined or ligated with another or the same nucleic acid strand. In some embodiments, a recombination site can be used for interstrand recombination (recombination with another nucleic acid strand). Alternatively or in addition to, a recombination site can be used for intrastrandrecombination (recombination with the same nucleic acid strand, either in the same place as the original cut or at another location along the strand).

[0025] In some embodiments, recombination sites can be naturally occurring (e.g., a part of the cer site in the ColEl origin of replication, or a part of the difm' the E. coli origin of replication, OriC). Naturally occurring recombination sites can include, but are not limited to RS1 / RS2 binding sites, MRS binding sites, res binding sites, ArgR binding sites, PepA binding sites, XerC / D binding sites, LoxP sites, FRT sites, att sites, Chi sites, Cre recombinase binding sites, FLP recombinase binding sites, and site-specific recombinase binding sites.

[0026] A nucleic acid can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more naturally-occurring recombination sites. A nucleic acid can comprise at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2 naturally-occurring recombination sites or one naturally-occurring recombination site.

[0027] Alternatively, nucleic acids can be modified or engineered to comprise a recombination site. A nucleic acid can be modified or engineered to comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more recombination sites. A nucleic acid can be modified or engineered to comprise most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2 recombination sites, or one recombination site.

[0028] Nucleic acids can be used as templates for biological methods. Engineering of a nucleic acid template can be achieved by a variety of genetic engineering or molecular biology techniques. The recombination site can be a homologous recombination site, or it can be a non-homologous recombination site. In some embodiments, the recombination site can be a site-specific recombination site. In some embodiments, multiple site-specific recombination sites can result from repeats (e.g., tandem repeats) of a single site-specific recombination site. In some embodiments, multiple site-specific recombination sites can result from one or more occurrences of two or more different site-specific recombination sites. In some embodiments, the recombination site can be an unspecific recombination site.

[0029] In some embodiments, the multiple site-specific recombination sites in the nucleic acid template can be arranged in a unidirectional (arranged in same orientation, or co-aligned) manner. In some embodiments, some of the multiple site-specific recombination sites in the nucleic acid template can be arranged in a unidirectional manner, while others can be arranged in the opposite orientation. In some embodiments, a site-specific recombination site can be recognized by a unidirectional, site-specific recombination protein (e.g., a recombinase).

[0030] Nucleic Acid Amplification

[0031] Nucleic acids can be amplified to increase the amount of nucleic acid. Nucleic acids can be amplified through reactions such as but not limited to ligase chain reaction, polymerase chain reaction, self-sustained sequence replication, amplification with Qb-replication, and isothermal amplification.

[0032] Isothermal amplification is a group of techniques that use a single temperature reaction to amplify nucleic acids. Types of isothermal amplification include but are not limited to loop- mediated isothermal amplification (LAMP), whole genome amplification, strand displacement amplification, helicase-dependent amplification, recombinase polymerase amplification, nucleic acid sequences-based amplification, rolling circle amplification (RCA), strand displacement amplification, and multiple displacement amplification (MDA).

[0033] In some embodiments, an amplification product can be circular (e.g., a circularized nucleic acid). Alternatively, an amplification product can be linear. In some embodiments, a linear amplification product can be circularized (e.g., to generate a circularized nucleic acid).

[0034] Amplification methods can use reagents such as primers, nucleic acid polymerase, and free nucleotides (for example, deoxyribonucleoside triphosphates (dNTPs)). The nucleic acid polymerase that is employed in the amplification reaction can be a proofreading nucleic acid polymerase. In some embodiments, each of the reagents used in the nucleic acid amplification reaction can be pre-treated to remove any contaminating nucleic acid sequences. In some embodiments, the pre-treatment of the reagents includes incubating the reagents in presence of Ultra-Violet radiation. In some embodiments, the reagents are de-contaminated by incubating the reagents in presence of a nuclease and its co-factor (for example, a metal ion). Suitable nucleases include, but are not limited to, exonucleases such as exonuclease I or exonuclease III. Proofreading DNA polymerases that can be used in a DNA amplification reaction can be de-contaminated by incubating with a divalent metal ion (for example, magnesium or manganese). The free nucleotides employed in nucleic acid template amplification may include natural nucleotides (for example, dATP, dGTP, dCTP or dTTP) or their modified analogues. Other components such as buffers, salts and the like can also be added to allow the nucleic acid amplification to occur efficiently.

[0035] In some embodiments, amplification of the nucleic acid template, and circularization of the amplified nucleic acid template to generate circular nucleic acids (e.g., in a recombination reaction) can be performed in a single vessel. Alternatively, amplification of the nucleic acid template, and circularization of the amplified nucleic acid template to generate circular nucleic acids (e.g., in a recombination reaction) can be performed in separate vessels. The amplification reaction and the recombination reaction can be performed sequentially, or they can be performedsimultaneously. For example, a reaction mixture for nucleic acid amplification can also comprise reagents required for the circularization of amplified nucleic acids. The methods for nucleic acid amplification and generation of circular nucleic acids can either be manually performed or be automated. In some embodiments, some steps of the methods can be manually performed while other steps can be automated.

[0036] Rolling circle amplification is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular nucleic acids. Rolling circle amplification methods can include but are not limited to linear rolling circle amplification, exponential rolling circle amplification, and multiply primed rolling circle amplification. Rolling circle amplification uses an initiator protein to nick a circular nucleic acid. A polymerase enzyme can then initiate nucleic acid synthesis using the circular nucleic acid as a template.

[0037] Amplification methods such as rolling circle amplification can produce nucleic acid concatemers, long continuous nucleic acid molecules that contain multiple copies of the same sequence linked in series, through continuous nucleic acid synthesis. Concatemers can comprise tandem repeat nucleic acids, which can further comprise at least one recombination site. Alternatively, rolling circle amplification can produce nucleic acid monomers, nucleic acid molecules that contain one copy of the desired sequence, through discrete, interrupted nucleic acid synthesis.

[0038] Multiple displacement amplification is a process of amplifying nucleic acids by annealing random short primers (e.g., a hexamer primer) to a template. Multiple displacement amplification can use short random primers which anneal to DNA at multiple sites followed by polymerase-mediated chain elongation. Amplification methods such as multiple displacement amplification can produce nucleic acid concatemers, long continuous nucleic acid molecules that contain multiple copies of the same sequence linked in series, through continuous nucleic acid synthesis. Concatemers can comprise tandem repeat nucleic acids, which can further comprise at least one recombination site. Alternatively, multiple displacement amplification can produce nucleic acid monomers, nucleic acid molecules that contain one copy of the desired sequence, through discrete, interrupted nucleic acid synthesis.

[0039] In some embodiments, nucleic acid amplification can produce concatemers. Alternatively, in some embodiments, nucleic acid amplification can produce monomers. In some embodiments, monomeric nucleic amplification products can be produced by resolving concatemer nucleic acids into monomer nucleic acids. In some embodiments, a template for amplification can be a plasmid or dsDNA comprising a single recombination site but the amplification products can comprise tandem repeats of the recombination site that can be collapsed into monomers by arecombinase (e.g., a resolvase) specific to the recombination site. Alternatively, amplification products can comprise tandem repeats of the recombination site that can be collapsed into monomers by a recombinase that is not specific to the recombination site.

[0040] In some embodiments, the resolution of concatemer amplification products into monomers can produce copies of the nucleic acid template that can themselves be used as a template for further amplification.

[0041] Nucleic acid monomers can be produced by adding recombinases to perform intrastrand recombination. In some embodiments, recombinases are resolvases (e.g., site-specific recombinases). In some cases, recombinases (e.g., resolvases) can excise DNA segments as circles, producing circular nucleic acid monomers. In some cases, recombinases (e.g., resolvases) can bind to recombination sites but are constrained by accessory factors binding nearby recombination sites (e.g., XerC / D acting on a cer plasmid site from ColEl along with ArgR and PepA as accessory factors). Alternatively or in addition to, recombinases (e.g., resolvases) (e.g. small serine recombinases such as CinH or ParA) can bind to recombination sites but are constrained by their own binding as recombinase extended binding sites can include one or more adjacent accessory binding sites recombination sites - these types of recombinases can also be called cognate resolvases.

[0042] Recombinases can include but are not limited to Xer recombinases (e.g., XerA, XerC, XerD, XerC / D, XerH, XerS, etc.), beta recombinases, gamma delta recombinases, Tn3 recombinases, Cre recombinases, Hin recombinases, serine recombinases (e.g., large serine recombinases (e.g., Bxbl, PhiC31) or small serine recombinases (e.g., gamma delta, CinH, ParA)) Tre recombinases, FLP recombinases, Rec Recombinases (e.g., RecA), other recombinases from bacteriophage (e.g., UvsX from bacteriophage T4, Dre (Bacteriophage D6)), CodV / RipX (Bacillus subtilis), Mob (Bacillus cereus group), KD (K. drosophilarum), B2 (Z. bailii), B3 (Z. bisporus), R (Z. rouxii), VCre (Vibrio species), SCre (Shewanella species), and Vika (V. coralliilyticus). In some cases, Cre recombinases can bind to loxP binding sites.

[0043] In some cases, recombinases can be mutated recombinases (e.g., a mutated Cre recombinase). In some embodiments, a L215P recombinase is used. L215P is a mutant Cre recombinases that is constrained by Pep and acts on LoxP sites within the context of the cer site from ColEl.

[0044] In some embodiments, recombinase extended binding sites can comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or more recombination binding site(s). Alternatively, recombinase extended binding sites can comprise at most about 10, at most about 9,at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 recombination binding site(s). In some embodiments, recombinases are not constrained by other recombinase binding sites.

[0045] In some embodiments no accessory factors are needed for the resolution and production of nucleic acid monomers. In some embodiments, accessory factors are not necessary for the resolution and production of nucleic acid monomers because the recombinase (e.g., gamma delta, CinH or ParA) can act as its own accessory factor, as described above.

[0046] In some embodiments, nucleic acid monomers can be produced by adding accessory factors that promote intrastrand recombination over interstrand recombination. Without wishing to be bound by theory, it is believed that accessory factors induce DNA coiling upon binding. Accessory factors can include but are not limited to arginine repressor (ArgR), aminopeptidase A (PepA), other factors which have binding sites proximal to recombinase sites, and other factors which can induce either positive or negative supercoiling upon binding (e.g., gyrase, nucleosome core particles, nucleoid-associated proteins, DNA helicase, DNA polymerase, RNA polymerase, topoisomerase, transcription factors, etc.). Accessory factors can also include Ss07d and other nonsequence specific groove binding proteins. Without wishing to be bound by theory, Sso7d and related factors are believed to induce negative supercoiling in a non-sequence specific manner.

[0047] In some embodiments, a recombinase can be constrained by accessory factors. In some embodiments, a recombinase can be constrained by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or more accessory factor(s). In some embodiments, a recombinase can be constrained by at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 accessory factor(s). Alternatively, a recombinase can be unconstrained by accessory factors. In some embodiments, a recombinase can act as an accessory factor.

[0048] In some embodiments, accessory factors are fused to a XerC recombinase. Alternatively or in addition to, accessory factors can be fused to a XerD recombinase. In some embodiments, the accessory factors are fused to the recombinase at the N terminus. Alternatively, in some embodiments, the accessory factors are fused to the recombinase at the C terminus. Alternatively, in some embodiments, the accessory factors are fused to the recombinase at both the N terminus and the C terminus. In some embodiments, an accessory factor is not fused to a recombinase.

[0049] In some embodiments, only one accessory factor (e.g., ArgR or PepA) is used to promote intrastrand recombination over interstrand recombination to form increased amounts of monomeric nucleic acids. Alternatively, two accessory factors (e.g., ArgR and PepA) are used topromote intrastrand recombination over interstrand recombination to form increased amounts of monomeric nucleic acids. Alternatively, three, four, five, six, seven, eight, nine, ten, or more accessory factors are used to promote intrastrand recombination over interstrand recombination to form increased amounts of monomeric nucleic acids.

[0050] In some embodiments, the accessory factor is a non-sequence specific groove binding protein (e.g., Sso7d). The non-sequence specific groove binding protein can fuse to recombinase polypeptides and form a recombination synapse with an intramolecular recombination bias.

[0051] In some embodiments, recombinases and / or accessory factors bind to specific binding sites. Alternatively, in some embodiments, recombinases and / or accessory factors bind to unspecific binding sites. In some embodiments, a nucleic acid molecule has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more binding sites. In some embodiments, a nucleic acid molecule has at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, or more binding sites. In some embodiments, a nucleic molecule has at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, or at most about 2 binding sites. In some embodiments, there are two binding sites on a nucleic acid molecule.

[0052] In some embodiments, all binding sites are in the same orientation (e.g., a binding site is in the same orientation as all other binding sites on the same molecule). Alternatively, in some embodiments, binding sites are in opposite orientations (e.g., at least one binding site is in an opposite orientation to at least one other binding site on the same molecule).

[0053] Nucleic acid amplification and replication enzymes used in amplification reactions can include but are not limited to polymerases, primases, helicases, ligases, gyrases, and topoisomerases. Amplification reactions of nucleic acids can use polymerase enzymes including but not limited to DNA polymerase I, DNA polymerase I modified with T7 DNA polymerase sequences, Klenow fragment of DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase III holoenzymes, DNA polymerase IV, DNA polymerase V, Pol y, Pol 9, Pol v, Pol a, Pol 5, Pol a, PolPol P, Pol X, Pol p, Pol q, Pol i, Pol K, terminal deoxynucleotidyl transferase, reverse transcriptase, telomerase, RNA polymerase (e.g., RNA polymerase I, RNA polymerase II, RNA polymerase III), RNA replicase, Taq DNA polymerase, EquiPhi29 DNA polymerase, bacteriophage Phi29 DNA polymerase, Phi29-like; polymerases (for example, Phage M2 DNA polymerase, Phage Bl 03 DNA polymerase; or Phage GA-1 DNA polymerase), phage Phi-PRDl polymerase, VENT DNA polymerase, DEEP VENT DNA polymerase, KlenTaq® DNA polymerase, T5 DNA polymerase, T4 DNA polymerase holoenzymes, T7 DNA polymerase; genetically engineered T7 DNA polymerase having reduced or insignificant 3 ' — 5' exonucleaseactivity (e.g., Sequenase DNA polymerase), DNA polymerase form Thermoanaerobacter thermohydrosulfuricus (Tts DNA polymerase), or fragments thereof, modified Tts DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, or TopoTaq DNA polymerase. Additional enzymes can be included in the amplification reaction mixture to minimize misincorporation events. For example, protein mediated error correction enzymes, such as, MutS, can be added to improve the polymerase fidelity either during or following the polymerase reaction. Additional mismatch recognizing endonucleases can be included in the amplification reaction mixture to cleave misincorporation events. Furthermore, additional enzymes can be included in the amplification mixture to open or relax the DNA structure to enhance amplification or enhance intramolecular recombination bias. For example, a helicase can be added to open the DNA and a gyrase or a topoisomerase can be added to relieve supercoiling. Additionally, enzymes to cleave branched DNA such as T7 endonuclease I may be included to reduce the complexity of the reaction product.

[0054] In some example embodiments, Phi29 DNA polymerase and / or Phi29-like polymerase are used for amplifying the circular DNA template (e.g., using a rolling circle amplification or multiple displacement amplification method). Alternatively, a Taq polymerase can be used for amplifying the circular DNA template. In some embodiments, a combination of a Phi29 DNA polymerase and a Taq DNA polymerase can be used for the circular DNA amplification.

[0055] In some embodiments, the polymerases and other enzymes constitute soluble forms of the enzymes. However, solid phase nucleic acid amplification reactions or solid phase recombination reactions may also be employed to streamline the generation of circular nucleic acids. Fusion proteins comprising, optimal regions of different enzymes (e.g., polymerases) that are designed to improve fidelity, efficiency and processing of the final product can be used. Recombinant forms of the enzymes containing one or more affinity tags (e.g., His-tag, S-tag, Calmodulin-binding peptide, maltose binding protein, or Protein A) may also be used. The tags may help in recovering the enzymes, immobilized on a solid matrix through the tag moiety, and can be used in subsequent enzymatic reactions.

[0056] In some embodiments, a method as described herein can produce a circular nucleic acid. A circular nucleic acid can be a plasmid (e.g., a BACS). Alternatively, a circular nucleic acid can be a nucleic acid minicircle (e.g., a DNA minicircle).

[0057] A generated circular nucleic acid can be at least about 100 base pairs (bp), at least about 150 bp, at least about 200 bp, at least about 250 bp, at least about 300 bp, at least about 350 bp, at least about 400 bp, at least about 450 bp, at least about 500 bp, at least about 550 bp, at least about 600 bp, at least about 650 bp, at least about 700 bp, at least about 750 bp, at least about 800 bp, atleast about 850 bp, at least about 900 bp, at least about 950 bp, at least about 1,000 bp, at least about 2,000 bp, at least about 3,000 bp, at least about 4,000 bp, at least about 5,000 bp, at least about 6,000 bp, at least about 7,000 bp, at least about 8,000 bp, at least about 9,000 bp, at least about 10,000 bp, at least about 11,000 bp, at least about 12,000 bp, at least about 13,000 bp, at least about 14,000 bp, at least about 15,000 bp, at least about 16,000 bp, at least about 17,000 bp, at least about 18,000 bp, at least about 19,000 bp, at least about 20,000 bp, at least about 25,000 bp, at least about 30,000 bp, at least about 35,000 bp, at least about 40,000 bp, at least about 45,000 bp, at least about 50,000 bp, at least about 60,000 bp, at least about 70,000 bp, at least about 80,000 bp, at least about 90,000 bp, at least about 100,000 bp, at least about 150,000 bp, at least about 200,000 bp, at least about 250,000 bp, at least about 300,000 bp, at least about 400,000 bp, at least about 500,000 bp, or more base pairs in length.

[0058] A generated circular nucleic acid can be at most about 500,000 bp, at most about 400,000 bp, at most about 300,000 bp, at most about 250,000 bp, at most about 200,000 bp, at most about 150,000 bp, at most about 100,000 bp, at most about 90,000 bp, at most about 80,000 bp, at most about 70,000 bp, at most about 60,000 bp, at most about 50,000 bp, at most about 45,000 bp, at most about 40,000 bp, at most about 35,000 bp, at most about 30,000 bp, at most about 25,000 bp, at most about 20,000 bp, at most about 19,000 bp, at most about 18,000 bp, at most about 17,000 bp, at most about 16,000 bp, at most about 15,000 bp, at most about 14,000 bp, at most about 13,000 bp, at most about 12,000 bp, at most about 11,000 bp, at most about 10,000 bp, at most about 9,000 bp, at most about 8,000 bp, at most about 7,000 bp, at most about 6,000 bp, at most about 5,000 bp, at most about 4,000 bp, at most about 3,000 bp, at most about 2,000 bp, at most about 1,000 bp, at most about 950 bp, at most about 900 bp, at most about 850 bp, at most about 800 bp, at most about 750 bp, at most about 700 bp, at most about 650 bp, at most about 600 bp, at most about 550 bp, at most about 500 bp, at most about 450 bp, at most about 400 bp, at most about 350 bp, at most about 300 bp, at most about 250 bp, at most about 200 bp, at most about 150 bp, at most about 100 bp, or fewer base pairs in length.

[0059] A circular nucleic acid template can comprise a specific nucleic acid sequence. Alternatively or in addition to, a circular nucleic acid template can comprise a random nucleic acid sequence.

[0060] A circular nucleic acid template can be at least about 100 base pairs (bp), at least about 150 bp, at least about 200 bp, at least about 250 bp, at least about 300 bp, at least about 350 bp, at least about 400 bp, at least about 450 bp, at least about 500 bp, at least about 550 bp, at least about 600 bp, at least about 650 bp, at least about 700 bp, at least about 750 bp, at least about 800 bp, at least about 850 bp, at least about 900 bp, at least about 950 bp, at least about 1,000 bp, at leastabout 2,000 bp, at least about 3,000 bp, at least about 4,000 bp, at least about 5,000 bp, at least about 6,000 bp, at least about 7,000 bp, at least about 8,000 bp, at least about 9,000 bp, at least about 10,000 bp, at least about 11,000 bp, at least about 12,000 bp, at least about 13,000 bp, at least about 14,000 bp, at least about 15,000 bp, at least about 16,000 bp, at least about 17,000 bp, at least about 18,000 bp, at least about 19,000 bp, at least about 20,000 bp, at least about 25,000 bp, at least about 30,000 bp, at least about 35,000 bp, at least about 40,000 bp, at least about 45,000 bp, at least about 50,000 bp, at least about 60,000 bp, at least about 70,000 bp, at least about 80,000 bp, at least about 90,000 bp, at least about 100,000 bp, at least about 150,000 bp, at least about 200,000 bp, at least about 250,000 bp, at least about 300,000 bp, at least about 400,000 bp, at least about 500,000 bp, or more base pairs in length.

[0061] A circular nucleic acid template can be at most about 500,000 bp, at most about 400,000 bp, at most about 300,000 bp, at most about 250,000 bp, at most about 200,000 bp, at most about 150,000 bp, at most about 100,000 bp, at most about 90,000 bp, at most about 80,000 bp, at most about 70,000 bp, at most about 60,000 bp, at most about 50,000 bp, at most about 45,000 bp, at most about 40,000 bp, at most about 35,000 bp, at most about 30,000 bp, at most about 25,000 bp, at most about 20,000 bp, at most about 19,000 bp, at most about 18,000 bp, at most about 17,000 bp, at most about 16,000 bp, at most about 15,000 bp, at most about 14,000 bp, at most about 13,000 bp, at most about 12,000 bp, at most about 11,000 bp, at most about 10,000 bp, at most about 9,000 bp, at most about 8,000 bp, at most about 7,000 bp, at most about 6,000 bp, at most about 5,000 bp, at most about 4,000 bp, at most about 3,000 bp, at most about 2,000 bp, at most about 1,000 bp, at most about 950 bp, at most about 900 bp, at most about 850 bp, at most about 800 bp, at most about 750 bp, at most about 700 bp, at most about 650 bp, at most about 600 bp, at most about 550 bp, at most about 500 bp, at most about 450 bp, at most about 400 bp, at most about 350 bp, at most about 300 bp, at most about 250 bp, at most about 200 bp, at most about 150 bp, at most about 100 bp, or fewer base pairs in length.

[0062] A nucleic acid primer can be a specified nucleic acid sequence. Alternatively, a nucleic acid primer can be a randomized sequence. Alternatively, in some examples, hexamer primers are used. In some reactions, one type of nucleic acid primer is used. Alternatively, multiple types of nucleic acid primers are used. For example, in some reactions, multiple random hexamers are used. In some embodiments, primers anneal at room temperature. Alternatively, primers can anneal at temperatures greater than 30°C. In some embodiments, one or more primers and template are combined, heat denatured, and allowed to anneal by reducing the temperature.

[0063] A nucleic acid primer can be at least about 2 bp, at least about 3 bp, at least about 4 bp, at least about 5 bp, at least about 6 bp, at least about 7 bp, at least about 8 bp, at least about 9 bp, atleast about 10 bp, at least about 11 bp, at least about 12 bp, at least about 13 bp, at least about 14 bp, at least about 15 bp, at least about 16 bp, at least about 17 bp, at least about 18 bp, at least about 19 bp, at least about 20 bp, at least about 21 bp, at least about 22 bp, at least about 23 bp, at least about 24 bp, at least about 25 bp, at least about 26 bp, at least about 27 bp, at least about 28 bp, at least about 29 bp, at least about 30 bp, at least about 31 bp, at least about 32 bp, at least about 33 bp, at least about 34 bp, at least about 35 bp, at least about 36 bp, at least about 37 bp, at least about 38 bp, at least about 39 bp, at least about 40 bp, at least about 41 bp, at least about 42 bp, at least about 43 bp, at least about 44 bp, at least about 45 bp, at least about 46 bp, at least about 47 bp, at least about 48 bp, at least about 49 bp, at least about 50 bp, at least about 55 bp, at least about 60 bp, at least about 65 bp, at least about 70 bp, at least about 75 bp, at least about 80 bp, at least about 85 bp, at least about 90 bp, at least about 95 bp, at least about 100 bp, at least about 105 bp, at least about 110 bp, at least about 115 bp, at least about 120 bp, at least about 125 bp, at least about 130 bp, at least about 135 bp, at least about 140 bp, at least about 145 bp, at least about 150 bp, at least about 160 bp, at least about 170 bp, at least about 180 bp, at least about 190 bp, at least about 200 bp, at least about 210 bp, at least about 220 bp, at least about 230 bp, at least about 240 bp, at least about 250 bp or more in length.

[0064] A nucleic acid primer can be at most about 250 bp, at most about 240 bp, at most about 230 bp, at most about 220 bp, at most about 210 bp, at most about 200 bp, at most about 190 bp, at most about 180 bp, at most about 170 bp, at most about 160 bp, at most about 150 bp, at most about 145 bp, at most about 140 bp, at most about 135 bp, at most about 130 bp, at most about 125 bp, at most about 120 bp, at most about 115 bp, at most about 110 bp, at most about 105 bp, at most about 100 bp, at most about 95 bp, at most about 90 bp, at most about 85 bp, at most about 80 bp, at most about 75 bp, at most about 70 bp, at most about 65 bp, at most about 60 bp, at most about 55 bp, at most about 50 bp, at most about 49 bp, at most about 48 bp, at most about 47 bp, at most about 46 bp, at most about 45 bp, at most about 44 bp, at most about 43 bp, at most about 42 bp, at most about 41 bp, at most about 40 bp, at most about 39 bp, at most about 38 bp, at most about 37 bp, at most about 36 bp, at most about 35 bp, at most about 34 bp, at most about 33 bp, at most about 32 bp, at most about 31 bp, at most about 30 bp, at most about 29 bp, at most about 28 bp, at most about 27 bp, at most about 26 bp, at most about 25 bp, at most about 24 bp, at most about 23 bp, at most about 22 bp, at most about 21 bp, at most about 20 bp, at most about 19 bp, at most about 18 bp, at most about 17 bp, at most about 16 bp, at most about 15 bp, at most about 14 bp, at most about 13 bp, at most about 12 bp, at most about 11 bp, at most about 10 bp, at most about 9 bp, at most about 8 bp, at most about 7 bp, at most about 6 bp, at most about 5 bp, at most about 4 bp, at most about 3 bp, or at most about 2 bp in length.

[0065] An amplification reaction as described herein can require at least about 0.5 picograms (pg), at least about 1 pg, at least about 10 pg, at least about 50 pg, at least about 100 pg, at least about 500 pg, at least about 1,000 pg, or more of circular template nucleic acid (e.g., DNA) that contains a recombinase (e.g., a XerC / D recombinase) site. Alternatively, an amplification reaction as described herein can requires at least about 0.5 picograms (pg), at least about 1 pg, at least about 10 pg, at least about 50 pg, at least about 100 pg, at least about 500 pg, at least about 1,000 pg, or more of linear DNA flanked by a recombinase (e.g., a XerC / D recombinase) site.

[0066] An amplification reaction as described herein can require at most about 1,000 pg, at most about 500 pg, at most about 100 pg, at most about 50 pg, at most about 10 pg, at most about 1 pg, at most about 0.5 pg or less of circular template nucleic acid (e.g., DNA) that contains a recombinase (e.g., a XerC / D recombinase) site. Alternatively, an amplification reaction as described herein can require at most about 1,000 pg, at most about 500 pg, at most about 100 pg, at most about 50 pg, at most about 10 pg, at most about 1 pg, at most about 0.5 pg or less of linear DNA flanked by a recombinase (e.g., a XerC / D recombinase) site.

[0067] Nucleic acid amplification can be performed in vivo. Alternatively, nucleic acid amplification can be performed in ex vivo. Alternatively, nucleic acid amplification can be performed in vitro (e.g., cell-free amplification, transcription-translation (TX-TL) systems, etc.).

[0068] Amplified nucleic acids can have at least about 80%, at least about 82%, at least about84%, at least about 86%, at least about 88%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or 100% sequence identity to the original nucleic acid sequence.

[0069] In some embodiments, methods as provided herein can produce generated nucleic acids. The generated nucleic acids as described herein can comprise concatemers. Alternatively or in addition to, the generated nucleic acids as described herein can comprise monomers. In some cases, the generated nucleic acids can be a mixture of concatemers and monomers. In some cases, the generated nucleic acids can be comprised of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or more monomers. In some embodiments, such monomers can be circular nucleic acids. Alternatively, such monomers can be linear nucleic acids.

[0070] In some cases, amplification reactions or other methods as described herein that use accessory factors can produce at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 1,500%, at least about 2,000%, at least about 5,000%, at least about 10,000% or more monomers than amplification reactions that do not use accessory factors.

[0071] In some cases, amplification reactions or other methods as described herein that use recombinases can produce at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 1,500%, at least about 2,000%, at least about 5,000%, at least about 10,000% or more monomers than amplification reactions that do not use recombinases.

[0072] Output nucleic acid concentrations from amplification can be at least about 0.0001 pg / pL, at least about 0.001 pg / pL, at least about 0.01 pg / pL, at least about 0.05 pg / pL, at least about 0.1 pg / pL, at least about 0.5 pg / pL, at least about 1 pg / pL, at least about 2 pg / pL, at least about 3 pg / pL, at least about 4 pg / pL, at least about 5 pg / pL, at least about 6 pg / pL, at least about 7 pg / pL, at least about 8 pg / pL, at least about 9 pg / pL, at least about 10 pg / pL, at least about 11 pg / pL, at least about 12 pg / pL, at least about 13 pg / pL, at least about 14 pg / pL, at least about 15 pg / pL, at least about 16 pg / pL, at least about 17 pg / pL, at least about 18 pg / pL, at least about 19 pg / pL, at least about 20 pg / pL, or more. Output nucleic acid concentrations can be at most about 20 pg / pL, at most about 19 pg / pL, at most about 18 pg / pL, at most about 17 pg / pL, at most about 16 pg / pL, at most about 15 pg / pL, at most about 14 pg / pL, at most about 13 pg / pL, at most about 12 pg / pL, at most about 11 pg / pL, at most about 10 pg / pL, at most about 9 pg / pL, at most about 8 pg / pL, at most about 7 pg / pL, at most about 6 pg / pL, at most about 5 pg / pL, at most about 4 pg / pL, at most about 3 pg / pL, at most about 2 pg / pL, at most about 1 pg / pL, at most about 0.5 pg / pL, at most about 0.1 pg / pL, at most about 0.05 pg / pL, at most about 0.01 pg / pL, at most about 0.001 pg / pL, at most about 0.0001 pg / pL, or less.

[0073] Amplified nucleic acid sequences can be further processed. One or more of the methods may further comprise steps of purifying, analyzing and / or quantifying the generated nucleic acids (e.g., circular nucleic acids, mini-circles, etc.). Any suitable techniques that are used for purification, analysis or quantification of nucleic acids can be employed. Non-limiting examplesinclude, filtration, affinity capture, gel electrophoresis, sequencing or HPLC analysis. For example, the purification of the circular nucleic acids can be achieved by affinity capture.

[0074] In some cases, nucleic acid sequences produced by the methods as described herein can be sequenced (e.g., single molecule sequencing, nanopore sequencing, whole-genome sequencing, targeting sequencing, RNA sequencing, DNA sequencing, methylation sequencing, etc.). In some cases, amplified nucleic acids can be further modified by adding exonucleases and / or endonucleases. Adding exonucleases and / or endonucleases can be used to remove residual nicked nucleic acids, to remove residual single-stranded DNA, to remove residual branched nucleic acids (e.g., branched DNA), or to remove residual linear double-stranded DNA (dsDNA). Exonucleases can include but are not limited to T5 exonuclease, T7 exonuclease, exonuclease I, exonuclease III, and lambda exonuclease. Endonucleases can include but are not limited to SI endonuclease, Pl endonuclease, FLAP endonucleases (e.g., FEN1), mung bean nucleases, and BAL 31 nuclease. In some cases, generated nucleic acids (e.g., circular nucleic acids) can be barcoded.

[0075] In some cases, amplified nucleic acids can be transformed or transfected into cells. Amplified nucleic acids can be transformed into bacteria or yeast or transfected into mammalian cells. Non-limiting examples of transformation, transfection, or transduction methods include viral transaction methods, non-viral transfection methods (e.g., cationic lipid-based nucleic acid transfection, polymer-based transfection), electroporation, or transformation via heat shock. Nucleic acids that have been transformed into cells can further be used for additional cell -based processing. In some embodiments, nucleic acids described herein (such as amplified nucleic acids) can be used in transcription-translation (TX-TL) methods.

[0076] Compositions comprising circular nucleic acids generated by the methods of one or more of the embodiments can comprise the generated circular nucleic acids alone, or their derivatives that are produced by post-processing of the circular nucleic acids, or a combination. In some embodiments, the circular nucleic acid of the composition can comprise an expression cassette. In some embodiments, the expression cassette can be a eukaryotic expression cassette. The expression cassette can comprise a sequence of interest, for example, a gene. In some embodiments, the expression cassette comprises a gene that encodes a polypeptide a polypeptide, which can generate an immune response in a host (e.g., human). For example, in some embodiments, the gene can encode an antigenic or immunogenic protein.

[0077] In some cases, the generated nucleic acids can be further used to generate large DNA constructs. In some cases, the large DNA constructs comprise extraneous sequences. In alternative cases, large DNA constructs do not comprise extraneous sequences. In some cases, the generated nucleic acids can be further used to generate genes. In some cases, the generated nucleic acids canbe further used to generate gene clusters. In some cases, the generated nucleic acids can be further used to generate chromosomes. In some cases, the generated nucleic acids can be further used to generate genomes.

[0078] In some cases, the generated nucleic acids, amplification products, or other nucleic acids as described herein can be used to generate RNA. In some cases, the generated nucleic acids and / or generated RNA can be used to generate proteins. In some cases, proteins can be therapeutic proteins. In some cases, a generated RNA can be used as an RNA therapeutic (e.g., a vaccine). In some cases, the RNA can be an mRNA. In some cases, the RNA (e.g., an mRNA) can be transfected or therapeutically administered in a buffer. Alternatively or in addition to, the RNA (e.g., an mRNA) can be formulated as a nanoparticle (e.g., a lipid nanoparticle (LNP)) and transfected into cells or administered to subjects. A DNA template for RNA transcription can encode a polyA tail, resulting in a polyA tail in the transcribed RNA. A DNA template for RNA can alternatively or additionally encode an IRES sequence, resulting in a transcribed RNA that can be translated in a cap-independent manner.

[0079] In some cases, the generated nucleic acids, amplification products, or other nucleic acids as described herein can be used as a DNA therapeutic. A DNA therapeutic can be formulated as a nanoparticle (e.g., a lipid nanoparticle (LNP)) and transfected into cells or administered to subjects.

[0080] In some embodiments, the methods as described herein can be performed in vivo. Alternatively, the methods as described herein can be performed in vitro. Alternatively, the methods as described herein can be performed ex vivo.

[0081] Vaccinations

[0082] An amplified nucleic acid composition, as described herein, can be used to produce a nucleic acid vaccination. Vaccinations are medications that allow a patient to gain immunity to disease. A vaccine can comprise a nucleic acid as an active ingredient. A nucleic acid vaccine can be a DNA vaccine. A nucleic acid vaccine can alternatively be an RNA vaccine.

[0083] A nucleic acid vaccine can comprise a nucleic acid encapsulated in a lipid nanoparticle. A lipid nanoparticle can comprise a cationic lipid (e.g., an ionizable lipid), a PEG-modified lipid, a sterol (e.g., cholesterol), a non-cationic lipid, or a combination thereof. A nucleic acid vaccine can comprise a viral vector such as an adeno-associated viral vector (AAV) or a vaccina viral vector.

[0084] A nucleic acid vaccine composition can comprise an excipient, a carrier, an adjuvant, an additive, a diluent, a stabilizer, a solubilizer, or combinations thereof.

[0085] A nucleic acid vaccine can treat a disease such as but not limited to Alzheimer’s disease, arthritis, encephalitis, asthma, cancer, rhinovirus, coronavirus, influenza, dengue, varicella, diphtheria, Ebola, hepatitis, HIV / AIDS, measles, papillomavirus, mumps, pneumococcal diseases,norovirus, polio, rotavirus, rabis, respiratory syncytial virus, tetanus, herpes viruses, rubella, or zika virus.

[0086] A nucleic acid vaccine can be administered through injection. Alternatively, a nucleic acid vaccine can be administered orally. A nucleic acid vaccine can be administered in a single dose. Alternatively, a nucleic acid vaccine can be administered in 2 doses, 3 doses, 4 doses or 5 doses.

[0087] A nucleic acid vaccine can immunize a patient from a disease for at least about 6 months, for at least about 1 year, for at least about 2 years, for at least about 3 years, for at least about 4 years, for at least about 5 years, for at least about 6 years, for at least about 7 years, for at least about 8 years, for at least about 9 years, for at least about 10 years, for at least about 11 years, for at least about 12 years, for at least about 13 years, for at least about 14 years, for at least about 15 years, for at least about 20 years, for at least about 25 years, for at least about 30 years, or more.

[0088] Kits

[0089] In some embodiments, a kit for generating circular nucleic acids in a cell-free system comprises reagents that are required for generating circular nucleic acid using the methods described herein. In some embodiments, the kit comprises a nucleic acid polymerase, a recombination protein, and an accessory factor. The nucleic acid polymerase in the kit is capable of amplifying a nucleic acid template to generate monomeric nucleic acid sequences.EXAMPLES

[0090] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

[0091] Example 1: Rolling Circle Amplification

[0092] Circular DNA targets are amplified using rolling circle amplification with phi29 DNA Polymerase and plasmids with a ColEl type origin of replication (ORI). Recombinase XerC / D and accessory factors ArgR and PepA are added to promote monomer resolution of the concatemer product.

[0093] The template is formed by taking plasmids with a ColEl ORI, which contains XerC / D, ArgR, and PepA binding sites. In other experiments, the template is formed using a DNA minicircle that contains the binding sites for XerC / D, ArgR, and PepA. In a third set of experiments, a linear template is used in which recombinase binding sites are repeated on both endsof the DNA such that the recombination of the two origins leads to a circular plasmid / BAC equivalent. For the linear template, only one end requires the ArgR and PepA sites as the sites distal to the XerC / D site are cleaved in the XerC / D reaction.

[0094] Priming is performed with random hexamers. In other experiments, priming is performed with specific primers that both promote rolling circle amplification and also amplify the reverse strand. The primers are modified to be 3’ exonuclease resistant to protect them from the 3’ exonuclease activity of the phi29 polymerase. Additionally, the primers are modified to be 5’ exonuclease resistant to protect the product in later steps.

[0095] The phi29 DNA polymerase is added for amplification of the product and the two polypeptides that form the XerC / D recombinase form the recombination enzyme that binds variants of its own recombinase site to variants of the recombinase site in the origins of the E. coli genome and in plasmids.

[0096] Accessory factors and their binding sites which are proximal to the XerC / D site promote monomeric resolution by XerC / D by favoring intrastrand rather than interstrand recombination. In some experiments, the accessory factor is ArgR. In some experiments, the accessory factor is PepA. In some experiments, both the ArgR and PepA accessory factors are used.

[0097] T5 Exonuclease is used to clean up single-stranded DNA and recombinant DNA. In some experiments, a single stranded exonuclease (e.g., SI, Pl or mug bean endonuclease) is used for clean-up.

[0098] Example 2: Rolling Circle Amplification

[0099] Circular DNA targets are amplified using rolling circle amplification with phi29 DNA Polymerase and plasmids with a XerC / D recombination site. Recombinase XerC / D and accessory factor Sso7d are added to promote monomer resolution of the concatemer product.

[0100] Priming is performed with specific primers that both promoted rolling circle amplification and also amplified the reverse strand. The primers are modified to be 3’ exonuclease resistant to protect them from the 3’ exonuclease activity of the phi29 polymerase. Additionally, the primers are modified to be 5’ exonuclease resistant to protect the product in later steps.

[0101] The phi29 DNA polymerase is added for amplification of the product and the two polypeptides that form the XerC / D recombinase form the recombination enzyme that binds variants of its own recombinase site to variants of the recombinase site in the origins of the E. coli genome and in plasmids. The accessory factor Sso7d promotes monomeric resolution by XerC / D by favoring intrastrand rather than interstrand recombination.

[0102] T5 Exonuclease is used to clean up single-stranded DNA and recombinant DNA. In some experiments, a single stranded exonuclease (e.g., SI, Pl or mug bean endonuclease) is additionally used for clean-up.

[0103] Example 3: Cell Free Rolling Circle Replication

[0104] The rolling circle replication (RCR) reaction provides a convenient cell free process to amplify DNA from 0.3 kilobases (kb) to 200 kb in size. The output DNA is high fidelity, monomeric and circular.

[0105] One picogram (pg) of circular DNA containing a XerC / D recombinase site or linear DNA flanked by XerC / D recombinase sites is used as template. The template uses a XerC / D recombinase site from the cer site in ColEl origin containing plasmids. Circular assembly reaction products are amplified directly in a cell-free reaction.

[0106] The lOx reaction buffer is thawed on ice and the enzyme mix is transferred from -20°C to ice. The template is thawed as a 0.1-1 nanogram (ng) solution of circular DNA containing a XerC / D recombinase site in a completed Gibson Assembly, or in a ligation reaction containing DNA with a recombinase similar to that of XerC / D. The lOx reaction buffer mix is vortexed immediately prior to use. The enzyme mixture is also briefly vortexed prior to use.

[0107] In a PCR tube, 5 microliters (pL) of lOx reaction buffer, 5uL of enzyme mixture, and up to 30pL of the template are added together. Water is added to create a final reaction volume of 50pL. The reaction is mixed by vortexing or pipetting and then spun down. The resulting reaction is incubated under the following conditions: amplification at 37°C for five hours and heat inactivation at 85°C for twenty minutes.

[0108] In some experiments, endonucleases and / or exonucleases are added to remove residual nicked, single-stranded, or linear double stranded DNA (dsDNA). For this, 10 units of mung bean nuclease and 10,000 units of exonuclease 1 are added and incubated at 37°C for 30 minutes. The resulting mixture is extracted with phenol and chloroform to inactivate the nucleases. After the incubations are complete, the reactions are stored at -20°C.

[0109] Output DNA concentrations are from 0. Ipg / pl to lOug / pl. The output DNA is subsequently used in sequencing and enzymatic processes such as RNA transcription and molecular biology methods like restriction digestion and cloning. The output DNA is also used for subsequent cell-based processes including transformation of bacteria and yeast and transfection of mammalian cells.

[0110] Example 4: Rolling Circle Replication Producing Monomeric Circles

[0111] The amplification reaction is based on RCA / MDA (using phi29 DNA polymerase) combined with XerC and XerD polypeptides to form the XerC / D recombinase. Either randomhexamers or longer, sequence-specific primers that anneal at >30°C are used to prime the reaction. An alternative amplification enzyme system using a jumbo bacteriophage is used for products over 35-40 kb.

[0112] Accessory factors are included to bias the recombination reaction towards intramolecular recombination over intermolecular recombination such that monomers are produced from multimeric circular templates. The accessory factors are polypeptides that interact with XerC, XerD, or both. Accessory factors, ArgR and Pep A, bind to binding sites on E. coh- Qv \rQ ColEl and pSClOl plasmids at a ~180bp site immediately adjacent to the XerC / D binding site in the origin.

[0113] Rolling circle replication proceeds as described in Example 3.

[0114] Example 5: Rolling Circle Replication Producing Monomeric Circles

[0115] The amplification reaction is based on RCA / MDA (using phi29 DNA polymerase) combined with XerC and XerD polypeptides to form the XerC / D recombinase. Either random hexamers or longer, sequence-specific primers that anneal at >30°C are used to prime the reaction.

[0116] A non-sequence specific groove binding protein, Sso7d, is fused to one or both of the recombinase polypeptides at either their N or C termini or both and leads to a formation of a recombination synapse with the desired intramolecular recombination bias.

[0117] Rolling circle replication proceeds as described in Example 3.

[0118] Example 6: Genome Construction

[0119] DNA Oligos to DNA Fragments

[0120] Full length oligonucleotide selection is used in oligonucleotide assembly of gene fragments that are synthesized by phosphonamidite chemistry. Phosphonamidite chemistry synthesizes from 3’ to 5’ direction, so the truncations occur on the 5’ strand. Oligo assembly methods are used to overlap and anneal in a tiling pattern such that a gene fragment is assembled from short oligos. If the oligo is sufficiently full length to bind other oligos or the growing amplicon, the 3’ is intact and is also extended.

[0121] Enzymatic selection of full length oligos allows efficient and accurate ligation based oligo assembly methods. Selection is accomplished by phosphorothioate modifying the 5’ of the oligos and using the modified oligos directly in an assembly with an exonuclease present.

[0122] Removal of oligo tails is accomplished by taking oligos modified with uracil and cleaving them to a sequence specific form when incubated with an uracil removing enzyme system comprised of Uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII. Cleavage results in a 5’ phosphorylation that facilitates downstream ligation. Oligos with non-templated tails form a 5’ flap when hybridized to template DNA or oligos. The 5’ flap is used as a substrate forand then removed by a flap endonuclease such as FEN1. Cleavage results in a 5’ phosphorylation that would facilitate downstream ligation.

[0123] DNA Fragments to DNA Constructs

[0124] Flap assembly uses ligation combined with FEN1. Ligation chain reaction (LCR) produces large and accurate DNA assemblies if DNA fragments have accurate end sequences. Perfectly prepared ends are used to create double-stranded products produced without nicks, gaps, or overlaps. Flap assembly uses a bridging oligo to hybridize to the ends or interior sites of DNA fragments to bring them together after denaturation and hybridization of the DNA and oligo(s). The resulting synapse(s) have a three stranded structure with one strand of DNA ligatable with DNA flaps on the other strand. The inclusion of FEN1 enzyme results in the systematic removal of the flaps, especially if the phosphorylation of the 5’ ends of the oligos or DNA fragments are designed to control the order of ligation or the 3’ specific sequence of the oligo is longer than the 5’ specific sequence. The reaction can be performed in one step as an isothermal reaction or thermocycled with a thermostable ligase and thermostable FEN1.

[0125] DNA fragments with homology at their ends or blunt ended DNA products bridged by an oligo with homology to both ends are ligated so that one strand is ligated while the other strands have DNA flaps. FEN1 systemically removes 5’ non-hybridized flaps until only a nick in one strand remains that is ligated.

[0126] Construct to Amplified Construct

[0127] Rolling circle amplification is performed as described in Example 1.

[0128] Construct Amplification to Adeno-associated Virus (AA V) Particles

[0129] AAV genomes are transfected into HEK293 cells and complemented with rep and cap genes either by co-transfection or as stably expressed genes in the cells. AAV capsid proteins from the single cap gene are VP1, VP2, and VP3. These are produced as a lysate or purified in mammalian or procaryotic cell systems and mixed with AAV genome at specific ratios. AAV capsid pseudotyping is facilitated by the modularity of the system. Rep proteins from the single rep gene (Rep78, Rep68, Rep52 and Rep40) are included as necessary and viral maturity and yield are demonstrated by the size of the virus particles, infectivity, and resistance to nucleases. Additional AAV derived factors such as AAP (Assembly-Activating Protein) or MAAP (Membrane-associated AAV Protein) are included either in the cell lysate or as purified proteins. Additionally, host factors such as Nucleolin, replication factor C, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), and high mobility group protein 1 are included in the cell lysate or as purified proteins. In some experiments, Adenovirus-derived proteins El A and E1B, E2A, E4, and VA are also included in the cell lysate or as purified proteins.

[0130] Example 7: Rolling Circle Amplification with CinH

[0131] In this example, a two-step MDA / RCA reaction was followed by de-concatenation and circularization using CinH.

[0132] A 50 pl MDA / RCA reaction contained 33.5 ng of SEQ ID NO: 1 plasmid as template, 5 pM of SEQ ID NO: 9 as primer, 5 pM of SEQ ID NO: 10 as primer, 50 mM Tris-HCl, 10 mM MgCh, 10 mM (NH4)2SO4, 4 mM DTT, 10 units of NEB Phi29 DNA polymerase, 10 pg of NEB Recombinant Albumin, 2.5 mM dNTPs, and water as needed. The reaction was incubated at 30°C for 8 hours and heat inactivated at 65 °C for 10 minutes. The SEQ ID NO: 1 plasmid used for template contained a single RS2 site (SEQ ID NO: 5) for CinH.

[0133] A 50 pl de-concatenation / circularization reaction contained 8 pls of the above MDA / RCA reaction product, 33 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 5 mM (NH4)2SO4, 2 mM DTT, 0.5 mM NAD+, 0.1 mM ATP, 0.1 units of T7 endonuclease I from NEB, 3 nM of MBP-CinH (SEQ ID NO: 20), and water as needed. The reaction was incubated at 30°C for 8 hours and heat inactivated at 65 °C for 10 minutes. The reaction was incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65°C for 10 minutes. Reactions were visualized by loading 25ul of DNA onto a 1.2% agarose gel with Tris-borate-EDTA buffer, a Ikb ladder (NEB) and Gel Green Stain (Biotium) (FIG. 2).

[0134] As shown in FIG. 2, an approximately 2.3 kb monomeric plasmid was produced, which matched the template (SEQ ID NO: 1).

[0135] Example 8: Plasmid Conversion with CinH

[0136] In this example, a plasmid containing two recombination cites was converted into two circles by CinH.

[0137] A 50 pl reaction contained 810 ng of a plasmid with dual RS2 sites (SEQ ID NO: 5) in the same orientation (SEQ ID NO: 25, FIG. 3), 33 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 5 mM (NH4)2SO4, 2 mM DTT, 0.5 mM NAD+, 0.1 mM ATP, either 3 nM, 9 nM, or 30 nM of MBP- CinH (SEQ ID NO: 20), and water as needed. The reaction was incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The reaction was incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65°C for 10 minutes.

[0138] Restriction digests were performed by combining 20 pls of the above reaction with 10 units of NEB BaeGl, 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 100 pg / ml Recombinant Albumin, and water to make a 50 pl reaction. Reactions were incubated at 37°C for 2 hours. Reactions were visualized by loading 24 pl of DNA onto a 1.5% agarose gel with Tris-borate- EDTA buffer with a Ikb ladder (NEB) and Gel Green Stain (Biotium). The parent vector was 2.6 kb (SEQ ID NO: 25, FIG. 3). Negative recombination would be expected to produce bands of 640and 1960 bp. Recombination followed by BaeGl digestion would be expected to produce 1071 and 1529 bp bands, as demonstrated in the second lane with 3nM MBP-CinH (FIG. 4).

[0139] Example 9: Rolling Circle Amplification with ParA

[0140] In this experiment, a two-step MDA / RCA reaction occurs, followed by deconcatenation and circularization with ParA.

[0141] A 50 pl MDA / RCA reaction contains 33.5 ng of SEQ ID NO: 2 plasmid as template, 5 pM of SEQ ID NO: 9 as primer, 5 pM of SEQ ID NO: 10 as primer, 50 mM Tris-HCl, 10 mM MgCh, 10 mM (NH4)2SO4, 4 mM DTT, 10 units of NEB Phi29 DNA polymerase, 10 pg of NEB Recombinant Albumin, 2.5 mM dNTPs, and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The SEQ ID NO: 2 plasmid used for template contains a single MRS site (SEQ ID NO: 6) for ParA.

[0142] A 50 pl de-concatenation / circularization reaction contains 8 pls of the above MDA / RCA reaction product, 33 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 5 mM (NH4)2SO4, 2 mM DTT, 0.5 mM NAD+, 0.1 mM ATP, 0.1 units of T7 endonuclease I from NEB, 24 nM of MBP-ParA (SEQ ID NO: 21), and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The reaction is incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65°C for 10 minutes.

[0143] Reactions are visualized by loading 25 pl of DNA onto a 1.2% agarose gel with Tris- borate-EDTA buffer.

[0144] Example 10: Rolling Circle Amplification with CreL215P, PepA and ArgR

[0145] In this experiment, a two-step MDA / RCA reaction occurs, followed by deconcatenation and circularization with Cre L215P, PepA, and ArgR.

[0146] A 50 pl MDA / RCA reaction contains 33.5 ng of SEQ ID NO: 3 plasmid as template, 5 pM of SEQ ID NO: 9 as primer, 5 pM of SEQ ID NO: 10 as primer, 50 mM Tris-HCl, 10 mM MgC12, 10 mM (NH4)2SO4, 4 mM DTT, 10 units of NEB Phi29 DNA polymerase, 10 ug of NEB Recombinant Albumin, 2.5mM dNTPs, and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The SEQ ID NO: 3 plasmid used for template contains a single, hybrid cer+LoxP site (SEQ ID NO: 7) for Cre L215P.

[0147] A 50 pl de-concatenation / circularization reaction contains 8 pls of the above MDA / RCA reaction, 33 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 5 mM (NH4)2SO4, 2 mM DTT, 0.5 mM NAD+, 0.1 mM ATP, 0.1 units of T7 endonuclease I from NEB, 6 nM of MBP-Cre L215P (SEQ ID NO: 22), 33 nM of ArgR-histag (SEQ ID NO: 19), 6.4 nM of histag-PepA (SEQ ID NO: 18), ImM L-Arginine, and water as needed. The reaction is incubated at 30°C for 8 hoursand heat inactivated at 65°C for 10 minutes. The reaction is incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65°C for 10 minutes.

[0148] Reactions are visualized by loading 25 pl of DNA onto a 1.2% agarose gel with Tris- borate-EDTA buffer.

[0149] Example 11: Rolling Circle Amplification with XerC, XerD, PepA, and ArgR

[0150] In this experiment, a two-step MDA / RCA reaction occurs, followed by deconcatenation and circularization with XerC, XerD, PepA, and ArgR.

[0151] A 50 pl MDA / RCA reaction contains 33.5 ng of SEQ ID NO: 4 plasmid as template, 5uM of SEQ ID NO: 9 as primer, 5uM of SEQ ID NO: 10 as primer, 50 mM Tris-HCl, 10 mM MgCh, 10 mM (NH4)2SO4, 4 mM DTT, 10 units of NEB Phi29 DNA polymerase, 10 ug of NEB Recombinant Albumin, 2.5mM dNTPs, and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The SEQ ID NO: 4 plasmid used for template contains a single cer site (SEQ ID NO: 8) for XerC / D.

[0152] A 50 pl de-concatenation / circularization reaction contains 8 pls of the aboveMDA / RCA reaction product, 33 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 5 mM (NH4)2SO4, 2 mM DTT, 0.5 mM NAD+, 0.1 mM ATP, 0.1 units of T7 endonuclease I from NEB, 5 nM of MBP- XerC (SEQ ID NO: 23), 4 nM of MBP-XerD (SEQ ID NO: 24), 33 nM of ArgR-histag (SEQ ID NO: 19), 6.4 nM of histag-PepA (SEQ ID NO: 18), ImM L-Arginine, and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The reaction is incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65°C for 10 minutes.

[0153] Reactions are visualized by loading 25 pl of DNA onto a 1.2% agarose gel with Tris- borate-EDTA buffer.

[0154] Example 12: Protein Purification

[0155] Histidine-Tagged Proteins

[0156] Histidine tagged proteins are expressed via pET 28, either as n-terminal fusions (ArgR, SEQ ID NO: 12 and SEQ ID NO: 19) or as c-terminal fusions (PepA, SEQ ID NO: 11, and SEQ ID NO: 18).Constructs are grown in NEB T7 Express cells with 50 pg / ml of kanamycin at 37°C, 250 RPM until they reached ODsoo:=:0.6-0. . IPTG is added to 250 pM and the cultures are further incubated at 16°C, 250 RPM for 16-20 hours. Cells are centrifuged at 1000 x G, 4°C for 20 minutes and the pellet is stored at -8°0C until lysis. The pellet is resuspended in 10 mis of lysis buffer for each pellet and incubated at room temperature for 5 minutes. Sonication is done on ice at level 5 (on for 10 seconds, off for 50 seconds x 8 cycles). Cellular debris is pelleted by centrifugation at 15,000 RPM for 30 minutes, 4°C. Ni-NTA resin (5 mis, 50% slurry) is added to the cleared lysate in a 15 mb falcon tube with gentle mixing by shaking at 200 RPM at 4°C for 60 minutes. The Ni-NTA-lysate mixture is transferred into a column and washed twice with 10 mb of equilibration buffer. The protein was eluted four times with 2 mb of four different elution buffers (buffers 1-4, respectively). Elution samples that are positive for the correct size protein are buffer exchanged into storage buffer using Amicon filter 50 kDa MWCO. Protein concentrations are determined by a Bradford assay.Table 1: Histidine-Tagged Protein Purification Buffers

[0157] Maltose-Binding Protein (MBP)-Tagged Proteins

[0158] MBP-tagged proteins are expressed via pMAL-c6T as c-terminal fusions (CinH, SEQ ID NO: 13 and SEQ ID NO: 20; ParA, SEQ ID NO: 14 and SEQ ID NO: 21; Cre L215P SEQ ID NO: 15 and SEQ ID NO: 22; XerC, SEQ ID NO: 16 and SEQ ID NO: 23; XerD, SEQ ID NO: 17 and SEQ ID NO: 24).Constructs are grown in NEB Express cells with 50 pg / ml of carbenicillin at 37°C, 250 RPM until they reach OD600 = 0.6-0.8. IPTG is added to 250 pM and the cultures are further incubated at 16°C, 250 RPM for 16-20 hours. Cells are centrifuged at 1000 x G, 4°C for 20 minutes and the pellet was stored at -80°C until lysis. Cells are resuspended in 20 mL of lysis buffer and incubated at room temperature for 5 minutes. Sonication is performed on ice at level 5 (on for 10 seconds, off for 50 seconds x 8 cycles). Cellular debris is pelleted by centrifugation at 15,000 RPM for 30 minutes, 4°C. Lysate is mixed with 2 mL amylose resin and mixed gently by shaking at 200 RPM at 4°C for 1 hour. The resin-lysate mixture is loaded onto a column and washed twice with 8 mL Equilibration Buffer. The protein is eluted four times with 1 mL of threedifferent elution buffers (Elution Buffers 1-3, respectively). Elution samples that are positive for the correct size protein are buffer exchanged into storage buffer using Amicon fdter 50 kDa MWCO. Protein concentrations are determined by a Bradford assay.Table 2: MBP-Tagged Protein Purification Buffers

[0159] Example 13: Rolling Circle Amplification with Gamma Delta Resolvase

[0160] In this example, a two-step MDA / RCA reaction is followed by de-concatenation and circularization using gamma delta resolvase (gamma delta).

[0161] A 50 pl MDA / RCA reaction contains 33.5 ng of SEQ ID NO: 26 plasmid as template, 5 pM of SEQ ID NO: 9 as primer, 5 pM of SEQ ID NO: 10 as primer, 50 mM Tris-HCl, 10 mM MgCh, 10 mM (N U)2SO4, 4 mM DTT, 10 units of NEB Phi29 DNA polymerase, 10 pg of NEB Recombinant Albumin, 2.5 mM dNTPs, and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The SEQ ID NO: 26 plasmid used for template contains a single res site (SEQ ID NO: 27) for gamma delta.

[0162] A 50 pl de-concatenation / circularization reaction contains 8 pls of the above MDA / RCA reaction product, 33 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 5 mM (NH4)2SO4, 2 mM DTT, 0.5 mM NAD+, 0.1 mM ATP, 0.1 units of T7 endonuclease I from NEB, 20 nM of MBP-gamma delta (SEQ ID NO: 28), and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The reaction is incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65 °C for 10 minutes.

[0163] Reactions are visualized by loading 25 pl of DNA onto a 1.2% agarose gel with Tris- borate-EDTA buffer.

[0164] Example 14: Rolling Circle Amplification with Tn3 Resolvase

[0165] In this example, a two-step MDA / RCA reaction is followed by de-concatenation and circularization using tn3 resolvase (tn3).

[0166] A 50 pl MDA / RCA reaction contains 33.5 ng of SEQ ID NO: 30 plasmid as template, 5 pM of SEQ ID NO: 9 as primer, 5 pM of SEQ ID NO: 10 as primer, 50 mM Tris-HCl, 10 mM MgCh, 10 mM (N U)2SO4, 4 mM DTT, 10 units of NEB Phi29 DNA polymerase, 10 pg of NEB Recombinant Albumin, 2.5 mM dNTPs, and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The SEQ ID NO: 30 plasmid used for template contains a single res site (SEQ ID NO: 31) for tn3.

[0167] A 50 pl de-concatenation / circularization reaction contains 8 pls of the above MDA / RCA reaction product, 33 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 5 mM (NH4)2SO4, 2 mM DTT, 0.5 mM NAD+, 0.1 mM ATP, 0.1 units of T7 endonuclease I from NEB, 20 nM of MBP-tn3 (SEQ ID NO: 32), and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The reaction is incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65°C for 10 minutes.

[0168] Reactions are visualized by loading 25 pl of DNA onto a 1.2% agarose gel with Tris- borate-EDTA buffer.

[0169] Example 15: Rolling Circle Amplification with Beta Resolvase.

[0170] In this example, a two-step MDA / RCA reaction is followed by de-concatenation and circularization using beta resolvase (beta).

[0171] A 50 pl MDA / RCA reaction contains 33.5 ng of SEQ ID NO: 34 plasmid as template, 5 pM of SEQ ID NO: 9 as primer, 5 pM of SEQ ID NO: 10 as primer, 50 mM Tris-HCl, 10 mM MgCh, 10 mM (NH4)2SO4, 4 mM DTT, 10 units of NEB Phi29 DNA polymerase, 10 pg of NEB Recombinant Albumin, 2.5 mM dNTPs, and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The SEQ ID NO: 34 plasmid used for template contains a single six site (SEQ ID NO: 35) for beta.

[0172] A 50 pl de-concatenation / circularization reaction contains 8 pls of the above MDA / RCA reaction product, 33 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 5 mM (NH4)2SO4, 2 mM DTT, 0.5 mM NAD+, 0.1 mM ATP, 0.1 units of T7 endonuclease I from NEB, 20 nM of MBP-beta (SEQ ID NO: 36), and water as needed. The reaction is incubated at 30°C for 8 hours and heat inactivated at 65°C for 10 minutes. The reaction is incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65°C for 10 minutes.

[0173] Reactions are visualized by loading 25 pl of DNA onto a 1.2% agarose gel with Tris- borate-EDTA buffer.

[0174] Example 16: Exemplary DNA SequencesTable 3: Exemplary DNA Sequences

[0175] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives tothe embodiments of the disclosure described herein can be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for generating circular nucleic acids, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises XerC / D, ArgR, and PepA binding sites; b) amplifying the nucleic acid template by providing an amplification enzyme and primers for rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR) amplification to form a tandem repeat nucleic acid sequence comprising at least two recombination sites; and c) generating an amplification product comprising a circular nucleic acid; wherein the method comprises incubating the tandem repeat nucleic acid sequence with: i) a recombination enzyme comprising XerC / D recombinase polypeptides, mutant Cre, or a resolvase; and ii) accessory factors or cognate resolvases that promote monomeric resolution.2 The method of claim 1, wherein the accessory factors that promote monomeric resolution comprise ArgR and PepA.3 The method of claim 1, wherein the accessory factors comprise a recombinase.4 The method of claim 1, wherein the nucleic acid template further comprises an expression cassette.5 The method of claim 1, wherein the resolvase is CinH, ParA, beta, Tn3, or gamma delta.6 The method of claim 1, wherein the amplification enzyme is phi29 DNA polymerase.7 The method of claim 1, wherein the primers for RCA comprise random hexamers or longer, sequence specific primers that anneal at greater than 30°C.8 The method of claim 1, wherein the primers for PCR amplification produce a linear product flanked by recombination sites and are compatible with the accessory factors and the recombination enzyme.9 The method of claim 1, further comprising sequencing the amplification product.10 The method of claim 9, wherein the sequencing comprises single molecule sequencing.11 The method of claim 9, wherein the sequencing comprises nanopore sequencing.12 The method of claim 1, further comprising employing the amplification product in cell-based processes such transformation of bacteria or yeast or transfection of mammalian cells.

13. The method of claim 1, further comprising adding an endonuclease and / or an exonuclease to remove a residual nicked dsDNA, a residual single stranded DNA, branched DNA or a residual linear dsDNA from the amplification product.

14. The method of claim 1, wherein the amplification product comprises at least 85% monomeric circular nucleic acids.

15. The method of claim 1, wherein the amplification product comprises at least 95% monomeric circular nucleic acids.

16. The method of claim 1, further comprising processing the amplification product to generate a nucleic acid vaccine.

17. The method of claim 1, further comprising generating a large DNA construct.

18. The method of claim 17, wherein the large DNA construct does not comprise extraneous sequences.

19. The method of claim 1, further comprising generating a gene.

20. The method of claim 1, further comprising generating a gene cluster.

21. The method of claim 1, further comprising generating a chromosome.

22. The method of claim 1, further comprising generating a genome.

23. The method of claim 1, wherein the method occurs in vitro.

24. The method of claim 23, further comprising transfecting a cell free-produced DNA into cells.

25. The method of claim 24, wherein the cells are mammalian cells.

26. The method of claim 23, wherein the in vitro method is a transcription-translation (TX-TL) method.

27. The method of claim 26, wherein the amplification product is used to generate an RNA.

28. The method of claim 27, wherein the RNA is used to generate a protein.

29. The method of claim 28, wherein the protein is a therapeutic protein.

30. The method of claim 27, wherein the RNA is used as an RNA therapeutic.

31. The method of claim 30, wherein the RNA therapeutic is formulated as a nanoparticle.

32. The method of claim 1, wherein the amplification product is used as a DNA therapeutic.

33. The method of claim 32, wherein the DNA therapeutic is formulated as a nanoparticle.

34. The method of claim 1, wherein the method occurs in vivo.

35. The method of claim 1, wherein the method occurs ex vivo.

36. The method of claim 1, further comprising barcoding the circular nucleic acid.

37. A method for generating an RNA vaccine, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises XerC / D, ArgR, and PepA binding sites; b) amplifying the nucleic acid template by providing an amplification enzyme and primers for rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR) amplification to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; and c) generating an RNA vaccine; wherein the method comprises incubating the tandem repeat nucleic acid sequence with: i) a recombination enzyme comprising XerC / D recombinase polypeptides, mutant Cre, or a resolvase; and ii) accessory factors or cognate resolvases that promote monomeric resolution.

38. A method for generating circular nucleic acids, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises a recombination site; b) amplifying the nucleic acid template by providing an amplification enzyme and primers for rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR) amplification to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; and c) generating an amplification product comprising a circular nucleic acid; wherein the method comprises incubating the tandem repeat nucleic acid sequence with: i) a recombination enzyme comprising a recombinase polypeptide; and ii) an accessory factor or cognate resolvase that promotes monomeric resolution.

39. The method of claim 38, wherein the recombination site comprises a loxP binding site.

40. The method of claim 39, wherein the recombinase polypeptide is a Cre recombinase polypeptide.

41. The method of claim 38, wherein the recombinase polypeptide is a resolvase polypeptide.

42. The method of claim 41, wherein the resolvase polypeptide is CinH, ParA, beta, Tn3, or gamma delta.

43. The method of claim 38, wherein the recombination site comprises a XerC / D binding site.

44. The method of claim 43, wherein the recombinase polypeptide comprises a XerC polypeptide.

45. The method of claim 44, further comprising a second recombination enzyme comprising a XerD polypeptide.

46. The method of claim 38, wherein the accessory factor that promotes monomeric resolution comprises ArgR.

47. The method of claim 38, wherein the accessory factor that promotes monomeric resolution comprises Pep A.

48. The method of claim 38, wherein the accessory factor that promotes monomeri c resolution comprises Sso7d.

49. The method of claim 38, further comprising a second accessory factor that promotes monomeric resolution.

50. The method of claim 49, wherein the accessory factor that promotes monomeric resolution comprises ArgR and wherein the second accessory factor that promotes monomeric resolution is PepA.

51. The method of claim 38, wherein the nucleic acid template further comprises an expression cassette.

52. The method of claim 38, wherein the amplification enzyme is phi29 DNA polymerase.

53. The method of claim 38, wherein the primers for RCA comprise random hexamers or longer, sequence specific primers that anneal at greater than 30°C.

54. The method of claim 38, wherein the primers for PCR amplification produce a linear product flanked by recombination sites and are compatible with the accessory factors and the recombination enzyme.

55. The method of claim 38, further comprising sequencing the amplification product.

56. The method of claim 55, wherein the sequencing comprises single molecule sequencing.

57. The method of claim 55, wherein the sequencing comprises nanopore sequencing.

58. The method of claim 38, further comprising employing the amplification product in cellbased processes such transformation of bacteria or yeast or transfection of mammalian cells.

59. The method of claim 38, further comprising adding an endonuclease and / or an exonuclease to remove a residual nicked dsDNA, a residual single stranded DNA, or a residual linear dsDNA from the amplification product.

60. The method of claim 38, wherein the amplification product comprises at least 85% monomeric circular nucleic acids.

61. The method of claim 38, wherein the amplification product comprises at least 95% monomeric circular nucleic acids.

62. The method of claim 38, further comprising processing the amplification product to generate a nucleic acid vaccine.

63. The method of claim 38, further comprising generating a large DNA construct.

64. The method of claim 63, wherein the large DNA construct does not comprise extraneous sequences.

65. The method of claim 38, further compri sing generating a gene.

66. The method of claim 38, further comprising generating a gene cluster.

67. The method of claim 38, further comprising generating a chromosome.

68. The method of claim 38, further comprising generating a genome.

69. The method of claim 38, wherein the method occurs in vitro.

70. The method of claim 69, further comprising transfecting a cell free-produced DNA into cells.

71. The method of claim 70, wherein the cells are mammalian cells.

72. The method of claim 69, wherein the in vitro method is a transcription-translation (TX-TL) method.

73. The method of claim 72, wherein the amplification product is used to generate an RNA.

74. The method of claim 73, wherein the RNA is used to generate a protein.

75. The method of claim 74, wherein the protein is a therapeutic protein.

76. The method of claim 73, wherein the RNA is used as an RNA therapeutic.

77. The method of claim 76, wherein the RNA therapeutic is formulated as a nanoparticle.

78. The method of claim 38, wherein the amplification product is used as a DNA therapeutic.

79. The method of claim 78, wherein the DNA therapeutic is formulated as a nanoparticle.

80. The method of claim 38, wherein the method occurs in vivo.

81. The method of claim 38, wherein the method occurs ex vivo.

82. The method of claim 38, further comprising barcoding the circular nucleic acid.

83. A method for generating an RNA vaccine, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises a recombination site; b) amplifying the nucleic acid template by providing an amplification enzyme and primers for either rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR) amplification to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; and c) generating an RNA vaccine; wherein the method comprises incubating the tandem repeat nucleic acid sequence with: i) a recombination enzyme comprising a recombinase polypeptide; and ii) an accessory factor or cognate resolvase that promotes monomeric resolution.

84. A method for generating circular nucleic acids, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises a recombination site; b) amplifying the nucleic acid template to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; c) generating an amplification product; d) providing a recombinase; and e) generating a circularized product comprising at least 85% monomeric circular nucleic acids.

85. The method of claim 84, wherein the amplification product comprises linear concatemers.

86. The method of claim 84, wherein the method does not comprise use of accessory factors.

87. The method of claim 84, further comprising use of an accessory factor or a cognate resolvase that promotes monomeric resolution.

88. The method of claim 87, wherein the accessory factor that promotes monomeric resolution comprises PepA.

89. The method of claim 87, wherein the accessory factor that promotes monomeric resolution comprises Sso7d.

90. The method of claim 87, further comprising a second accessory factor that promotes monomeric resolution.

91. The method of claim 90, wherein the accessory factor that promotes monomeric resolution comprises ArgR, and wherein the second accessory factor that promotes monomeric resolution is Pep A.

92. The method of claim 84, wherein the recombinase is a resolvase.

93. The method of claim 84, wherein the recombinase is beta, a beta recombinase peptide, Tn3, a Tn3 recombinase peptide, gamma delta, or a gamma delta recombinase peptide.

94. The method of claim 84, wherein the recombinase comprises a serine recombinase.

95. The method of claim 94, wherein the serine recombinase is a small serine recombinase.

96. The method of claim 95, wherein the small serine recombinase is CinH or a CinH polypeptide.

97. The method of claim 95, wherein the small serine recombinase is ParA or a ParA polypeptide.

98. The method of claim 84, wherein the recombinase comprises a Cre polypeptide.

99. The method of claim 84, wherein the recombinase comprises a XerC polypeptide.

100. The method of claim 99, further comprising a second recombinase comprising a XerD polypeptide.

101. The method of claim 84, wherein the recombination site comprises an RS1 / RS2 binding site.

102. The method of claim 84, wherein the recombination site comprises an MRS binding site.

103. The method of claim 84, wherein the recombination site comprises a res binding site.

104. The method of claim 84, wherein the nucleic acid template further comprises an expression cassette.

105. The method of claim 84, wherein the amplification comprises rolling circle amplification (RCA), multiple displacement amplification (MDA), or polymerase chain reaction (PCR).

106. The method of claim 84, further comprising providing an amplification enzyme.

107. The method of claim 106, wherein the amplification enzyme is phi29 DNA polymerase.

108. The method of claim 84, further comprising sequencing the amplification product or the circularized product.

109. The method of claim 108, wherein the sequencing comprises single molecule sequencing.

110. The method of claim 108, wherein the sequencing comprises nanopore sequencing.

111. The method of claim 84, further comprising employing the circularized product in cell-based processes such transformation of bacteria or yeast or transfection of mammalian cells.

112. The method of claim 84, further compri sing adding an endonuclease and / or an exonuclease to remove a residual nicked dsDNA, a residual single stranded DNA, or a residual linear dsDNA from the amplification product or the circularized product.

113. The method of claim 84, wherein the circularized product comprises at least 90% monomeric circular nucleic acids.

114. The method of claim 113, wherein the circularized product comprises at least 95% monomeric circular nucleic acids.

115. The method of claim 84, further comprising processing the circularized product to generate a nucleic acid vaccine.

116. The method of claim 84, further comprising generating a large DNA construct.

117. The method of claim 1 16, wherein the large DNA construct does not comprise extraneous sequences.

118. The method of claim 84, further comprising generating a gene.

119. The method of claim 84, further comprising generating a gene cluster.

120. The method of claim 84, further comprising generating a chromosome.

121. The method of claim 84, further comprising generating a genome.

122. The method of claim 84, wherein the method occurs in vitro.

123. The method of claim 122, further comprising transfecting the cell free-produced DNA into cells.

124. The method of claim 123, wherein the cells are mammalian cells.

125. The method of claim 122, wherein the in vitro method is a transcription-translation (TX-TL) method.

126. The method of claim 125, wherein the circularized product is used to generate an RNA.

127. The method of claim 126, wherein the RNA is used to generate a protein.

128. The method of claim 127, wherein the protein is a therapeutic protein.

129. The method of claim 126, wherein the RNA is used as an RNA therapeutic.

130. The method of claim 129, wherein the RNA therapeutic is formulated as a nanoparticle.

131. The method of claim 84, wherein the amplification product is used as a DNA therapeutic.

132. The method of claim 131, wherein the DNA therapeutic is formulated as a nanoparticle.

133. The method of claim 84, wherein the method occurs in vivo.

134. The method of claim 84, wherein the method occurs ex vivo.

135. The method of claim 84, further comprising barcoding the circularized product.

136. A method for generating an RNA vaccine, comprising: a) providing a nucleic acid template, wherein the nucleic acid template comprises a recombination site; b) amplifying the nucleic acid template to form a tandem repeat nucleic acid sequence comprising multiple recombination sites; c) generating an amplification product; d) providing a recombinase; and e) generating an RNA vaccine.

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