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WO2025120370A1 - Compositions et procédés de synthèse d'acides nucléiques sans phosphoramidite en phase soluble - Google Patents

Compositions et procédés de synthèse d'acides nucléiques sans phosphoramidite en phase soluble Download PDF

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Publication number
WO2025120370A1
WO2025120370A1 PCT/IB2024/000703 IB2024000703W WO2025120370A1 WO 2025120370 A1 WO2025120370 A1 WO 2025120370A1 IB 2024000703 W IB2024000703 W IB 2024000703W WO 2025120370 A1 WO2025120370 A1 WO 2025120370A1
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sequence
dna
iisre
bases
nickase
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Corinne ARNOLD
Derek L. Stemple
Gabrielle CHRISTIE
James Allen
James Richardson
Maria PRIMO
Nadin HOLBROOK
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Camena Bioscience Ltd
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Camena Bioscience Ltd
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the invention described herein describes a gene assembly method, in particular double stranded DNA assembly methods referred to as geometric synthesis (gSynth) methods.
  • Environmentally-friendly (e.g., “green”) double-stranded DNA synthesis methods are described herein using double-stranded DNA constructs referred to as “addamers”.
  • Addamers are short, exonuclease resistant, double-stranded double-hairpin structures that carry a DNA payload as well as a variety of control elements that allow the manipulation of the sequence.
  • the methods have previously relied on solid supports to generate new sequences in a phosphoramidite-free way.
  • the invention described herein discloses a cost effective, efficient, and environmentally conscious process for phosphoramidite synthesis.
  • compositions and methods allow for the generation of sequences of DNA at any arbitrary’ length in solution. Furthermore, described herein are addamer reagents generated from bacterial plasmids as well as bacterially expressed, purified enzymes used to generate a sequence.
  • composition comprising: (a) a double stranded DNA sequence comprising: (i) an N-mer sequence; (ii) at least one offset-cutting nickase DNA binding sequence configured to bind a nickase that cleaves a first strand or a second strand of the double stranded DNA sequence; and (iii) at least one Type II S restriction endonuclease (IISRE) DNA binding sequence configured to bind a IISRE that cleaves the first stand and the second strand of the N-mer sequence, wherein the nickase DNA binding sequence is located between the N-mer sequence and the IISRE DNA binding sequence.
  • a double stranded DNA sequence comprising: (i) an N-mer sequence; (ii) at least one offset-cutting nickase DNA binding sequence configured to bind a nickase that cleaves a first strand or a second strand of the double stranded DNA sequence; and (iii) at least one
  • nickase In some aspects, a nickase.
  • the nickase and the double stranded DNA sequence is in a single volume.
  • the double stranded DNA sequence is not coupled to a solid support.
  • IISRE Type II S restriction endonuclease
  • the nickase cleaved site is within or adjacent to the N-mer sequence.
  • the nickase DNA binding sequence comprises enzymes: BbvCI,
  • the IISRE DNA binding sequence comprises: Mlyl, NgoAVII, SspD5I, Alwl, Ajul, Alol, BccI, Bcefl, Piel, BceAI, BceSIV, BscAI, BspD6I, Faul, Earl, BspQI, BfuAI, PaqCI, Esp3I, BbsI, Bbvl, BtgZI, FokI, BsmFI, Bsal, BcoDI, Hgal, or SfaNI.
  • the IISRE DNA binding sequence comprises Earl.
  • At least one hairpin structure is at least one hairpin structure.
  • At least one hairpin structure comprises an aptamer sequence.
  • a second IISRE DNA binding sequence is provided.
  • the second IISRE DNA binding sequence comprises a BspQI DNA binding sequence.
  • a core melting temperature of the double stranded DNA sequence is at least 60°C. In some aspects, a core melting temperature of the double stranded DNA sequence is between 50°C to 70°C. In some aspects, a core melting temperature of the double stranded DNA sequence is about 50°C. 51 °C. 52°C, 53°C, 54°C. 55°C, 56°C, 57°C. 58°C, 59°C, 60°C. 61°C, 62°C, 63°C. 64°C, 65°C, 66°C, 67°C. 68°C, 69°C, or 70°C.
  • a method of synthesizing a target sequence in solution comprising: (a) providing a plurality of DNA sequences in solution, wherein a DNA sequence of the plurality of DNA sequences comprises aN-mer, a IISRE sequence, and at least one hairpin structures; (b) providing at least one IISRE to the solution, wherein the at least IISRE cleaves the IISRE sequence, thereby exposing an M-base overhang; (c) ligating at least two DNA sequences of the plurality of the DNA sequences having the M-base overhang to generate one or more ligated DNA sequences, wherein a ligated DNA sequence comprises a sequence having a length of 2N-M; and (d) repeating (a)-(c) using the one or more ligated DNA sequences to generate a final DNA sequence comprising a target sequence.
  • M is 3. In some aspects, M is at least 3.
  • the N-mer is at least a 4-mer. In some aspects, the N-mer is no more than the 4-mer.
  • the target sequence comprises a length of about 50 bases to 5000 bases. In some aspects, the target sequence comprises the length of about 100 bases to 5000 bases. In some aspects, the length is about 50 bases, 60 bases, 70 bases, 80 bases, 90 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1000 bases, 1100 bases, 1200 bases, 1300 bases, 1400 bases, 1500 bases, 1600 bases, 1700 bases, 1800 bases, 1900 bases, 2000 bases, 2100 bases, 2200 bases, 2300 bases, 2400 bases, 2500 bases, 2600 bases, 2700 bases, 2800 bases, 2900 bases, 3000 bases, 3100 bases, 3200 bases, 3300 bases, 3400 bases, 3500 bases, 3600 bases, 3700 bases, 3800 bases, 3900 bases, 4000 bases, 4100 bases, 4200 bases, 4300 bases, 4400 bases, 4500 bases, 4600 bases, 4700 bases, 4800 bases, 4900 bases, or 5000 bases.
  • the target sequence comprises a length of about 50 bases to 300 bases. In some aspects, the length is about 50 bases. 60 bases. 70 bases. 80 bases, 90 bases, 100 bases, 110 bases. 120 bases, 130 bases, 140 bases, 150 bases. 160 bases, 170 bases, 180 bases. 190 bases, 200 bases, 210 bases, 220 bases, 230 bases, 240 bases. 250 bases, 260 bases, 270 bases, 280 bases, 290 bases, or 300 bases.
  • the DNA sequence further comprises a nickase DNA binding sequence.
  • the target sequence is generated w ith an error rate of less than 1 in 100,000.
  • the solution comprises: (a) 50 mM Potassium acetate, 20 mM Trisacetate, 10 mM Magnesium acetate, 100 pg/mL rAlbumin, pH 7.9 at 25°C, (b) 100 mM NaCl, 50 mM Tris-HCL 10 mM MgCh, 100 pg/mL rAlbumin, pH 7.9 at 25°C, or (c) 66 mM Potassium acetate, 33 mM Tris-acetate, 10 mM Magnesium acetate, 100 pg/mL Bovine Serum Albumin, pH 7.9 at 37°C.
  • the solution further comprises 10 mM DTT and either 1 mM ATP or 1 pM ATP.
  • DTT is about 5mm, 6mm, 7mm, 8mm, 9mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm.
  • ATP is about 0.5 mM, 06 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM.
  • ATP is about 0.5 pM, 06 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 1.1 pM, 1.2 pM, 1.3 pM, 1.4 pM, or 1.5 pM.
  • (a), (b), or (c) is performed at a temperature of at least 10°C.
  • the temperature is about 10°C, 16°C, 25°C, 37°C, 50°C, 55°C, 60°C or 65°C.
  • the temperature is about 10°C, 11°C, 12°C. 13°C, 14°C, 15°C. 16°C, 17°C . 18°C, 19°C, 20°C, 21°C. 22°C, 23°C, 24°C, 25°C. 26°C, 27°C, 28°C. 29°C, 30°C, 31°C. 32°C, 33°C, 34°C, 35°C, 36°C, 37°C.
  • (a), (b), or (c) is performed cycling between one or more temperatures: about 10°C, 16°C, 25°C, 37°C, 50°C, 55°C, 60°C or 65°C.
  • the temperature is about 5°C, 6°C, 7°C, 8°C, 9°C. 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C,
  • the DNA sequence is formed using a ligase enzyme.
  • the ligase enzyme is T4 DNA ligase, T7 DNA ligase, T3 DNA ligase, or human DNA ligase III.
  • the synthesized target sequence has a purity of at least 80%. In some aspects, the synthesized target sequence has the purity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%. 82%, 83%. 84%. 85%. 86%. 87%. 88%. 89%. or 90.
  • the DNA sequence further undergoes heat inactivation of the enzymes directly or through protease treatment, wherein the protease treatment is inactivated by heat.
  • a composition comprising: (a) a double stranded DNA sequence, wherein the double stranded DNA sequence comprises: (i) an N-mer sequence; (ii) a nickase sequence, wherein the nickase binding sequence is at most 4 bases from a nick site; and (iii) a first Type II S restriction endonuclease (IISRE) binding sequence configured to bind a IISRE that cleave the N-mer sequence, wherein the nickase binding sequence is located between the N-mer sequence and the IISRE binding sequence.
  • IISRE Type II S restriction endonuclease
  • nickase In some aspects, a nickase. [0036] In some aspects, the nickase and the double stranded DNA sequence is in a single volume.
  • the double stranded DNA sequence is not coupled to a solid support.
  • IISRE Type II S restriction endonuclease
  • the nickase cleaved site is within or adjacent to the N-mer sequence.
  • the nickase binding sequence comprises enzymes: BbvCI, Bsml,
  • BsrDI, BssSI, BtsI, Alwl, BbvCI, BsmAI, BspQI, or BstNBI are examples of BsrDI, BssSI, BtsI, Alwl, BbvCI, BsmAI, BspQI, or BstNBI.
  • the IISRE binding sequence comprises: Mlyl, NgoAVII, SspD5I, Alwl, Ajul, Alol, BccI, Bcefl, Piel, BceAI, BceSIV, BscAI, BspD6I, Faul, Earl, BspQI, BfuAI, PaqCI, Esp3I, BbsI, Bbvl, BtgZI, FokI, BsmFI, Bsal, BcoDI, Hgal, or SfaNI.
  • the IISRE sequence comprises Earl.
  • At least one hairpin structure is at least one hairpin structure.
  • At least one hairpin structure comprises an aptamer sequence.
  • a second IISRE binding sequence is provided.
  • the second IISRE binding sequence comprises a BspQI sequence.
  • a core melting temperature of the double stranded DNA sequence is at least 60°C. In some aspects, a core melting temperature of the double stranded DNA sequence is between 50°C to 70°C. In some aspects, a core melting temperature of the double stranded DNA sequence is about 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, or 70°C.
  • a system for synthesizing a target sequence in solution comprising: (a) a plurality of DNA sequences in solution, wherein a DNA sequence of the plurality of DNA sequences comprises a N-mer, a IISRE sequence, and at least one hairpin structures; (b) at least one IISRE in the solution, wherein the at least IISRE is configured to cleave the IISRE sequence, thereby exposing an M-base overhang; (c) at least two DNA sequences of the plurality of the DNA sequences configured to have the M-base overhang are configured to generate one or more ligated DNA sequences, wherein a ligated DNA sequence comprises a sequence having a length of 2N-M; and (d) one or more ligated DNA sequences are configured to generate a final DNA sequences comprising the target sequence.
  • M is 3. In some aspects, M is at least 3.
  • the N-mer is at least a 4-mer. In some aspects, the N-mer is no more than the 4-mer.
  • the target sequence comprises a length of about 50 bases to 5000 bases. In some aspects, the target sequence comprises the length of about 100 bases to 5000 bases. In some aspects, the length is about 50 bases, 60 bases, 70 bases, 80 bases, 90 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 600 bases, 700 bases, 800 bases, 900 bases, 1000 bases, 1100 bases, 1200 bases, 1300 bases, 1400 bases, 1500 bases, 1600 bases, 1700 bases, 1800 bases, 1900 bases, 2000 bases, 2100 bases, 2200 bases, 2300 bases, 2400 bases, 2500 bases, 2600 bases, 2700 bases, 2800 bases, 2900 bases, 3000 bases, 3100 bases, 3200 bases, 3300 bases, 3400 bases, 3500 bases, 3600 bases, 3700 bases, 3800 bases, 3900 bases, 4000 bases, 4100 bases, 4200 bases, 4300 bases, 4400 bases, 4500 bases, 4600 bases, 4700 bases, 4800 bases, 4900 bases, or 5000 bases.
  • the target sequence comprises a length of about 50 bases to 300 bases.
  • the length is about 50 bases, 60 bases, 70 bases, 80 bases, 90 bases, 100 bases, 110 bases, 120 bases, 130 bases, 140 bases, 150 bases, 160 bases, 170 bases, 180 bases, 190 bases, 200 bases, 210 bases, 220 bases, 230 bases, 240 bases, 250 bases, 260 bases, 270 bases, 280 bases, 290 bases, or 300 bases.
  • an exonuclease in the solution configured to remove any DNA sequence of the plurality of DNA sequences having the M-base overhang.
  • the DNA sequence further comprises a nickase sequence.
  • at least one nickase in the solution wherein the at least one nickase cleaves the nickase sequence.
  • the target sequence is generated with an error rate of less than 1 in 100,000.
  • the solution comprises: (a) 50 mM Potassium acetate, 20 mM Trisacetate, 10 mM Magnesium acetate, 100 pg/mL rAlbumin, pH 7.9 at 25°C, (b) 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCh, 100 pg/mL rAlbumin, pH 7.9 at 25°C, or (c) 66 mM Potassium acetate, 33 mM Tris-acetate, 10 mM Magnesium acetate, 100 pg/mL Bovine Serum Albumin, pH 7.9 at 37°C.
  • the solution further comprises 10 mM DTT and either 1 mM ATP or 1 pM ATP.
  • DTT is about 5mm, 6mm, 7mm, 8mm, 9mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm.
  • ATP is about 0.5 mM, 06 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, or 1.5 mM.
  • ATP is about 0.5 pM, 06 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 1.1 pM, 1.2 pM, 1.3 pM, 1.4 pM, or 1.5 pM.
  • the temperature is about 10°C, 16°C, 25°C, 37°C, 50°C, 55°C, 60°C or 65°C.
  • the temperature is about 10°C, 1 1 °C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C , 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C,
  • the ligase enzyme is T4 DNA ligase, T7 DNA ligase, T3 DNA ligase, or human DNA ligase 111.
  • the synthesized target sequence has a purity of at least 80%. In some aspects, the synthesized target sequence has the purity of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90.
  • the DNA sequence further undergoes heat inactivation of the enzymes directly or protease treatment, wherein the protease treatment is inactivated by heat.
  • a composition comprising: (a) a double stranded DNA sequence, wherein the double stranded DNA sequence comprises: (i) an N-mer sequence; (ii) a first enzyme binding sequence, wherein the first enzyme binding sequence is at most 4 bases from a nick site; and (iii) a second binding sequence configured to bind a IISRE that cleave the N-mer sequence, wherein the nickase binding sequence is located between the N- mer sequence and the IISRE binding sequence.
  • the first enzyme binding sequence is a nickase binding sequence.
  • the second binding sequence is a first Type II S restriction endonuclease (IISRE) binding sequence.
  • IISRE Type II S restriction endonuclease
  • FIG. 1 depicts an exemplary schematic of a pay load addamer.
  • FIG. 2 depicts an exemplary schematic of control addamers including 3-base overhang systems using a TTCG linker.
  • FIG. 3 depicts an exemplary schematic of control addamers including 3-base overhang systems using a TCG or GTG linker.
  • FIG. 4 depicts an exemplary schematic of control addamers including 4-base overhang systems using a TTCG linker.
  • FIG. 5 depicts an exemplary' schematic of control addamers including Bsal and BsmBI sites using a TCG linker.
  • FIG. 6 depicts an exemplary' schematic of generic libraries of control payload addamer sets.
  • FIG. 7 depicts an exemplary' schematic of a method of addamer excision.
  • FIG. 8 depicts an exemplary' schematic of addamer excision systems.
  • FIGs. 9A-C depicts an exemplary schematic of building addamers.
  • FIG. 10 depicts an exemplary schematic using gSynth to assemble addamer arrays.
  • FIG. 11 depicts an exemplary schematic of combining a pay load addamer with a control addamer.
  • FIG. 12 depicts an exemplary schematic combining two control payload addamers to make a 5 bp payload.
  • FIG. 13 depicts an exemplary 7 schematic combining two 5 bp payload addamers to make a 7 bp payload.
  • FIG. 14 depicts an exemplary 7 schematic combining two 7 bp payload addamers to make an 11 bp payload.
  • FIG. 15 depicts an exemplary schematic combining two 11 bp payload addamers to make a 19 bp payload.
  • FIG. 16 depicts an exemplary schematic illustrating the generation of a Level 0 fragment assembly using a mixture of 3- and 4-base overhangs.
  • FIG. 17 depicts an exemplary schematic illustrating the assembly from reagents to Level £ through Level 0.
  • FIG. 18 depicts an exemplary schematic of GreenSynth Assembly using only 3-base overhangs.
  • FIG. 19 depicts an exemplary embodiment of the assembly pathways for Fragment 1.
  • FIG. 20 depicts an exemplary embodiment of the assembly pathways for Fragment 2.
  • FIG. 21 depicts an exemplary embodiment of the assembly pathway for Fragment 3.
  • FIG. 22 depicts an exemplary embodiment of the assembly pathway for Fragment 4.
  • FIG. 23 depicts an exemplary embodiment of the final pooled assembly to Level 0.
  • FIG. 24 depicts an example of the addamers that earn 7 4 bp pay loads.
  • FIG. 25 depicts an example of the pay load-free reagent addamer.
  • FIG. 26 depicts a partial assembly path diagram for generation of a Level 0 addamer.
  • FIG. 27 depicts in a diagram the sequence verification of the Level 0 pay load.
  • FIG. 28 depicts an exemplary schematic for the plasmid insert design and recovery method.
  • FIG. 29 depicts the stages of plasmid digestion and treatment to recover the addamer.
  • FIG. 30 depicts an exemplar ⁇ 7 schematic two enzy me-system for generating addamers for 3’ base overhangs.
  • FIG. 31 depicts an exemplar ⁇ 7 schematic two enzy me-system for generating addamers for 3’ base overhangs.
  • FIGs. 32A-B shows an exemplary 7 schematic of the tests performed to validate the two-enzy me systems.
  • FIG. 33 depicts exemplary 7 schematics of blunt cutting two-enzyme system addamers.
  • FIG. 34 depicts exemplary 7 schematics of bi-active addamer generic library designs.
  • Solution phase GreenSynth is a non-templated DNA synthesis system capable of generating any arbitrary DNA sequence.
  • small DNA fragments carrying 4 bp payloads are pieced together pairwise by a process of cutting with a Type II S restriction endonuclease (IISRE), combining the pair of cut Addamers, ligating them together, then digesting away unused reactants using an exonuclease, which degrades any DNA with a nick or free end.
  • IISRE Type II S restriction endonuclease
  • a small group of fragments for example five fragments each with payload just over 18 bp long, are pooled and combined to generate a Level 0 Addamer of 50-100 bp in length.
  • the system may generate accurate, specific lengths of Level 0 Addamer sequence for further assembly using gSynth. Addamer-based methods and compositions of the present disclosure
  • an Addamer (FIG. 1) comprises a double stranded DNA capped at both ends with a hairpin, which is a stemloop structure, like the sequence 5’-GCGCAGC-3’ that forms a small stem-loop with a 3-base loop.
  • An Addamer also carries a payload, which is an original small insert payload (such as NNNN), a linker sequence, or a target sequence in mid-synthesis.
  • Addamers have DNA binding site sequences for offset cutting IISREs systems that flank the payload, such as BtgZI / Nt.BstNBI and BspQI as shown in FIG. 1.
  • an Addamer may also contain other elements such as primer sequences for amplification (e.g., Payload Primer) and the remnants of a plasmid excision system, such as PacI / Nb.BssSI adjacent to the hairpins in FIG. 1.
  • primer sequences for amplification e.g., Payload Primer
  • remnants of a plasmid excision system such as PacI / Nb.BssSI adjacent to the hairpins in FIG. 1.
  • FIG. 1 shows three different payload Addamer libraries.
  • any element of these libraries can be combined with a Control Addamer to generate a new Addamer with a new Control element on the left side and BspQI on the right.
  • the combination of two enzy mes BtgZI and Nt.BstNBI may be used to reveal the 3 base overhang linker TCG (See FIG. 3 and 5 for control Addamers), which will allow the new Control element to have access to the payload sequence.
  • NNNN (pl-p256) b BbsI digestion reveals the 4 base overhang linker TTCG (See FIG. 2 and 4 for control Addamers).
  • NNNN (pl-p256) c the combination of two enzymes BtgZI and Nt.BstNBI is used to reveal the 3 base overhang linker GTG (See FIG. 3 and 5 for control Addamers).
  • combining another class of restriction endonucleases, with other IISREs can generate 3-base overhangs in 6 additional ways (FIG. 2 and 3).
  • the other class of restriction endonucleases comprises nickases.
  • the nickases comprise nickases that are offset cutting.
  • this system has the advantage of making the remaining half of the cut Addamer unable to be ligated and sensitive to exonuclease activity', which may be used to purify one or more final reaction products.
  • Nt. Top strand cutters
  • Nb. Bottom strand cutters
  • the DNA binding sequences are oriented 5’-3’.
  • Nb.BbvCI and its recognition sequence CCTCAGC (none/-2).
  • CCTCAGC recognition sequence
  • ‘none’ means that the enzy me does not cut the top strand
  • ‘-2’ means that the enzy me will cut 2 bases before the 3’ most base of the recognition sequence, i.e., between the A and the G.
  • an Addamer can comprise, consists essentially of, or consist of DNA.
  • the Addamers considered in this disclosure are detailed in FIGs. 1 - 6.
  • the Addamers are grouped into several categories, payload Addamers (FIG. 1), 3-base overhang control Addamers (FIGs. 2 and 3), 4-base overhang control Addamers (FIG. 4 and 5) and generic control: :payload Addamer sets (FIG 6).
  • payload Addamers FIG. 1
  • 3-base overhang control Addamers FIG. 2 and 3
  • 4-base overhang control Addamers FIG. 4 and 5
  • generic control: : payload Addamer sets FIG. 6
  • the linker sequences are TTCG, TCG, CGTG or GTG.
  • Level 0 was designated as Addamers, generally with payloads under 100 bp, which are generated by hybridization and ligation of phosphoramidite synthesized oligonucleotides.
  • Level 1 assembly a group of Level 0 Addamers are assembled into a longer Addamer. up to about 500 bp. through a cycling assembly using a IISRE and DNA ligase, followed by exonuclease digestion.
  • Level 1 Addamers can be cloned into plasmids, in which case Levels 2 and 3 are assembled by a Golden-Gate process into destination vectors.
  • assemble DNA is assembled from DNA fragments with generic DNA reagents generated from plasmids harvested from bacteria. It is sufficient to have one library of 256 4-base long payload Addamers that can be combined with at least 16 different control Addamers as needed. In this way, any DNA sequence can be built from generic reagent Addamers.
  • the first step is to put together specific 4 bp payload Addamers each together with prescribed Control Addamers (FIG. 12).
  • the combination of payloads and controls is determined beforehand by an algorithm that identifies a set of Level 0 gene fragments, then specifies a set of overlapping 5 bp fragments, for each Level 0 gene fragment.
  • the overlaps between the 5 bp fragments can be 3- or 4-bases.
  • the first extension reaction is carried out.
  • a 3- base overhang cutting enzyme BspQI exposes the proximal 3 bases each of a left and right Addamer.
  • ligase is added after digestion is complete and the exposed 3-base overhangs hybridize and are ligated together. After ligation, the entire reaction is further processed with an exonuclease that degrades all DNA except the desired reaction product, which is a new Addamer with a 5 bp payload with distinct left and right side Control elements, including specific left and right restriction enzyme system DNA binding sites and specific left and right side primer binding sites.
  • Plasmid Construction and Excision Provided herein is a system for manufacture at scale of any reagent Addamer. Addamers are cloned as a pre- Addamer construct, either as a single Addamer or an array of identical Addamers. In three preferred embodiments, using a two-enzyme system, where by first nicking one strand at a specific position then full double-stranded digestion of the Addamer with an IISRE, a long single stranded overhang is generated that encodes a strong stem-loop structure (FIG. 7).
  • target Addamers may be excised from larger Addamers using three different combinations of Nickase’s and IISRE’s
  • Addamers may also be excised from plasmids.
  • FIG. 7 The sequence of events in Addamer excision is depicted in FIG. 7.
  • a larger Addamer that contains the target Addamer is generated.
  • This larger Addamer is cloned into a plasmid as a single copy insert.
  • the larger Addamer is digested with Nb.
  • BssSI then digested with BseRI and held at a temperature below that of the Core melting temperature and above the melting temperature of the long overhang generated by the restriction enzy mes.
  • the melting temperature of the formed hairpins is well above the melting temperature of the overhang.
  • a quick ligation at an elevated temperature favors the healing of the nicks in the newly formed addamer.
  • the other reaction products are then degraded by exonuclease leaving the excised target Addamer.
  • ExA uses the nickase Nt.BbvCl and the IISRE Btsl to generate a long overhang that will fold to be the hairpin of the target Addamer.
  • ExB the enzyme combination Nb.BssSI and BseRI is used to generate the long overhang.
  • the combination of Nb.BsmI and Acul is used for ExC.
  • plasmids may have a single pre-Addamers.
  • to maximize Addamer production it is desirable to have as many copies of the same pre- Addamer as possible in one plasmid.
  • FIG. 10 depicts the assembly of an array of 4 copies of the same pre- Addamer, which can be inserted into a plasmid backbone and propagate in bacteria. The copy number can be doubled with each new round of gSynth.
  • FIG. 2 shows schematics for three 3-base overhang generating Control Addamers.
  • the digestion with BbsI leads to the generation of a TTCG overhang linker which will allow ligation with predigested Generic Payload Addamers NNNN (pl-p256) b (FIG. 1).
  • the Controls elements are: 3a (Earl), which confers an Earl IISRE control to the specific Payload Addamer; 3b (BsaXI), which confers a BsaXI IISRE to the payload Addamer, to generate 3’ 3-base overhangs; and 3c (BbvI/Nt.BstNBI), which confers a combination of Bbvl and Nt.BstNBI control to the Payload Addamer to generate a 5’ 3-base overhang using a two-enzyme system.
  • FIG. 3 shows five Control Addamers that can each generate 3-base overhangs for their respective payloads.
  • Earl is used to generate either a TCG overhang linker for combination with the NNNN (pl-p256) a Pay load Addamer (FIG. 1) or to generate a GTG overhang linker for combination with the NNNN (pl-p256) c Payload Addamer (FIG. 1).
  • FIG. 4. in some embodiments, shows three 4-base overhang generating Control Addamers.
  • digestion with BbsI leads to the generation of a TTCG overhang linker which will allow 7 ligation with predigested Generic Payload Addamers NNNN (pl-p256) b (FIG. 1).
  • Each of the five Addamers confers a different control element, which include a IISRE and a control-specific primer, to the specific payload.
  • FIG. 5 shows 3 Control Addamers that can each generate 4-base overhangs for their respective payloads.
  • BspQI is used to generate a TCG overhang linker for combination with the NNNN (pl-p256) a Pay load Addamer (FIG. 1).
  • the BsmBI Control Addamers are of two flavors, with distinct primer sequences so that synthesized Addamers with the distinct T3 or T7 primer sites can be amplified by PCR or an equivalent in vitro amplification system.
  • FIG. 6 show s a pre-configured control payload Addamer libraries.
  • each of these libraries carries a distinct IISRE and primer sequence.
  • Table 2 disclosed herein shows a list of addamer designs.
  • FIG. 7 shows the process of Addamer excision from a construct that can be recovered from a plasmid.
  • the Addamer 3a AAAA may be contained within a construct that possesses a IISRE and Nickase site on either side of the Addamer sequence.
  • the Nickase, Nb.BssSI may be first used to cut the bottom strand on the left side and the top strand on the right.
  • the IISRE, BseRI may be used to generate double stranded cuts that leave the hairpin sequences to properly fold under the appropriate temperature conditions.
  • the resulting nicks in the reformed Addamer may be sealed by T4 DNA ligase treatment.
  • the exonuclease is used to remove all non-Addamer DNA.
  • FIG. 8 shows three different Addamer excision systems. These systems can be used to generate plasmid inserts for production scale Addamer formation.
  • FIGs. 9A-C shows addamer arrays. These addamer arrays can be built from the use of three different Building Addamers. Each of these Addamer may have a pair of 4-base overhang sites, which can be exposed by BsmFI, PaqCI or Bbsl. The arrays of reagent addamers can be generated using a Golden-Gate like assembly.
  • described herein is a process for generating a four element array of Addamer using the constructs described in FIG. 10.
  • a similar process can be used to generate arbitrarily long (i.e., > 4) Addamer arrays.
  • FIG. 11 may depict combining a payload Addamer pl 5a (AATG) with a Control Addamer 3a (Earl).
  • the final product can be used in subsequent steps to assemble a de novo DNA sequence.
  • two loaded Addamers may be combined (FIG. 12).
  • BspQI is used to cut the two 4 bp payload Addamer to form a 5 bp payload Addamer mediated by a 3-base overhang.
  • the Addamers 3a_AATG and 3d_CCAT (whose reverse complement is ATGG) may be combined to form 3a_AATGG_3d.
  • two loaded Addamers may be combined (FIG. 13).
  • Nt.BsmAI and BsmFI may be used to cut the two 5 bp payload Addamer to form a 7 bp payload Addamer mediated by a 3-base overhang.
  • the Addamers 3a_AATGG_3d (FIG. 13) and 3d_TGGAC_3c may be combined to form 3a_AATGGAC_3c.
  • two loaded Addamers may be combined (FIG. 14).
  • Nt.BstNBI and Bbvl may be used to cut the two 7 bp payload Addamer to form an 11 bp payload Addamer mediated by a 3-base overhang.
  • the Addamers 3a_AATGGAC_3c (FIG. 14) and 3c_GACCGTG_3d may be combined to form 3a_AATGGACCGTG_3d.
  • two loaded Addamers may be combined (FIG. 15).
  • Nt.BsmAI and BsmFI may be used to cut the two 11 bp payload Addamer to form a 19 bp pay load Addamer mediated by a 3-base overhang.
  • the Addamers 3a_AATGGACCGTG_3d (FIG. 15) and 3d_GTGACCGCTAC_3c may be combined to form 3a AATGGACCGTGACCGCTAC 3c.
  • a 65 bp Level 0 Addamer can be assembled using a mixture of 3- and 4-base overhangs (FIG. 16). The sequence may be divided into four fragments with 3-base overlaps. In some embodiments, each of the four fragments are individually assembled then ultimately mixed and assembled together in a Golden-Gate like reaction. In some embodiments, 5 bp payloads contribute to each of the four fragments.
  • the overall assembly strategy may be depicted in FIG. 17 in Fragment 1 assembly at sub-Level 0.
  • the four fragments going into the final step to Level 0 may be all Level a fragments.
  • the 31 5 bp pay load Addamer may be formed from 62 original reagent Addamers or 124 reactions combining the payload and control Addamers.
  • the assembly of Fragment 1 (FIG. 19), Fragment 2 (FIG. 20), Fragment 3 (FIG. 21), and Fragment 4 (FIG. 22) may have intermediate Addamers.
  • the four fragments may be combined to form the 65 bp payload Level 0 Addamer (FIG. 23).
  • the NEB ligation fidelity calculator can predict the ligation fidelity to be 97% after 1 hour at 25°C with T4 DNA ligase.
  • FIG. 24 may depict addamers that cany 4 bp payloads, wherein each has a BspQI control element that allows combinations using 3 base overhangs in initial assembly reactions.
  • each class of addamer carries a unique binding site for the IISRE control and a unique primer sequence for amplification.
  • each Addamer may carry a nickase DNA binding site and a binding site for the restriction endonuclease Pad.
  • FIG. 25 may show two examples of this type of payload-free reagent Addamer. In some embodiments, they carry' a TCT sequence that allows them to be combined with any other reagent Addamer with a payload sequence of the form NAGA. In some embodiments, the combined use of the reagent Addamer and the adapter Addamer will only contribute 1 bp to the final assembly product.
  • FIG. 26 may depict a diagram of two of the eight way pools are highlighted. In some embodiments, in the assembly a total of 80 reagent Addamers and adapter Addamers were used. The assembly may level Epsilon through Level zero, corresponding to the maximum possible payload length for each column, i.e., step, in the assembly process.
  • FIG. 25 may show two examples of this type of payload-free reagent Addamer. In some embodiments, they carry' a TCT sequence that allows them to be combined with any other reagent Addamer with a payload sequence of the form NAGA. In some embodiments, the
  • FIG. 27 may depict multiple clones of the cloned final product.
  • the clones were sequenced using capillary Sanger sequencing. The results may verify that the expected sequence was generated.
  • FIG. 28 may show the plasmid insert design and the recovery' method.
  • FIG. 29 shows a schematic shows stages of plasmid digestion and treatment to recover the addamer from a mini-prep.
  • FIG. 30 show four of the two enzyme systems.
  • the IISRE cut site are denoted by the colored line and the Nickase cut site by a colored arrowhead.
  • FIG. 31 may show four of the two enzyme systems.
  • the IISRE cut site are denoted by the colored line and the Nickase cut site by a colored arrowhead.
  • FIGs. 32A-B may show the two Addamer for each of the two enzyme systems. Each pair may combine the payload TCGA and CGAG to generate the BspQI CTCGA BspQI Addamer product.
  • FIG. 32A may show are a schematic and a before and after analytical gel of the results of cutting, ligation and exonuclease treatments for each of the three enzy me systems.
  • the 3d system both BsmFI and its isoschizomer FaqI were tested.
  • FIG. 32B may show before and after analytical gel of the results of cutting, ligation and exonuclease treatments for each of the three enzyme systems. Table 3 described herein provides a list of Addamer designs describing Addamers in terms of their constituent parts.
  • FIG. 33 may show Addamer designs in which the payload can be exposed with a blunt end.
  • each of the Addamer designs comprise a different pair of nickase and IISREs are used to generate the blunt ends.
  • FIG. 34 may show the Bi-active Addamer Generic Library Designs.
  • the two enzyme systems use the same pair of enzymes.
  • BtgZI and Nt.AlwI are used to generate both 3’ overhangs (3k) and 5’ overhangs (31).
  • Bbvl and Nt.BstNBI are used to generate 3’ (3m) or 5‘ (3n) overhangs.
  • the methods described herein produce deeper pooling of Addamers at the start of Addamer synthesis.
  • gSynth GreenSynth (gSynth) reactions.
  • the gSynth reaction generates a 19 bp pay load.
  • the 19 bp pay load is generated using eight, 3 -base overlapping 5 bp fragments. Each of the eight, 3 -base overlapping 5 bp fragment are combined using the Control and Payload Addamers as described herein.
  • a reaction is depicted as in FIG. 11. As shown in FIG. 11. the payload Addamer pl 5 (AATG) is combined with the control Addamer 3a (Earl).
  • the enzyme BtgZI is further used for digestion to expose the linker sequence TTCG on both the control and payload Addamers described herein.
  • BspQI acts as the payload primer.
  • the digestion products are further recombined by annealing and DNA ligation.
  • the reaction product is designated as 3a_AATG (Earl).
  • 3a_AATG (Earl) is further purified using exonuclease treatment and Proteinase K treatment.
  • nickase DNA binding sequence is an enzyme.
  • the enzyme described herein is used to expose the overlapping 3 base ATG for each of the Addamers.
  • the enzyme is BspQI.
  • the enzy me is BbvCI.
  • the enzyme is BsmI.
  • the enzyme is BsrDI. In some instances, the enzyme is BssSI. In some instances, the enzyme is Btsl. In some instances, the enzyme is AlwI. In some instances, the enzy me is BbvCI. In some instances, the enzyme is BsmAI. In some instances, the enzyme is BstNBI.
  • the Addamers are further recombined by annealing.
  • the Addamers further undergo DNA ligation.
  • the Addamers are further purified to produce the product 3a_AATGG_3d.
  • the two 5 bp Addamers are cut with a serial combination of Nt. BsmAI followed by BsmFI.
  • the two 5 bp Addamers are further recombined to form a 7 bp Addamer.
  • the 7 bp Addamer are designated as 3a_AATGGAC_3c (FIG. 13). In some instances, the 7 bp Addamers are 3a_AATGGAC_3c and 3c_GACCGTG_3d.
  • the 7 bp Addamers, 3a_AATGGAC_3c and 3c_GACCGTG_3d, are cut with a nickase enzyme and a IISRE enzyme.
  • the nickase enzyme is Nt.BstNBl and the IISRE enzyme is Bbvl.
  • the nickase enzyme is BspQI.
  • the nickase enzyme is BbvCI.
  • the nickase enzyme is BsmI.
  • the nickase enzyme is BsrDI.
  • the nickase enzyme is BssSI.
  • the nickase enzyme is Btsl.
  • the nickase enzyme is AlwI. In some instances, the nickase enzyme is BbvCI. In some instances, the nickase enzyme is BsmAI. In some instances, the nickase enzyme is BstNBI. In some instances, the IISRE sequence is Mlyl. In some instances, the IISRE sequence is NgoAVII. In some instances, the IISRE sequence is SspD5I. In some instances, the IISRE sequence is AlwI. In some instances, the IISRE sequence is Ajul. In some instances, the IISRE sequence is Alol. In some instances, the IISRE sequence is Bed. In some instances, the IISRE sequence is Bcefl.
  • the IISRE sequence is Piel. In some instances, the IISRE sequence is BceAI. In some instances, the IISRE sequence is BceSIV. In some instances, the IISRE sequence is BscAI. In some instances, the IISRE sequence is BspD6I. In some instances, the IISRE sequence is Faul. In some instances, the IISRE sequence is Earl. In some instances, the IISRE sequence is BspQI. In some instances, the IISRE sequence is BfuAI. In some instances, the IISRE sequence is PaqCI. In some instances, the IISRE sequence is Esp3I. In some instances, the IISRE sequence is Bbsl. In some instances, the IISRE sequence is BtgZI.
  • the IISRE sequence is Fokl. In some instances, the IISRE sequence is BsrnFI. In some instances, the IISRE sequence is Bsal. In some instances, the IISRE sequence is BcoDI. In some instances, the IISRE sequence is Hgal. In some instances, the IISRE sequence is SfaNI. [00160] The 7 bp Addamers are further recombined to generate the 11 bp Addamer. In some instances, the 11 bp Addamer is 3a_AATGGACCGTG_3d (FIG. 14). In the final step, two 11 bp Addamers are recombined to form the final 19 bp product. In some instances, the final 19 bp product is 3a_AATGGACCGTGACCGCTAC_3c (FIG. 15).
  • Example 2 GreenSynth Assembly: Generation of a Level 0 Sequence Fragment
  • the known Level 0 gene is generated using a mix of 4-base and 3-base overhang cutting (FIG. 16). There are three types of Level 0 gene fragments.
  • the generation of Level 0 Addamers from Green Synth uses adapter Addamers. In some instances, the adaptor Addamers attach Bsal or BsmBI site to the ends of the Level 0 Addamer.
  • the leftmost Level 0 fragment of a Level 1 gene carries a left-hand BsmBI site and a left-hand Bsal site [4f (Bsal)].
  • the left-hand BsmBI site has a T7 primer sequence [4gT7 (BsmBI)].
  • the central Level 0 fragments of a Level 1 gene carry Bsal sites. The Bsal sites exist on either side of the pay load.
  • the rightmost Level 0 fragment carries a left-hand Bsal site and a right-hand BsmBI site with a T3 primer sequence [4gT3 (BsmBI)].
  • a central Level 0 gene fragment is assembled.
  • the payload needs the linker sequence CTCG added at either end for addition of Bsal (4f) caps.
  • the gene is divided into 4 fragments at the 4-base overhangs.
  • the 4-base overhangs are CTCC, TCAT, and CGTC as described herein.
  • the 4-base overhangs are used for Level 0 assembly.
  • the ligation fidelity for the mixed ligation reaction of these three, 4-base overhangs appears to be about 100%.
  • the sequence contains two 4-base self-complementary' sub-sequences.
  • the two 4-base self-complementary sub-sequences are TCGA and CCGG.
  • the two 4-base self-complementary' sub-sequences need to be avoided in this assembly' as 4-base overhangs are being used (FIG. 16).
  • Levels of synthesis for this assembly are depicted in FIG. 17. In some instances, the Levels of synthesis are Level s through Level a. The Addamers assembly proceeds in a pairwise fashion. In some instances, the Level a to Level 0 assembly is carried out by digestion with Bsal. The Addamer further undergoes ligation to generate the Level 0 Addamer. [00165] Example 3. GreenSynth Assembly Using Only 3-base Overhangs
  • FIG. 18 shows the list of 5 bp Addamers and their control elements with overlaps of 3-bases. A total of 31 5 bp Addamers are needed to generate the Level 0 sequence of 65 bases.
  • FIGs. 19-22 show the assembly pathways for each of the 4 sub-Level 0 Addamers that are pooled (FIG. 23) and assembled to the Level 0 addamer using the two enzyme system Nt.BstNBI / Bbvl to generate the 3-base overhangs. Thereafter, ligation is performed.
  • the generic reagent addamers are used to synthesize an arbitrary DNA sequence. Described herein the first 56 bp of a modified PhiX174 genome are generated as an addamer payload. The genomic sequence is divided into fragments to prepare a full assembly of the PhiX174 genome. In the first step, each fragment possesses compatible 4-base sites at each end. Each level has a different average payload length. The base level for geometric synthesis is designated Level 0. In some instances, this level of Addamers carry payloads of 50- 100 bp.
  • Step 1 described herein outlines BspQI digestion and Ligation Within Pools. Reagent Addamers are joined in a pairwise process with a 3 base overhang, generated by cleavage by BspQI. In the first block of eight the Addamers and adaptors are described herein in Table IX:
  • the 4c TCT Addamer is an adaptor and the TCT sequence is used to connect the two reagent Addamers.
  • the two reagent Addamers are 4c TCT and 3a CAGA.
  • the 3a CAGA Addamer is equivalent to the TCTG 3a Addamer.
  • the next step further involves pairwise hybridization and ligation.
  • the Addamer further undergoes exonuclease treatment. As a result, the process yields the following 5 bp payload Addamers as described herein in Table 2X:
  • Step 2 outlines Earl (3a) digestion and ligation.
  • the pool of four 5 bp Addamers is cut by the IISRE.
  • IISRE sequence is Earl.
  • Earl generates 3 base overhangs as described herein.
  • To generate the following ⁇ 7 bp payload Addamers see Table 3X described herein (FIG. 3).
  • Step 3 outlines BsmBI (4g) digestion and ligation.
  • the final reaction cycle uses the IISRE sequence to cut the two ⁇ 7 bp payload Addamers.
  • the IISRE sequence is the BsmBI sequence.
  • the 4 base overhangs can hybridize and ligate to generate the following ⁇ 10 bp Addamer (FIG. 3) as described herein in Table 4X: [00177] Table 4X
  • steps 4 and 5 outline digestion and litigation of the first enzyme followed by the second enzyme.
  • the enzyme is BbsI (4h).
  • the enzyme is BsmFI (4d). Pairs of ⁇ 10 bp Addamers are combined using BbsI to generate 4 base overhangs. The Addamers are used to form ⁇ 16 bp Addamers. The pairs of ⁇ 16 bp
  • Addamers are further combined using BsmFI to generate 4 base overhangs.
  • the final product will form ⁇ 28 bp Addamers (FIG. 3) as described herein in Table 5X.
  • Step 6 involve combining pool of ⁇ 28 bp Addamers using Bsal (4f).
  • a pool of arbitrary size in this case three fragment Addamers, are combined in a Golden- gate like reaction using the IISRE Bsal to generate the Level 0 Addamer (FIG. 3).
  • IISRE Bsal the IISRE Bsal
  • the method described herein provides a cost effective method for producing reagent Addamers in bulk.
  • the reagent Addamers can be produced through fermentation or in vitro amplification methods. Given that these methods do not involve organic-phase chemical synthesis, the whole gene synthesis pathway using reagent Addamer gSynth is an environmentally friendly process producing little in non-compostable waste.
  • An example reagent Addamer 3a AAAA was assembled into a special construct as described in FIG. 5 (e.g., a pUC19 based plasmid vector). The construct was designed so that a Nickase, a Nb.BssSI and a IISRE BseRI are used together.
  • the Addamer is released from the plasmid backbone.
  • the Addamer can refold the hairpins at either end.
  • the hairpins can be refold by a temperature change. In some instances, cleanup inactivates the enzymes.
  • the remaining nicks are further a refolded Addamer.
  • the refolded Addamer are healed by ligation using a ligase enzyme (e.g., T4 DNA ligase). This is followed by exonuclease treatment.
  • the final Addamer is recovered (FIG. 6).
  • Described herein is a generation of a 3 ’base overhangs using a two-enzyme system.
  • Addamer usage efficiency is increased as the length of the overhangs decreases while 4-base overhangs.
  • the 4-base overhangs are generated by some IISREs.
  • the IISREs are robust and efficient for ligation. In some instances, they have some technical difficulties. For example, there is a huge reduction in ligation efficiency for those 4 base overhangs that are palindromic.
  • sequences such as CGCG or ACGT can pose a challenge during assembly. Palindromes force the assembly path to use adapter Addamers to shift the sequence phase to avoid palindromes.
  • each of the enzyme systems uses an offset cutting Nickase, which is deployed first, followed by a double strand cutting IISRE (FIG. 7).
  • Described herein is the process of generating blunt ends using the two-enzyme system. Blunt ends are needed for DNA synthesis. In anticipation of DNA synthesis, the two- enzyme system is used to generate blunt ends. The two-enzyme system relies on two enzymes to generate blunt ends. In some instances, DNA binding sites for distinct pairs of nickase and IISREs w ere designed. Described herein are unique combinations of nickase sequences and IISRE sequences (FIG. 33). In some instances, the nickase enzyme is BspQI. In some instances, the nickase enzyme is BbvCI. In some instances, the nickase enzy me is BsmI.
  • the nickase enzyme is BsrDI. In some instances, the nickase enzy me is BssSI. In some instances, the nickase enzyme is Btsl. In some instances, the nickase enzy me is AlwI. In some instances, the nickase enzyme is BbvCI. In some instances, the nickase enzy me is BsmAI. In some instances, the nickase enzy me is BstNBI. In some instances, the IISRE sequence is Mlyl. In some instances, the IISRE sequence is NgoAVII. In some instances, the IISRE sequence is SspD5I.
  • the IISRE sequence is AlwI. In some instances, the IISRE sequence is Ajul. In some instances, the IISRE sequence is Alol. In some instances, the IISRE sequence is Bccl. In some instances, the IISRE sequence is Bcefl. In some instances, the IISRE sequence is Piel. In some instances, the IISRE sequence is BceAI. In some instances, the IISRE sequence is BceSIV. In some instances, the IISRE sequence is BscAI. In some instances, the IISRE sequence is BspD6I. In some instances, the IISRE sequence is Faul. In some instances, the IISRE sequence is Earl. In some instances, the IISRE sequence is BspQI.
  • the IISRE sequence is BfuAI. In some instances, the IISRE sequence is PaqCI. In some instances, the IISRE sequence is Esp31. in some instances, the IISRE sequence is Bbsl. In some instances, the IISRE sequence is BtgZI. In some instances, the IISRE sequence is Fokl. In some instances, the IISRE sequence is BsmFI. In some instances, the IISRE sequence is Bsal. In some instances, the IISRE sequence is BcoDI. In some instances, the IISRE sequence is Hgal. In some instances, the IISRE sequence is SfaNI. In some instances, the IISRE enzy me is BbvI.
  • the two enzyme system can further be generated with a 3’ overhang or a 5‘ overhang.
  • the two enzyme system generated with a 5’ overhang can rely on a variety' of pairs of enzy mes (FIG. 34) as described herein.

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Abstract

L'invention divulgue des compositions et des procédés de synthèse d'acides nucléiques sans phosphoramidite en phase soluble, appelés addamers. Les addamers peuvent être synthétisés pour n'importe quelle séquence arbitraire d'ADN, quelle que soit la longueur, avec un surplan de paire de bases en position 3' en solution. Par conséquent, l'invention divulgue des réactifs addamers générés à partir de plasmides bactériens ainsi que des enzymes purifiées exprimées de manière bactérienne utilisées pour générer n'importe quelle séquence d'acide nucléique (NA) donnée dans un processus appelé GreenSynth.
PCT/IB2024/000703 2023-12-08 2024-12-06 Compositions et procédés de synthèse d'acides nucléiques sans phosphoramidite en phase soluble Pending WO2025120370A1 (fr)

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