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WO2025151664A1 - Formation de constructions d'adn - Google Patents

Formation de constructions d'adn

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Publication number
WO2025151664A1
WO2025151664A1 PCT/US2025/010977 US2025010977W WO2025151664A1 WO 2025151664 A1 WO2025151664 A1 WO 2025151664A1 US 2025010977 W US2025010977 W US 2025010977W WO 2025151664 A1 WO2025151664 A1 WO 2025151664A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
amplification
recombinase
generating
monomeric
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/010977
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English (en)
Inventor
Kent Stewart BOLES
Phu Khat Nwe
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Bespoke Bioworks Inc
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Bespoke Bioworks Inc
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Publication date
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Publication of WO2025151664A1 publication Critical patent/WO2025151664A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • nucleic acid template comprises XerC/D, ArgR, and PepA binding sites
  • 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
  • PCR polymerase chain reaction
  • amplification product comprising a circular nucleic acid
  • 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.
  • 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 XerCZD recombinase polypeptides, mutant Cre, or a resolvase; and ii) accessory factors or cognate resolvases that promote monomeric resolution.
  • RCA rolling circle amplification
  • MDA multiple displacement amplification
  • PCR polymerase chain reaction
  • the recombination site comprises a loxP binding site.
  • the recombinase polypeptide is a Cre recombinase polypeptide.
  • the recombinase polypeptide is a resolvase polypeptide.
  • the resolvase polypeptide is Cin l, ParA, beta, Tn3, or gamma delta.
  • the recombination site comprises a XerC/D binding site.
  • the recombinase polypeptide comprises a XerC polypeptide.
  • the method further comprises a second recombination enzyme comprising a XerD polypeptide.
  • the accessory factor that promotes monomeric resolution comprises ArgR.
  • the accessory factor that promotes monomeric resolution comprises PepA.
  • the accessory factor that promotes monomeric resolution comprises Sso7d.
  • the method further comprises a second accessory factor that promotes monomeric resolution.
  • the accessory factor that promotes monomeric resolution comprises ArgR and wherein the second accessoiy factor that promotes monomeric resolution is PepA.
  • the nucleic acid template further comprises an expression cassette.
  • the amplification enzyme is phi29 DNA polymerase.
  • 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.
  • the amplification product comprises at least 85% monomeric circular nucleic acids.
  • the amplification product comprises at least 95% monomeric circular nucleic acids.
  • the method further comprises processing the amplification product to generate a nucleic acid vaccine.
  • the method further comprises generating a large DNA construct. In some embodiments, the large DNA construct does not comprise extraneous sequences.
  • 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.
  • RCA rolling circle amplification
  • MDA multiple displacement amplification
  • PCR polymerase chain reaction
  • 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).
  • RCA rolling circle amplification
  • MDA multiple displacement amplification
  • PCR polymerase chain reaction
  • the method further comprises providing an amplification enzyme.
  • the amplification enzyme is phi29 DNA polymerase.
  • the method further comprises sequencing the amplification product or the circularized product.
  • the sequencing comprises single molecule sequencing.
  • the sequencing comprises nanopore sequencing.
  • the method further comprises employing the circularized product in cell-based processes such transformation of bacteria or yeast or transfection of mammalian cells.
  • 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.
  • the circularized product comprises at least 90% monomeric circular nucleic acids.
  • the circularized product comprises at least 95% monomeric circular nucleic acids.
  • the method further comprises processing the circularized product to generate a nucleic acid vaccine.
  • the method further comprises generating a large DNA construct. In some embodiments, the large DNA construct does not comprise extraneous sequences.
  • 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.
  • TX-TL transcription-translation
  • 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.
  • FIG. 1A depicts an exemplary ColEl plasmid with a ColEl origin site (ColEl cer).
  • FIG. IB diagrams an exemplary target site for the XerC/D system, ColEl cer, showing the XerC, XerD, ArgR, and PepA binding sites.
  • FIG. 3 shows a plasmid map of SEQ ID NO: 25 with direct repeats of RS2 sites.
  • 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 nu
  • 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).
  • 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.
  • reactions such as but not limited to ligase chain reaction, polymerase chain reaction, self-sustained sequence replication, amplification with Qb-replication, and isothermal amplification.
  • 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.
  • each of the reagents used in the nucleic acid amplification reaction can be pre-treated to remove any contaminating nucleic acid sequences.
  • the pre-treatment of the reagents includes incubating the reagents in presence of Ultra-Violet radiation.
  • amplification of the nucleic acid template, and circularization of the amplified nucleic acid template to generate circular nucleic acids can be performed in a single vessel.
  • amplification of the nucleic acid template, and circularization of the amplified nucleic acid template to generate circular nucleic acids can be performed in separate vessels.
  • the amplification reaction and the recombination reaction can be performed sequentially, or they can be performed simultaneously.
  • 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.
  • 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.
  • 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.
  • 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.
  • nucleic acid amplification can produce concatemers.
  • nucleic acid amplification can produce monomers.
  • monomeric nucleic amplification products can be produced by resolving concatemer nucleic acids into monomer nucleic acids.
  • 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 a recombinase (e.g., a resolvase) specific to the recombination site.
  • 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.
  • 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.
  • Nucleic acid monomers can be produced by adding recombinases to perform intrastrand recombination.
  • recombinases are resolvases (e.g., site-specific recombinases).
  • recombinases e.g., resolvases
  • recombinases e.g., resolvases
  • 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)),
  • recombinases can be mutated recombinases (e.g., a mutated Cre recombinase).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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 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 100
  • 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, at least 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
  • 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.
  • a recombinase e.g., a XerC/D recombinase
  • 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.
  • 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,
  • 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.).
  • 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).
  • the generated nucleic acids can be further used to generate large DNA constructs.
  • the large DNA constructs comprise extraneous sequences. In alternative cases, large DNA constructs do not comprise extraneous sequences.
  • the generated nucleic acids can be further used to generate genes.
  • the generated nucleic acids can be further used to generate gene clusters.
  • 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.
  • the generated nucleic acids, amplification products, or other nucleic acids as described herein can be used to generate RNA.
  • the generated nucleic acids and/or generated RNA can be used to generate proteins.
  • proteins can be therapeutic proteins.
  • a generated RNA can be used as an RNA therapeutic (e.g., a vaccine).
  • the RNA can be an mRNA.
  • the RNA e.g., an mRNA
  • the RNA can be transfected or therapeutically administered in a buffer.
  • 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.
  • LNP lipid nanoparticle
  • 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.
  • 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.
  • 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.
  • 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, t
  • 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.
  • 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.
  • 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.
  • 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.
  • ORI ColEl type origin of replication
  • 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 ends of 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.
  • 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.
  • 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.
  • the accessory factor is ArgR.
  • the accessory factor is PepA.
  • both the ArgR and PepA accessory factors are used.
  • T5 Exonuclease is used to clean up single-stranded DNA and recombinant DNA.
  • a single stranded exonuclease e.g., SI, Pl or mug bean endonuclease
  • SI single stranded exonuclease
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • endonucleases and/or exonucleases are added to remove residual nicked, single-stranded, or linear double stranded DNA (dsDNA).
  • dsDNA linear double stranded DNA
  • 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.
  • 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.
  • An alternative amplification enzyme system using a jumbo bacteriophage is used for products over 35-40 kb.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 640 and 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).
  • 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.
  • Reactions are visualized by loading 25 pl of DNA onto a 1.2% agarose gel with Tris- borate-EDTA buffer.
  • Example 13 Rolling Circle Amplification with Gamma Delta Resolvase
  • 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.
  • 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 (NH 4 ) 2 SO4, 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.
  • reaction is incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65 °C for 10 minutes.
  • Reactions are visualized by loading 25 pl of DNA onto a 1.2% agarose gel with Tris- borate-EDTA buffer.
  • 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.
  • 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.
  • 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 (NH 4 ) 2 SO4, 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.
  • reaction is incubated at 30°C for 2 hours, 37°C for 2 hours, and heat inactivated at 65°C for 10 minutes.
  • Reactions are visualized by loading 25 pl of DNA onto a 1.2% agarose gel with Tris- borate-EDTA buffer.
  • Example 16 Exemplary DNA Sequences

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Abstract

L'invention concerne des procédés de génération de molécules d'acide nucléique circulaire comprenant la fourniture d'un modèle d'acide nucléique, l'amplification du modèle d'acide nucléique, et la génération d'un produit d'amplification comprenant des acides nucléiques circulaires.
PCT/US2025/010977 2024-01-11 2025-01-09 Formation de constructions d'adn Pending WO2025151664A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055744A1 (en) * 2008-09-02 2010-03-04 General Electric Company Dna mini-circles and uses thereof
US20210363570A1 (en) * 2016-12-16 2021-11-25 Roche Sequencing Solutions, Inc. Method for increasing throughput of single molecule sequencing by concatenating short dna fragments
US20230151367A1 (en) * 2020-04-23 2023-05-18 The J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Glads Therapeutic interfering particles for corona virus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100055744A1 (en) * 2008-09-02 2010-03-04 General Electric Company Dna mini-circles and uses thereof
US20210363570A1 (en) * 2016-12-16 2021-11-25 Roche Sequencing Solutions, Inc. Method for increasing throughput of single molecule sequencing by concatenating short dna fragments
US20230151367A1 (en) * 2020-04-23 2023-05-18 The J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Glads Therapeutic interfering particles for corona virus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ALéN CLAUDIA, SHERRATT DAVID J., COLLOMS SEAN D.: "Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination", THE EMBO JOURNAL / EUROPEAN MOLECULAR BIOLOGY ORGANIZATION, IRL PRESS, OXFORD, vol. 16, no. 17, 1 September 1997 (1997-09-01), Oxford , pages 5188 - 5197, XP093336802, ISSN: 0261-4189, DOI: 10.1093/emboj/16.17.5188 *

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