WO2023086109A1 - Procédés de synthèse de molécules d'acide nucléique - Google Patents

Procédés de synthèse de molécules d'acide nucléique Download PDF

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Publication number
WO2023086109A1
WO2023086109A1 PCT/US2021/059422 US2021059422W WO2023086109A1 WO 2023086109 A1 WO2023086109 A1 WO 2023086109A1 US 2021059422 W US2021059422 W US 2021059422W WO 2023086109 A1 WO2023086109 A1 WO 2023086109A1
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Prior art keywords
sequence
dsdna
sequences
variable
oligonucleotides
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PCT/US2021/059422
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English (en)
Inventor
John E. Gill
Lixia Fu
Sydney KERR
Michelle VARGAS
Daniel G. Gibson
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Codex Dna, Inc.
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Application filed by Codex Dna, Inc. filed Critical Codex Dna, Inc.
Priority to AU2021473848A priority Critical patent/AU2021473848A1/en
Priority to PCT/US2021/059422 priority patent/WO2023086109A1/fr
Priority to CN202180104210.3A priority patent/CN118355127A/zh
Priority to CA3238653A priority patent/CA3238653A1/fr
Priority to EP21964265.9A priority patent/EP4433605A1/fr
Publication of WO2023086109A1 publication Critical patent/WO2023086109A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • C12N15/10—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

Definitions

  • the invention provides compositions, methods, and kits for synthesizing any possible DNA molecule from a limited library of oligonucleotides.
  • Enzymatic methods of synthesizing oligonucleotides also exist and involve the use of enzymes such as terminal deoxynucleotidyl transferase (TdT), a template-independent polymerase that catalyzes the incorporation of deoxyribonucleotides into the 3 '-hydroxyl end of DNA templates.
  • TdT terminal deoxynucleotidyl transferase
  • a template-independent polymerase that catalyzes the incorporation of deoxyribonucleotides into the 3 '-hydroxyl end of DNA templates.
  • the enzyme shows strong bias for specific nucleotide bases and does not reliably add nucleotides in the desired order and length.
  • Figures 1 A-1B Figure 1 A provides a schematic illustration of the synthesis of a DNA molecule of desired sequence according to one embodiment of the invention.
  • Figure IB provides further reactions from the product of a schematic illustration of the synthesis of a DNA molecule of desired sequence
  • Figures 2A-2B Figure 2A provides a schematic illustration containing details of an embodiment of the DNA synthesis reaction of the invention.
  • Figure 2B shows an additional part of the reaction. The illustrations show complementary 3’ and 5’ overhang sequences between dsDNA molecules and fragments.
  • Figure 3 provides a schematic illustration of the overall scheme for hierarchal assembly using the methods of the invention.
  • Assembly can also include the addition of dsDNA fragments with a 5’ and 3’ overhang, which can be added to the final assembly. Lengths of variable sequences are for illustration only.
  • Figures 4A-4D Figures 4A-4D;
  • Figure 4A provides a gel image of PCR1 products after the first ligation step (L0) where three oligos from the library are combined, ligated and PCR amplified using a single universal primer pair. Contained within each of the 98bp PCR products is lObp of synthetic DNA that is leveraged for downstream assembly.
  • Figure 4B provides a gel image showing PCR2 products after the first digest and ligation step (DL1). These PCR products resulted from combining two PCR1 products, removal of one flank on each product (by enzymatic digestion) and then ligation of the products.
  • FIG. 4C provides a gel image showing PCR3 products after the second digestion and ligation step (DL2). These PCR products resulted from combining two PCR2 products, digesting away one flank on each product followed by ligation. Note that the 1st and 4th PCR product contain a capped termini, which aids in reducing downstream mis-ligation events, plus these also provide a universal priming sequence for the subsequent PCR4 amplification.
  • the variable sequence of the dsDNA molecule is 28 bp in length in all the sequences on this gel.
  • Figure 4D provides a gel image showing PCR4 products after the third digestion and ligation step (DL3). These PCR products result from combining four PCR3 products, digesting away one or two flanks on each product and then ligated them together.
  • the variable sequence of the dsDNA is 100 bp in length in all the sequences on this gel, and they are flanked by 40bp of flank sequence on both sides, which can be digested away by using BsmBI, thus enabling further assembly into even larger pieces of DNA.
  • the flank sequence can be digested away by using BsmBI, thus enabling further assembly into even larger pieces of DNA.
  • Figure 5 provides a schematic illustration of an embodiment of the invention for storing digital information in DNA
  • a 16 bp product DNA molecule is produced encoding four bytes of information.
  • the examples shows how a non-genetic message (here "cat in a hat") can be encoded into DNA using the methods of the invention.
  • Figure 6 discloses SEQ ID NOS 51-53, 55 and 62-68.
  • FIG. 6 is a schematic illustration of an embodiment of the invention applied to synthesizing a 120 bp product DNA, which is an initial guide structure having the transcriptional elements of a promoter, a guide RNA, a Cas9 handle, and a terminator.
  • the first cycle of PCR utilizes two primers having two variable bases on their 3' ends. This converts the otherwise 16 bp product DNA molecule into a 20 bp product. Later step(s) of PCR incorporate transcriptional elements.
  • the invention provides methods for synthesizing a product DNA molecule of any possible DNA sequence from a universal library of overlapping oligonucleotides.
  • the method involves combining a plurality of the overlapping oligonucleotides in a reaction pool, where the sequences of the plurality of oligonucleotides comprise at least a sub-sequence of the product DNA molecule.
  • the method also involves annealing the plurality of oligonucleotides, performing a ligation step, and performing an amplification step to thereby synthesize a sub-sequence of the product DNA molecule.
  • the invention can be used to synthesize a DNA molecule of any possible sequence from the universal library, which can be accomplished through a hierarchal assembly scheme.
  • the universal library comprises fewer than 10,000 pre-manufactured oligonucleotides that can be synthesized into the any possible DNA sequence.
  • the product DNA molecule can be more than 150 base pairs long. When subsequent DNA assembly techniques are employed DNA molecules of thousands of base pairs can be synthesized. In any embodiment the product DNA molecule has an error rate of less than 1 error per 2,000 nucleotides.
  • the invention provides methods of synthesizing a DNA molecule having a desired sequence.
  • the methods involve annealing at least two oligonucleotides to an anchor strand so that the at least two oligonucleotides annealed to the anchor strand abut one another on the anchor strand.
  • the oligonucleotides can abut on the anchor strand at their variable sequences.
  • the at least two oligonucleotides can each comprise a universal primer binding site on a 3’ or 5’ end, and a variable sequence on the opposing 5’ or 3’ end, and a conserved flanking sequence in between the universal primer binding site and the variable sequence.
  • the anchor strand can have conserved flanking sequences complementary to those on the at least two oligonucleotides, and further can have at least one variable sequence.
  • At least one portion of the at least one variable sequence on the anchor strand is complementary to at least a portion of the variable sequences on the at least two oligonucleotides.
  • the invention involves a step of ligating the at least two oligonucleotides annealed to the anchor strand to produce a first dsDNA molecule, performing an amplification step on the first dsDNA molecule having a desired sequence and comprising universal primer binding sites at the 3’ and 5’ ends, a conserved flanking sequence inside each of the 3’ and 5’ ends, and a variable sequence inside the conserved flanking sequences.
  • the first dsDNA molecule has a variable sequence that is about 10 nucleotides long.
  • the method can also involve contacting the first dsDNA molecule with a restriction endonuclease to produce first dsDNA fragments having 3’ and/or 5’ overhang sequences comprising a portion of the variable sequence from the first dsDNA molecule, providing at least one additional dsDNA fragment comprising a 3’ and/or 5’ overhang sequence that is at least partially complementary to an overhang sequence of at least one of the first dsDNA fragments.
  • the 3’ and/or 5’ overhang sequences can contain at least a portion of the variable sequence.
  • the methods also involve annealing the first dsDNA fragments and at least one additional dsDNA fragment by the 3’ and/or 5’ overhang sequences, and ligating the annealed dsDNA fragments to produce a second dsDNA molecule having a conserved flanking sequence inside each of the 3’ and 5’ ends, and a variable sequence inside the 3’ and 5’ conserved flanking sequences that is longer than the variable sequence on the first dsDNA molecule.
  • the variable sequence of the second dsDNA molecule is about 16 base pairs in length.
  • the method can also involve performing an amplification step on the second dsDNA molecule.
  • the restriction endonuclease can be a Type IIS restriction endonuclease.
  • the at least one additional dsDNA fragment can be the product of a parallel DNA synthesis reaction.
  • the method can further involve contacting the at least one second dsDNA molecule with a restriction endonuclease to produce a plurality of second dsDNA fragments comprising 3’ and/or 5’ overhang sequences and a conserved flanking sequence inside each of the 3’ or 5’ ends.
  • the 3’ and/or 5’ overhang sequences can contain at least a portion of the variable sequence.
  • the method can further involve a step of providing at least one additional dsDNA fragment comprising a 3’ and/or 5’ overhang sequence that is at least partially complementary to an overhang sequence of at least one of the second dsDNA fragments, annealing the plurality of second dsDNA fragments to the one or more additional dsDNA fragment(s) by the 3’ and/or 5’ overhang sequence(s); and performing a step of ligation to produce a third dsDNA molecule having a conserved flanking sequence on the 3’ and 5’ ends, and a variable sequence inside the conserved flanking sequences that is longer than the variable sequence of the second dsDNA molecule. In one embodiment the variable sequence is about 28 base pairs long.
  • the method can include performing an amplification step on the third dsDNA molecule.
  • the at least one additional dsDNA fragment can be the product of a parallel DNA synthesis reaction.
  • the method can further involve contacting the at least one third dsDNA molecule with a restriction endonuclease to produce a plurality of third dsDNA fragments comprising 3’ and/or 5’ overhang sequences and a conserved flanking sequence inside each of the 3’ or 5’ ends; the fragments can contain at least a portion of the variable sequence on the 3’ and/or 5’ overhangs.
  • the methods can also involve providing at least one additional dsDNA fragment comprising a 3’ and/or 5’ overhang sequence that is at least partially complementary to an overhang sequence of at least one of the third dsDNA fragments.
  • the overhang sequences can contain at least a portion of the variable sequence.
  • the methods can also involve annealing the plurality of third dsDNA fragments to the one or more additional dsDNA fragment(s) by the 3’ and/or 5’ overhang sequence(s).
  • the method can further involve performing a step of ligation to produce a fourth dsDNA molecule having a conserved flanking sequence on the 3’ and 5’ ends, and a variable sequence inside the conserved flanking sequences that is longer than the variable sequence of the third dsDNA molecule.
  • the methods can also involve performing an amplification step on the fourth dsDNA molecule.
  • the variable sequence is about 100 base pairs long.
  • step a) further involves annealing at least two paired oligonucleotides to a paired anchor strand so that the at least two paired oligonucleotides bound to the paired anchor strand abut one another on the paired anchor strand, which can occur at their variable sequences.
  • the at least two paired oligonucleotides can have a universal primer binding site on a 3’ or 5’ end, and a variable sequence on the opposing 5’ or 3’ end, and a conserved flanking sequence in between the universal primer binding site and the variable sequence.
  • the paired anchor strand can have conserved flanking sequences complementary to those on the at least two paired oligonucleotides, and further have at least one variable sequence.
  • a portion of the variable sequence on the paired anchor strand can overlap with a portion of the variable sequence on the first anchor strand.
  • the variable sequence can be located in between the two sequences complementary to the conserved flanking sequences. At least a portion of the variable sequence differs between the first and second anchor strands.
  • the method can further involve ligating the at least two paired oligonucleotides annealed to the anchor strand, performing an amplification step to produce a paired dsDNA molecule of desired sequence and comprising a universal primer binding site at a 3’ and 5’ end, a conserved flanking sequence inside each of the 3’ and 5’ ends, and a variable sequence inside the conserved flanking sequences that partially overlaps with the variable sequence of the first dsDNA molecule.
  • the at least two oligonucleotides and first anchor strand, and the at least two paired oligonucleotides and paired anchor strand can be annealed in a simultaneous reaction in the same pool.
  • the method can further involve contacting the first dsDNA molecule and the paired dsDNA molecule with a restriction endonuclease to produce at least one dsDNA fragment and at least one paired dsDNA fragment, each comprising at least one 3’ and/or 5’ overhang sequence; and at least a portion of a 3’ or 5’ overhang sequence from the first dsDNA fragment can be complementary to at least a portion of a 5’ or 3’ overhang sequence from the paired dsDNA fragment.
  • the method can further involve annealing the first and paired dsDNA fragments by their complementary overhang sequences and performing a step of ligation to produce a second dsDNA molecule having a conserved flanking sequence inside each of the 3’ and 5’ ends, and a variable sequence inside the 3’ and 5’ conserved flanking sequences that is longer than the variable sequence on the first dsDNA molecules.
  • the method can also involve performing an amplification step on the second dsDNA molecule.
  • the method can further involve contacting the at least one second dsDNA molecule and an at least one paired second dsDNA molecule with a restriction endonuclease to produce a plurality of second dsDNA fragments and paired second dsDNA fragments, each comprising a 3’ and/or 5’ overhang sequence(s). At least two of the plurality comprise, a conserved flanking sequence inside each of the 3’ or 5’ ends. At least a portion of the 3’ or 5’ overhang sequence from a second dsDNA fragment can be complementary to at least a portion of the 5’ or 3’ overhang sequence from a paired second dsDNA fragment.
  • the method can further involve annealing the second and paired second dsDNA fragments by their complementary overhang sequences, and performing a step of ligation to produce a third dsDNA molecule comprising a conserved flanking sequence inside each of the 3’ and 5’ ends, and a variable sequence inside the 3’ and 5’ conserved flanking sequences that is longer than the variable sequence on the second dsDNA molecules. At least a portion of the variable sequence on the third dsDNA molecule can overlap with a portion of the variable sequence on the paired third dsDNA molecule.
  • the methods can also involve performing a step of amplification on the third dsDNA molecule.
  • the methods further involve contacting the at least one third dsDNA molecule and an at least one paired third dsDNA molecule with a restriction endonuclease to produce a plurality of third dsDNA fragments and paired third dsDNA fragments, each comprising a 3’ and/or 5’ overhang sequence(s); the third dsDNA fragments can have at least a portion of the variable sequence on the 3’ and/or 5’ overhangs. At least two of the plurality can have a conserved flanking sequence inside each of the 3’ or 5’ ends.
  • At least a portion of the 3’ or 5’ overhang sequence from a third dsDNA fragment can be complementary to at least a portion of the 5’ or 3’ overhang sequence from a paired third dsDNA fragment.
  • the methods can further involve a step of annealing the third and paired third dsDNA fragments by their complementary overhang sequences, and performing a step of ligation to produce a fourth dsDNA molecule having a conserved flanking sequence inside each of the 3’ and 5’ ends, and a variable sequence inside the 3’ and 5’ conserved flanking sequences that is longer than the variable sequence on the third dsDNA molecule.
  • the methods can also involve performing a step of amplification on the fourth dsDNA molecule.
  • the first dsDNA molecule can have a variable region of 8- 12 base pairs.
  • the paired dsDNA molecule can have a variable region of 8-12 base pairs.
  • the second dsDNA molecule can have a variable sequence of 14-18 base pairs.
  • the third dsDNA molecule can have a variable sequence of 24-32 base pairs.
  • the fourth dsDNA molecule can have a variable sequence of 90-110 base pairs.
  • the at least two oligonucleotides can have a variable sequence of 4-6 nucleotides.
  • the anchor strands can have the sequences complementary to the conserved flanking sequences on the at least two oligonucleotides on the 3’ and 5’ ends. In any embodiment the anchor strands can have the sequences that are complementary to the conserved flanking sequences on the at least two oligonucleotides on the 3’ and 5’ ends. In any embodiment the amplification step can be performed by the polymerase chain reaction (PCR). In any embodiment the variable sequence can be equal to the lengths of the variable sequences on the at least two oligonucleotides. In any embodiment the anchor strand can have a variable sequence present in between the two sequences complementary to the conserved flanking sequences on the at least two oligonucleotides.
  • the anchor strand can have a variable sequence present in between the two sequences complementary to the conserved flanking sequences on the at least two oligonucleotides.
  • the at least two oligonucleotides bound to the anchor strand can abut one another on the anchor strand at their variable sequences.
  • the portion of the variable sequence on the anchor strand that is complementary to the conserved flanking sequence on the at least two oligonucleotides can be 2-6 nucleotides.
  • the at least two oligonucleotides and anchor strand are programmed so that the dsDNA molecule has at least one recognition site for a restriction endonuclease.
  • restriction endonuclease can be a Type IIS endonuclease.
  • the anchor strand can have 4-6 degenerate nucleotides.
  • the at least one additional dsDNA fragment can be from a parallel synthesis reaction.
  • the 3’ and/or 5’ overhang sequences can have the portion of the variable sequence from the first dsDNA molecule.
  • the step of ligation can occur spontaneously.
  • the at least one additional dsDNA fragment can have a variable sequence at least partially complementary to the variable sequence from the first dsDNA molecule.
  • the methods can further involve a step of ligating the at least two oligonucleotides bound to the anchor strand.
  • the invention provides a composition of at least two oligonucleotides, each comprising a universal primer binding site on a 3’ or 5’ end, and a variable sequence on the opposing 5’ or 3’ end, and a conserved flanking sequence in between the universal primer binding site and the variable sequence.
  • an anchor strand can have sequences complementary to the conserved flanking sequences on the at least two oligonucleotides, and further have at least one variable sequence, which can be located in between the two sequences complementary to the conserved flanking sequences.
  • At least a portion of the variable sequence on the anchor strand can be complementary to at least a portion of the variable sequences on the at least two oligonucleotides.
  • the anchor strand can have the sequences complementary to the conserved flanking sequences on the at least two oligonucleotides at its 3’ and 5’ ends.
  • the anchor strand can have the variable sequence in between the two sequences complementary to the conserved flanking sequence.
  • the invention provides methods of storing data in a DNA sequence.
  • the methods involve determining a sequence of DNA that encodes a non-genetic message according to a coding scheme that translates the non-genetic message from a reference language into a DNA sequence and vice versa; synthesizing the sequence of DNA that encodes the non-genetic message according to any method disclosed herein; and thereby store data in a DNA sequence.
  • the invention provides methods of synthesizing a DNA sequence encoding a guide RNA.
  • the methods involve determining a sequence of DNA that encodes a guide RNA; synthesizing the sequence of DNA that encodes the guide RNA according to any method disclosed herein.
  • the invention provides methods of assembling DNA molecules of any sequence with high fidelity using a universal library of oligonucleotides.
  • the methods involve the use of an oligonucleotide library having DNA molecule members such that all possible DNA sequences can be assembled from the library using the methods.
  • the library of oligonucleotides has less than 10,000 members.
  • Many efforts have been made towards achieving methods of assembling any possible DNA sequence from a library having a limited number of members.
  • the present inventors discovered that any possible DNA sequence can be conveniently assembled using the materials and methods disclosed herein.
  • the invention therefore enables creation of a library of less than 10,000 oligonucleotides, from which all possible oligonucleotide sequences can be assembled.
  • the library of less than 10,000 oligonucleotides can be conveniently provided on a small device (e.g. a DNA chip), and devices and instrumentation provided to selectively assembly any DNA sequence using only the members of the oligonucleotide library.
  • the oligonucleotide members in the library can be DNA of various lengths.
  • the library of oligonucleotides can have less than 20,000 members or less than 15,000 or less than 12,000 members, or less than 10,000 members, or less than 9,000 members or less than 6,000 members.
  • the methods are able to synthesize all possible polynucleotide sequences using the oligonucleotide members in the library.
  • the invention permits the assembly of over 4 billion (for a 16mer) and up to over 1 trillion (for a 20mer) polynucleotides of distinct sequence beginning only with those oligonucleotides in the library.
  • each oligonucleotide in the library can be used from 100 to 10,000 times in the synthesis of product DNA molecules.
  • the product DNA molecule assembled can be of any size, for example it can be longer than 1 kb, or longer than 1.5 kb, or longer than 2 kb, or longer than 5 kb or longer than 10 kb or 2-10 kb or 5-15 kb or 5-20 kb less than 500 kb or less than 1 Mbp or less than 5 Mbp or less than 10 Mbp or less than 12 Mbp, or less than 13 Mbp, or less than 14 Mbp, or less than 15 Mbp or 1-10 Mbp, or 1-12 Mbp, or 1-15 Mpb.
  • oligo and “oligonucleotide” are used interchangeably herein, and indicates a polymer of nucleotides of generally shorter length.
  • Polynucleotide is a general term denoting a polymer of nucleotides of any length.
  • the methods can also be used with even smaller libraries to assemble a significant number of sequences that may be desired, for example to assemble a more limited and directed number of sequences in a defined category where such sequences are needed.
  • Examples of a defined category can include a set of genes related to a specific biological function, or genes from a particular organism.
  • the product DNA molecule synthesized in the method can be synthesized entirely from and only using oligonucleotides from the oligonucleotide library.
  • a “universal library” is a library of polynucleotide molecules from which any possible DNA sequence can be assembled. At a broad lever a universal library can be any possible DNA sequence.
  • sequences of interest for example a universal library of DNA sequences for RNA metabolism, or for genes or sequences related to transcription, regulation, RNA metabolism, translation, protein folding, protein export, RNA (rRNA, tRNA, small RNAs), ribosome biogenesis, rRNA modification, DNA replication, DNA repair, DNA topology, DNA metabolism, chromosome segregation, cell division, and tRNA modification.
  • RNA rRNA, tRNA, small RNAs
  • ribosome biogenesis ribosome biogenesis
  • rRNA modification DNA replication, DNA repair, DNA topology, DNA metabolism, chromosome segregation, cell division, and tRNA modification.
  • the at least two oligonucleotides can be DNA of any convenient length to the purposes.
  • the at least two oligonucleotides can be greater than 12 nucleotides in length or, without limitation, about 20-35 nucleotides, or 35-55 nucleotides, or 30-60 nucleotide, or 40-50 nucleotides or about 42-48 nucleotides, or about 44 or about 45 nucleotides.
  • Anchor strands used in the method can be from 20-60 nucleotides, or from 20-70 nucleotides, or from 30-60 nucleotides or from 40-60 nucleotides or from 40- 50 nucleotides.
  • the at least two oligonucleotides are from 40-50 nucleotides and the anchor strand is from 35-45 or from 45-55 nucleotides.
  • Primer binding sites can be added to or included in these oligonucleotide lengths.
  • Oligonucleotides can be present in any combination or sub-combination of the lengths provided herein. In any embodiment the oligonucleotides can have only nucleotides having no non-standard bases. But in any embodiment the oligonucleotides can have only nucleotides having standard bases, i.e.
  • nucleotides in the oligonucleotide have a base that is either A (adenine), T (thymine), C (cytosine), or G (guanine). In other embodiments any of the oligonucleotides can contain non-standard bases.
  • the oligonucleotides and/or anchor strands can have sequences for binding a primer, which can be used in PCR or another DNA amplification procedure. In sizing nucleotides for the library the person of ordinary skill with resort to this disclosure will realize optimal sizes of oligonucleotides to use in the methods by considering the ability of oligo lengths to anneal to other oligos.
  • any of the oligonucleotides can be programmed to comprise a recognition site for a restriction endonuclease in a resulting dsDNA molecule.
  • the restriction enzyme can be one that recognizes asymmetric DNA sequences and cleaves a number of nucleotides outside of their recognition sequence (e.g. within 1-5 or 1-10 or 1-20 nucleotides).
  • the methods of the invention synthesize a product dsDNA molecule having a desired sequence, which can be a pre-determined sequence, i.e. one decided by the user prior to beginning the method.
  • the product DNA molecule can be any molecule produced by the method including but not limited to the first dsDNA molecule, the second dsDNA molecule, the third dsDNA molecule the fourth dsDNA molecule, the dsDNA fragments, and the additional dsDNA molecule and fragments.
  • Figure 1 A depicts a method of synthesizing a product DNA molecule according to a method of the invention.
  • 01 and 02 are the at least two oligonucleotides, and 03 is the anchor strand.
  • O1-O2 each have a universal primer binding site 101 on the 5’ or 3’ end, and a variable sequence 105 on the opposing 3’ or 5’ end.
  • 01-02 also have a conserved flanking sequence 110, in this embodiment depicted in between the universal primer binding site 101 and the variable sequence 105.
  • the “conserved flanking sequences” serve to assist the oligos in annealing to complementary CFSs on target oligos, and can also provide primer binding sites for use later in the method.
  • the CFSs can be a sequence of 15-20 or 15-30 nucleotides, but any convenient length can be used that is able to aid in annealing and provide a primer binding site.
  • the anchor strand 03 has, at the 3’ and 5’ ends, conserved flanking sequences 110 complementary to the conserved flanking sequences on the at least two oligonucleotides.
  • 03 also has at least one variable sequence 105, here situated in between the two conserved flanking sequences 110.
  • the variable sequence is in between the two conserved flanking sequences.
  • the variable sequence can be moved, as long as sufficient space is left for a CFS able to facilitate annealing and/or provide a primer binding site (if utilized).
  • at least 10 or at least 15 or at least 18 nucleotides of a conserved flanking sequence are present on both sides of the variable sequence of the anchor strand.
  • variable sequence 105 comprises degenerate nucleotides N, here six degenerate nucleotides as depicted in Figure 1. At least a portion of the at least one variable sequence 105 on the anchor strand is complementary to at least a portion of the variable sequences 105 on the at least two oligonucleotides, and in one embodiment can be complementary across the whole variable sequence.
  • One or more of the at least two oligonucleotides can further be programmed to have a recognition site for a restriction endonuclease when assembled into a dsDNA molecule; the anchor strand can also be programmed to contain a recognition site for a restriction endonuclease so that, when bound to the at least two oligonucleotides the recognition sites are formed by base pairs.
  • the restriction endonuclease can be a Type IIS restriction endonuclease.
  • the recognition site on the at least two oligonucleotides and anchor strand can be programmed to lie outside of the variable sequence on the assembled molecule, but the restriction endonuclease can cut inside of the variable sequence.
  • the recognition sites are comprised within the conserved flanking sequence; and in one embodiment the restriction endonuclease cleaves within the variable sequence of a dsDNA molecule. Sequences or nucleotides are “complementary” when they are able to anneal to each other and form base pairs, for example by standard Watson-Crick base pairing.
  • the method involves steps of annealing the at least two oligonucleotides 01- 02 to an anchor strand 03 so that the at least two oligonucleotides bound to the anchor strand abut one another on the anchor strand.
  • the at least two oligonucleotides can abut at their variable sequences.
  • the variable sequences 105 of the at least two oligonucleotides are annealed to the variable sequence of the anchor strand 105. It is noted in the embodiment illustrated that each of 01-02 have a variable sequence of 5 nucleotides and 03 has a variable sequence of ten nucleotides. Upon annealing the respective variable sequences anneal and form base pairs.
  • binding or “annealing” are used interchangeably and refer to formation of a double-stranded polynucleotide sequence by standard Watson-Crick base pairing.
  • Two oligonucleotides abut one another when a first oligonucleotide contains a nucleotide that is present adjacent to a nucleotide on a second oligonucleotide when both oligonucleotides are bound to the same (third) complementary oligonucleotide (e.g. an anchor strand) by Watson-Crick base pairing, at least at their variable sequences.
  • Figures 1 and 2 illustrate this concept.
  • the methods also involve a step of ligating the at least two oligonucleotides annealed to the anchor strand to produce a (“first”) dsDNA molecule.
  • the step of ligation or “ligating” can mean allowing ligation to occur spontaneously, or by contacting the annealed dsDNA fragments or dsDNA molecules with a ligase.
  • a ligase is an enzyme that catalyzes the joining of two polynucleotide molecules by forming a new chemical bond.
  • the ligase can ligate polynucleotides bound to the same complementary polynucleotide strand.
  • any DNA ligase can be used, for example T4 DNA ligase and E. coli DNA ligase are just two examples, but another DNA ligase can also be used.
  • the methods involve a step of performing an amplification step.
  • amplification can be, for example, PCR, isothermal amplification, rolling circle amplification, loop-mediated isothermal amplification, or another DNA amplification method) on the dsDNA molecules (e.g. O1-O3 and 04-06 when present) to produce a first dsDNA molecule 07 (and/or 08).
  • the variable sequence of 07 is a lOmer as an example, but persons of ordinary skill with resort to this disclosure will realize that any appropriate length of variable sequence can be used in the methods.
  • variable sequences of 6-20 or 6-12 or 10-14 or 10-16 nucleotides or other numbers of nucleotides can be used on anchor strands in any embodiment and, optionally, correspond in length to the sum of the variable sequences on the at least two oligonucleotides.
  • the at least two oligonucleotides can have variable sequences of different lengths.
  • one of the two oligonucleotides can have a variable sequence of 4 nucleotides and the second oligo a variable sequence of 6 nucleotides, or other combinations.
  • the first dsDNA molecule e.g.
  • DNA sequences “inside” refers to a feature present further towards the center of the DNA sequence (and further away from the 5’ or 3’ ends) than a reference feature.
  • a plurality of product DNA molecules can be “multiplexed,” i.e. synthesized in the same reaction pool. In other embodiments where desired DNA molecules can be synthesized individually (or “in parallel”) in their own reaction pools (and combined subsequently).
  • Reactions can be multiplexed with two or more binding sets of the at least two oligonucleotides and at least one anchor strand.
  • the method depicted in Figure 1 can be performed as an individual synthesis of one dsDNA molecule, or as a multiplexed synthesis of at least two dsDNA molecules, which can later, optionally, be joined as illustrated in Figures 1-2.
  • multiplexing is utilized more than one DNA molecule or more than two DNA molecules are synthesized in a simultaneous reaction in the same pool.
  • multiplexing is depicted as paired oligos 04-05 and paired anchor strand 06 forming a separate paired dsDNA molecule 08, as a pair to the first dsDNA molecule 07.
  • a paired dsDNA molecule 08 can be synthesized in a parallel and separate reaction pool, and the first and paired dsDNA molecules (or fragments therefrom) combined in a subsequent step.
  • the dsDNA molecules (or dsDNA fragments) are “paired” when they have an overlapping sequence at the variable sequence.
  • the overlap can be at least 1 bp, or at least 2 bp or at least 3 bp or at least 4 bp, or at least 5 bp or at least 6 bp.
  • Any two dsDNA molecules (or dsDNA fragment), whether first dsDNA molecule, second, third, fourth, etc., can be dsDNA molecules (or dsDNA fragments) that are paired.
  • dsDNA fragments can be produced (e.g. by restriction enzyme action on a dsDNA molecule or, in any embodiment, separately synthesized) to produce paired dsDNA fragments that have overhanging 3’ and/or 5’ sequences, which overhangs can be at their variable sequences and can at least partially overlap.
  • Such dsDNA fragments can therefore be annealed at the 3’ and/or 5’ overhangs to form a larger dsDNA molecule.
  • Overlapping sequences are those that comprise the same sequence for a series of nucleotides.
  • the methods can utilize polynucleotides that overlap by 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides, or by at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8, or by more than 4 or more than 5 or more than 6 consecutive nucleotides.
  • the overlap can be at their variable sequences.
  • “Overhangs” or “overhanging” sequences refers to 3’ or 5’ single-stranded DNA sequences that extend from a double-stranded DNA sequence. In various embodiments the overhangs can be at least 2 or at least 3 or at least 4 nucleotides.
  • the methods can involve further steps towards synthesizing a larger product DNA molecule.
  • the methods can involve a step of contacting paired (e.g. the first and paired) dsDNA molecule(s) (e.g. 07-08) with a restriction enzyme to produce first and paired dsDNA fragments that have 3’ and/or 5’ complementary overhang sequences and a portion of the variable sequence from the first dsDNA molecule(s).
  • the first and paired dsDNA molecules can have variable sequences that overlap, and can have conserved flanking sequences containing a recognition site for a restriction endonuclease (e.g. a Type IIS restriction endonuclease).
  • the restriction enzyme can cleave within the variable sequence of the first dsDNA molecule (and paired dsDNA molecule when present) to produce complementary overhanging 3’ and/or 5’ sequences of the first dsDNA fragment and paired dsDNA fragment.
  • the methods can involve a step of providing at least one additional dsDNA fragment that has a 3’ and/or 5’ overhang sequence complementary to an overhang sequence of at least one other dsDNA fragment in the synthesis reaction (e.g. the first and/or paired dsDNA fragment).
  • the overhang sequence of the additional dsDNA fragment can contain at least a portion of the variable sequence (depicted in Figure 2).
  • the overhang sequences can comprise at least a portion of the variable sequence of the first dsDNA molecule (and paired dsDNA molecule when present), and therefore the fragments can have overlapping sequences at the variable sequence.
  • the at least one additional dsDNA fragment can be from a restriction endonuclease reaction on the dsDNA molecule(s) or its pair (e.g. 07 and 08), or can be a DNA fragment produced in another, parallel reaction, and can also be separately synthesized.
  • Each of these dsDNA fragments have a complementary 3’ or 5’ overhang sequence to at least one other dsDNA fragment in the synthesis reaction.
  • a plurality of additional dsDNA fragments can be imported and assembled into the product DNA molecule at the same time.
  • Some dsDNA fragments can have both a 3’ and a 5’ overhang sequence complementary to two other dsDNA fragments in the reaction, and can therefore be inserted in between two fragments to lengthen the product DNA molecule - these dsDNA fragments therefore will have 3’ and 5’ overhangs, each complementary to one other 3’ or 5’ overhang of a dsDNA fragment in the synthesis.
  • the additional dsDNA fragment(s) can be used in any embodiment. “Additional dsDNA fragment” (or additional dsDNA molecule) is a general term, not necessarily specific to any particular step in the methods.
  • Such additional dsDNA fragments can be annealed to another dsDNA fragment at any step in the methods at 3’ and/or 5’ overhangs.
  • the additional dsDNA fragment can be at least partially complementary to the 3’ and/or 5’ overhang on at least one other dsDNA fragment in the method, which can be the first dsDNA fragment(s), or second dsDNA fragment(s), or third dsDNA fragment(s), or fourth dsDNA fragment(s), or other additional dsDNA fragments.
  • additional dsDNA fragments can be derived from additional dsDNA molecules.
  • the additional dsDNA fragment can therefore have at the 3’ and/or 5’ overhangs the next series of nucleotides to be synthesized into the final product dsDNA molecule to form the dsDNA molecule of pre-determined sequence.
  • the methods can involve a step of annealing a first dsDNA fragment with at least one additional dsDNA fragment by their complementary overhang sequences.
  • the method also can involve performing a step of ligation to produce a second dsDNA molecule (09) (here depicted having a 16mer variable sequence) having a conserved flanking sequence (CFS) 110 inside each of the 3’ and 5’ ends, and a variable sequence 105 inside the 3’ and 5’ conserved flanking sequences that is longer than the variable sequence on the first dsDNA molecule.
  • a second dsDNA molecule (09) (here depicted having a 16mer variable sequence) having a conserved flanking sequence (CFS) 110 inside each of the 3’ and 5’ ends, and a variable sequence 105 inside the 3’ and 5’ conserved flanking sequences that is longer than the variable sequence on the first dsDNA molecule.
  • CFS conserved flanking sequence
  • the methods therefore can further involve a step of contacting at least one second dsDNA molecule with a restriction enzyme to produce a plurality of second dsDNA fragments comprising 3’ and/or 5’ overhang sequences. At least two of the plurality can have a conserved flanking sequence inside each of the 3’ or 5’ overhangs.
  • the method can further involve a step of annealing the plurality of second dsDNA fragments to one or more additional dsDNA fragments having a complementary 3’ or 5’ overhang sequence (which can be at the variable sequence), and performing a step of ligation to produce at least one third dsDNA molecule having a conserved flanking sequence on the 3’ and 5’ ends, and a variable sequence inside the conserved flanking sequences that is longer than the variable sequence of the second dsDNA molecule.
  • the at least one third dsDNA molecule has a variable sequence of 28 bp and a 4 bp overlap with an at least one paired third dsDNA molecule.
  • the methods can further involve a step of reacting the at least one third dsDNA molecule with a restriction enzyme to produce at least one third dsDNA fragment 125 having 3’ and/or 5’ overhang sequences, optionally annealing the at least one third dsDNA fragment 125 to one or more additional dsDNA fragments 130 having a complementary 3’ or 5’ overhang sequence, and performing a step of ligation to produce a fourth dsDNA molecule.
  • Two of the dsDNA fragments in the mixture can have a conserved flanking sequence inside the variable sequence, and a variable sequence at the 3’ or 5’ overhang.
  • the fourth dsDNA molecule can therefore have conserved flanking sequences inside the 3’ and 5’ ends, an optional universal primer binding sequence, and a variable sequence (optionally between the CFSs) that is longer than the variable sequence of the third dsDNA molecule.
  • the at least one fourth dsDNA molecule has a variable sequence of 100 bp.
  • one or more additional dsDNA fragments can be included into the reaction to further lengthen the variable sequence of the product dsDNA molecule.
  • the at least one additional dsDNA fragment can be derived from parallel (or multiplexed) reactions and, as depicted in Figure IB by fragments 125 and 130, can have overhangs at both the 3’ and 5’ ends with a sequence complementary to the overhang sequences of two other dsDNA fragments in the reaction.
  • These additional dsDNA fragments can be produced by including two restriction recognition sites on the dsDNA molecule (optionally within the CFSs) contacted with the restriction endonuclease, which can then cleave the dsDNA molecule into at least three fragments.
  • the dsDNA fragments can therefore be joined in annealing and ligation reactions to form a longer product dsDNA molecule.
  • two or more dsDNA fragments can be included in the reaction, and the dsDNA fragments can have a 3’ and 5’ overhang sequence and not have a CFS, i.e. these dsDNA fragments can be all variable sequence.
  • the methods offer great versatility in synthesizing a product dsDNA molecule.
  • at least one dsDNA fragment can be included in the annealing reaction to place a desired sequence on the product dsDNA molecule.
  • a 5’ cap and universal priming sequence 120 can be added to the 3’ and 5’ ends of the product dsDNA molecule.
  • the 5’ cap can assist in preventing degradation of the ends of the DNA molecule, and the priming sequence is convenient for amplification when desired.
  • Additive reactions can also be performed.
  • the fourth dsDNA molecule has a variable sequence of 100 bp.
  • Parallel reactions can produce a plurality of additional dsDNA molecules having complementary and/or overlapping sequences with the fourth dsDNA molecule.
  • the additional dsDNA can also have a variable sequence of, for example, 100 bp or any suitable length.
  • Any of the dsDNA molecules can be cut with one or two restriction endonuclease(s) to produce a plurality of dsDNA fragments having 3’ and/or 5’ overhangs that contain complementary sequences with the 3’ or 5’ overhang of one or two other dsDNA fragments.
  • the overhangs can comprise at least a portion of the variable sequence of each dsDNA molecule.
  • the dsDNA fragments can be combined to synthesize a much longer variable sequence in a product dsDNA molecule.
  • Figure 2 provides a more detailed illustration of methods of the invention. Again are present at least two oligonucleotides O1-O2 and anchor strand 03, and paired oligonucleotides 04-05 and paired anchor strand 06.
  • Figure 2B shows the complementary overlapping 3’ and 5’ overhang sequences that occur after restriction endonuclease digestion of the first and paired dsDNA molecules (depicted as 07 and 08). The variable sequence is again depicted as a lOmer and forming part of the 3’ or 5’ overhang sequences.
  • the second dsDNA molecule (again illustrated with a variable sequence of 16 nucleotides) that is synthesized after annealing and amplification (PCR2) of the first and paired dsDNA fragments.
  • PCR2 annealing and amplification
  • annealing, ligation 0 (LO), and PCR1 occur between the at least two oligonucleotides and anchor strands, here depicted in binding sets 01-03 and 04-06, in multiplex mode, to form the first (07) and paired (08) dsDNA molecules.
  • Digestion with restriction endonuclease is then performed to produce first and paired dsDNA fragments followed by ligation 1 to additional dsDNA fragments (here the paired dsDNA fragment), then PCR2 to form the second dsDNA molecule, here depicted as having a variable sequence that is a 16mer (09).
  • the second dsDNA molecule can then be digested with restriction endonuclease to form second dsDNA fragments, and ligated (L2) with additional dsDNA fragments followed by PCR3 to form the third dsDNA molecule, which is depicted as having a 28mer variable sequence (010).
  • the third dsDNA molecule can in turn be digested with restriction endonuclease and to form third dsDNA fragments 125, which can be combined and ligated (L3) with additional dsDNA fragments 130 and PCR3 performed to yield the fourth dsDNA molecule (014), which is depicted as having a lOOmer variable sequence.
  • a plurality of additional dsDNA fragments 130 can be included in the reaction, which can derive from multiplexed or parallel synthesis reactions.
  • first dsDNA molecule “second dsDNA molecule,” “third dsDNA molecule,” “fourth dsDNA molecule,” “dsDNA fragments,” “additional dsDNA,” and “paired dsDNA molecule” are relative terms that are provided to assist in tracking a molecule through any step(s) in the method, and do not necessarily refer to any absolute point or DNA molecule or fragment in the reaction.
  • the first dsDNA molecule contains a variable sequence provided by the at least two oligonucleotides and first anchor strand; its paired dsDNA molecule can also contain a variable sequence that at least partially overlaps with the variable sequence of the first dsDNA molecule.
  • variable sequence of the first dsDNA molecule will at least partially overlap with the variable sequence of at least one additional dsDNA molecule.
  • the second dsDNA molecule contains a variable sequence of the first and paired dsDNA molecule, and in turn can overlap with a paired dsDNA fragments or additional dsDNA fragments.
  • the third dsDNA molecule contains a variable sequence of the at least one second dsDNA molecule, and can further contain a variable sequence of the first dsDNA molecule, and can also have a variable sequence of one or more additional dsDNA molecules.
  • the fourth dsDNA molecule can contain a variable sequence from the first, paired, second dsDNA molecule (and its pair), and third dsDNA molecule (and its pair); in some embodiments the fourth dsDNA molecule contains a variable sequence of a plurality of third dsDNA molecules. Such can continue and five to ten dsDNA molecules can be synthesized in hierarchal fashion, as generally depicted in Figure 3. When digested by a Type IIS restriction endonuclease the dsDNA molecules will produce a dsDNA fragment having 3’ and/or 5’ overhang sequences that are complementary to at least one other dsDNA fragment in the mixture (or produced by a parallel synthesis reaction) at their variable sequences.
  • the product DNA molecule can be optionally assembled having flanking sequences, useful for continuing procedures (e.g. PCR or other DNA amplification). Flanking sequences can be of any length appropriate for the continuing procedures contemplated, for example about 12 nucleotides, or about 18 nucleotides, or about 18-22 nucleotides or 18-30 nucleotides or 18-60 nucleotides. Flanking sequence can include a 5’ cap to discourage degradation of the dsDNA molecule, and a universal primer binding sequence to aid amplification.
  • the methods therefore allow the production of a product DNA molecule having a variable sequence of any length without the need for a conventional oligonucleotide synthesizer, which typically relies on chemical synthesis (e.g. phosphorami di te chemistry). Instead, the methods rely on enzymatic-based synthesis, and therefore the DNA molecules or polynucleotides can be produced on demand.
  • DNA molecules can refer to single-stranded polynucleotides or double-stranded DNA bound by Watson-Crick base pairing.
  • the methods can also involve performing multiple cycles of PCR or another DNA amplification procedure on any product DNA molecule.
  • the methods or any step of the methods can be performed without cloning or the need for cloning. In any embodiment the methods or any step of the methods can be performed entirely in vitro. In any embodiment the methods or any step of the methods can be performed without the use of live cells. In any embodiment the methods or any step of the methods can produce a scarless product DNA molecule.
  • scarless DNA is meant DNA that does not have any nucleotide(s) introduced by or from the process of synthesizing the DNA (e.g. residue nucleotides from a linker or flanking sequence). In any embodiment the methods or any step of the methods can produce a product DNA molecule that is barcode free, or free of a nucleotide sequence placed for identification purposes.
  • a barcode can be a sequence that is not otherwise needed but has a particular sequence and is used to identify a sequence of DNA.
  • a barcode sequence is 6-8 nucleotides in length, or 4-10 nucleotides in length.
  • the methods or any step of the methods can be performed without any part of any oligonucleotide used in the method being immobilized, i.e. bound to a solid phase or solid support (e.g. a bead, DNA chip, microfluidic surface, etc).
  • the oligonucleotides can be annealed in solution, and can be ligated in solution, i.e.
  • the methods or any step of the methods can synthesize the product DNA molecule without the use of and without performing chemical assembly techniques (e.g. phosphoramidite chemistry).
  • the methods or any step of the methods can assemble the product DNA molecule using only enzymatic assembly of oligonucleotides.
  • the methods or any step of the methods can be performed by drawing the at least two oligonucleotides and anchor strands from a library comprising less than 20,000 members, or from any library described herein.
  • the at least two oligonucleotides and anchor strands can be selected from an oligonucleotide library having less than 10,000 members, or from any oligo library described herein. In any embodiment the methods or any step of the methods do not utilize or require the use of a vector in the methods.
  • the product DNA molecule can optionally be formed having conserved flanking sequences and, optional universal primer binding sites on the 3' and 5' ends of the product DNA molecule.
  • the at least two oligonucleotides can be formed with one or more primer binding sites, which can provide binding sites for primers in amplification procedures (e.g. by PCR). Once the anchor strands are no longer necessary (e.g. a sufficiently long product DNA molecule has been synthesized), amplification can be done using primers that bind to the conserved flanking sequences and the universal primer binding sites are not needed.
  • the method can be facilitated by the use of recognition sites for a restriction endonuclease that can be effectively activated or inactivated.
  • recognition sites for a restriction endonuclease can have an inactive recognition site and 02 an active site programmed into the sequence when bound to the anchor strand 03.
  • This recognition site can be utilized to digest the formed dsDNA molecules 07 and 08 and digest the dsDNA molecules at one location leaving 5’ and 3’ overhangs.
  • the restriction sites can be formulated within the sequences so that the restriction site is active on one side of the dsDNA molecule and inactive on the other, and vice versa for the other member of the pair.
  • each dsDNA when digested each dsDNA will produce two dsDNA fragments, which can then be annealed.
  • a larger dsDNA e.g. one having at least a 20mer or 28mer or similar variable sequence
  • the dsDNA molecule can be formulated so that it has active restriction recognition sites on both sides of the dsDNA molecule.
  • digested it will be cut into at least three fragments, at least one having both a 3’ and 5’ overhang sequence, which can be at the variable sequence.
  • This additional dsDNA fragment can then be included within an annealing and ligation reaction with at least one 3’ end and at least one 5’ end of the dsDNA molecule, per Figure IB.
  • the recognition (and restriction) sites can be turned “on” or “off’ by utilizing a primer having a nucleotide mismatch, so the product dsDNA molecule no longer has an active recognition site (or does have one where formerly it did not).
  • the restriction site for Bsal is 5'-GGTCTC(Nl)-3' (SEQ ID NO: 17).
  • restriction site By changing one nucleotide in the sequence on can turn the restriction site “off’ (or vice versa) by utilizing a primer with a single mismatch.
  • This can be utilized for any restriction endonuclease and can be used to place or remove a recognition site on either or both ends of the DNA.
  • the methods can include a step of removing conserved flanking sequences and/or universal primer binding sites on one side or both sides of the DNA molecule after amplification to yield a product DNA molecule.
  • Methods of removing flanking sequences are known in the art.
  • the conserved flanking sequences and/or universal primer binding sites can be utilized to add length to the product DNA molecule, or to surround the product DNA molecule with transcriptional elements or other beneficial sequences that will be utilized in the final desired sequence.
  • the flanking sequences can be set to provide a promoter in front of the product DNA molecule, and/or to provide a terminator (i.e. regulatory sequences).
  • the product DNA molecule is a gRNA sequence (e.g.
  • flanking sequences can optionally be set to provide a promoter in front of the gRNA sequence, and a Cas9 handle and terminator after it.
  • the product DNA molecule can be expanded to encompass the universal primer binding sites and/or flanking sequences and/or one or more regulatory sequences and/or a Cas9 handle, any of which can provide more utility than being only binding sites for primers.
  • Any of the methods disclosed herein can be performed in an automated method, for example by an automated instrument.
  • An automated method is one where no human intervention is necessary after the method is initiated - the method goes to completion from that point without a human having to perform any action.
  • the automated instrument can contain components for selecting oligonucleotide members from the oligo library.
  • a DNA sequence to be assembled can be uploaded, recorded on, or stored on a non-transitory computer-readable medium.
  • a non-transitory computer-readable medium can be programmed to execute automated steps when inserted into or otherwise in electronic communication with a processor attached to or comprised within the automated instrument.
  • the automated steps can be any disclosed herein for performing any method disclosed herein.
  • the invention also provides a non-transitory computer-readable medium that is programmed with the locations of each member of an oligonucleotide library described herein, where the oligonucleotide library is present on a suitable support structure for the oligo library.
  • the non-transitory computer-readable medium is programmed with the locations of at least 6,000 or at least 9,000 oligonucleotide library members.
  • the medium can also be programmed with instructions to combine 4-6 members of a binding set from the library and to assemble the members of the binding set into a product DNA molecule according to the methods described herein.
  • a “member” of a library is one or more polynucleotides at a location.
  • An oligo library can be comprised on any type of medium, for example a multi-well plate or plurality of plates.
  • kits having an oligo library described herein located on a medium can be any suitable medium, for example one or more of a DNA chip, one or more bead(s), microtubes, one or more of a 96 well plate, one or more of a 384 well plate(s), one or more 1536-well plate(s), one or more microfluidic reaction support(s), one or more microtiter plate(s), one or more nanotiter plate(s), one or more picotiter plate(s), or other solid support or solid phase surface that can retain oligonucleotide members of the library.
  • the media can be present in numbers sufficient to accommodate the oligo library.
  • the medium containing the oligonucleotide library can contain members in any suitable volume, and examples include volumes of 1 nl up to 100 ul, or 10 nl up to 100 ul.
  • a DNA chip (or DNA microarray) is a solid surface having a collection of microscopic locations, to which oligonucleotides can be attached and/or stored.
  • any of the methods of the invention can synthesize a product DNA molecule with a very low error rate.
  • the methods can produce any product DNA molecule described herein with error rates of less than 1 in 1,000 nucleotides, or less than 1 in 2,000 nucleotides, or less than 1 in 2,400 nucleotides, or less than 1 in 2,500 nucleotides, or less than 1 in 3,000 nucleotides, or less than 1 in 8,000 nucleotides.
  • the methods can begin with a pooling of at least two oligonucleotides and an anchor strand from the oligo library.
  • a general embodiment is depicted in Figure 1 A-B. In the embodiment described here multiplexing will be utilized, and the at least two oligos and anchor strand have been prepared with restriction sites for Bsal, although any Type IIS restriction enzyme can be utilized.
  • the pool of oligos can be subjected to a step of annealing and a step of ligation (e.g. L0 and PCR1).
  • the ligation step can be performed by contacting the pool of oligonucleotides with a ligase, for example T4 DNA ligase. But any ligase can be utilized at any step in the invention.
  • Ligation involves the annealing of complementary 5’ and 3’ overhang sequences on the dsDNA fragments produced by the digestion with restriction endonuclease. Ligation can also involve contacting the oligos with a ligase.
  • the polymerase chain reaction (PCR) is a common reaction in biology known to persons of ordinary skill.
  • PCR can be used in the invention according to normal procedures and well known techniques.
  • PCR results in amplification of the oligos, depicted in the example in Figure 1 A as 07 and 08 and having variable sequences that are 16mers.
  • the methods can involve a step of digestion with restriction endonuclease and annealing with additional dsDNA fragments and ligation, followed by a step of PCR (DI and PCR2).
  • DI and PCR2 a step of PCR
  • the oligo set can be digested with the restriction enzyme.
  • Another digestion (DL2) can be performed on the product to result in dsDNA fragments, steps of annealing, ligation, and PCR3 to form dsDNA molecules, depicted in Figure 1 A as having variable sequences that are 28mers ( Figure IB).
  • a step of digestion with restriction endonuclease, annealing with additional dsDNA fragments and ligation (DL3) can then be utilized.
  • the ligation can optionally involve dsDNA fragments from parallel reactions or otherwise synthesized that have a 3’ and/or 5’ overhang complementary to at least one other dsDNA fragment in the mixture. In this manner the length of the variable sequence can be quickly increased.
  • the product dsDNA molecule of desired sequence in this embodiment is a lOOmer variable sequence depicted in Figure IB.
  • the universal primer binding sites can be present on some DNA molecules in some embodiments of the methods. In some embodiments the sites can be present on the at least two oligonucleotides and on the at least one first dsDNA molecule. However, in some embodiments these sites can be eliminated in any step after the anchor strand is no longer utilized. For example, the sites can be eliminated after formation of the at least one first or second dsDNA molecule, and the conserved flanking sequences used as primer binding sites thereafter.
  • the at least two oligonucleotides and the anchor strand can have universal primer binding sites, which then are present in the at least one first dsDNA molecule, but any one or more of the second, third, and fourth dsDNA molecules can not have a universal primer binding site.
  • Universal primer binding sites can also be added to dsDNA molecules at any step where convenient in the methods, e.g. on forming the final product dsDNA molecule it may be found desirable to have a convenient methods of amplifying the product.
  • the length of the universal primer binding site can be at least 6 nucleotides or at least 10 or at least 15 or at least 18 or at least 20 nucleotides or at least 25 nucleotides, or less than 15 nucleotides, or less than 12 nucleotides or less than 10 nucleotides or less than 8 nucleotides, but no particular length is necessary, only that the site allow for binding of a primer and amplification of the molecule.
  • the universal primer binding sites have the same sequence on all molecules in a mixture, enabling amplification of the mixture from a single set of primers. In any step of amplification all dsDNA molecules to be amplified can have a universal primer binding site of the same sequence.
  • variable sequence in the dsDNA molecule can grow longer as the methods proceed due to progressively combining more DNA containing a variable sequence that will be part of the product dsDNA molecule.
  • variable sequence in the first dsDNA molecule can equal the variable sequences from the at least two oligonucleotides combined.
  • variable sequence in the first dsDNA molecule is 6-14 nucleotides, or 8-12 nucleotides or about 10 nucleotides, which can be adjusted depending on the dsDNA molecule to be synthesized.
  • the second dsDNA molecule can have a variable sequence of 8-24 or 10-22 or 14-18 or 15-17 base pairs.
  • the third dsDNA molecule comprises a variable sequence of 18-38 or 20-36 or 24-32 or 26-30 or 27-29 base pairs.
  • the fourth dsDNA molecule can have a variable sequence of 70-130 or 80-120 or 90-110 base pairs. But the length of the variable sequence in any step is not fixed and can be varied to whatever is convenient or desirable in the application.
  • variable sequences can be sequences that will be present in the product dsDNA molecule. Thus, the variable sequences will vary in each construct depending on what portion of the final product DNA molecule it is carrying and what product dsDNA molecule is being synthesized.
  • the product DNA molecule can be the DNA molecule having the desired sequence. In one embodiment all of the variable sequences in the at least two oligonucleotides will be present in the product dsDNA molecule produced at the end of whichever method is performed.
  • variable sequence in the at least two oligonucleotides can be at least 4 nucleotides, or at least 5 nucleotides, or at least 6 nucleotides, or at least 10 or at least 12 or at least 15 or at least 18 or at least 20 nucleotides, or 3-7 nucleotides or 4-6 nucleotides, or 4-8 nucleotides, or 6-10 nucleotides, or 6-12 nucleotides, or 12-16 nucleotides, or 14-18 nucleotides.
  • the variable sequence for an anchor strand can be equal to the lengths of the variable sequences in the at least two oligonucleotides.
  • the variable sequence on the anchor strand can anneal entirely with the variable sequences on the at least two oligonucleotides.
  • variable sequence can be present as one consecutive sequence, or the nucleotides of the variable sequence can be separated singly or in groups of two or three or four or more consecutive nucleotides throughout the oligo sequence to comprise a variable region.
  • the variable sequence can be at least a portion of the desired sequence or product dsDNA molecule to be synthesized in the methods.
  • a variable sequence can represent a distinct sequence for each possibility presented by the length of the variable sequence, and each distinct sequence can be present at a distinct location in the oligo library.
  • each oligo having a distinct variable sequence can be located at a distinct location in the oligo library. For example, 01 of the at least two oligonucleotides has a variable sequence.
  • variable sequence is five nucleotides
  • 01 can have 1024 possible nucleotide sequences, i.e. 4x4x4x4 equals 1024 variable sequences for 01.
  • 4x4x4x4x4 equals 1024 variable sequences for 01.
  • 02-06 depicted in Figure 1.
  • Variable nucleotides can be dispersed in the sequence, singly or in groups as explained above.
  • variable sequences of two dsDNA molecules can overlap, i.e. have a common sequence for two or more nucleotides.
  • any of the dsDNA molecules can contain variable sequences that overlap by at least 1 or at least 2 or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8 nucleotides, or by 1-6 nucleotides or 2-5 nucleotides, or by 3-10 nucleotides, or by about 4 nucleotides, or by at least 10 nucleotides, or by more than 8 nucleotides.
  • the first or second or third dsDNA molecules, or additional dsDNA molecules described herein can have variable regions that overlap with other dsDNA molecules as described.
  • the first dsDNA molecule can overlap with its paired dsDNA molecule
  • the second dsDNA molecule, third dsDNA molecule, or additional dsDNA molecules can all overlap with their paired dsDNA molecule.
  • dsDNA molecules can also overlap with any other dsDNA molecule (e.g. a second dsDNA can be made to overlap with a third dsDNA from a parallel synthesis reaction.
  • dsDNA fragments can also have a 3’ and/or 5’ overhang sequence that contains a variable sequence that overlaps by at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8 nucleotides, or by at least 10 nucleotides, or by more than 8 nucleotides with other (paired) dsDNA fragments.
  • a first dsDNA fragment can have a variable sequence on the 3’ and/or 5’ overhang that overlaps with that of a paired dsDNA fragment on its 5’ or 3’ overhang.
  • a second dsDNA fragment can have 3’ and/or 5’ overhang sequence that contains a variable sequence that overlaps with that of its paired dsDNA fragment, or an additional dsDNA fragment.
  • the 3’ and/or 5’ overhangs can be produced by restriction endonuclease action on a dsDNA molecule, and can also be synthesized separately and provided to any of the reactions.
  • dsDNA fragments can have 3’ and/or 5’ overhang sequences that are part of the variable sequence, and which can be used to anneal and combine them with one or more other dsDNA fragments at their variable sequences.
  • One or more of the at least two oligonucleotides and the anchor strands used in the methods can, optionally, have one or more degenerate nucleotides.
  • Degenerate nucleotide refers to degenerate positions in the oligo sequence. In any embodiment the degenerate nucleotides can be present within and as part of the variable sequence of the anchor strand or other oligonucleotide in the methods.
  • a degenerate nucleotide in an oligo is a nucleotide that can be any of A, C, T, or G, i.e. a nucleotide position in a library member that has been randomized.
  • Randomization can be performed by simply supplying all four bases during oligo synthesis producing a randomized oligonucleotide.
  • degenerate nucleotides can be a universal base. Examples include deoxyinosine, 2-deoxyinosine, nitroindole, 2’-deoxynebularine, 3 -nitropyrrole, dP, dK, or other universal bases can be used to reduce degeneracy. 3 -nitropyrrole 2’ -deoxynucleoside, and 5- nitroindole 2’-deoxynucleoside can also be used as degenerate bases.
  • An oligo having one or more degenerate nucleotides is a degenerate oligonucleotide.
  • Degenerate oligos can be colocated at the same (degenerate oligonucleotide) location in an oligo library. Degenerate oligos can thus be present at a location as a group of slightly different sequences, with each degenerate oligo having a distinct sequence due to the degenerate nucleotides, yet all colocated at the same location.
  • degenerate nucleotides on one oligo can anneal to nucleotides of a variable sequence on another (target) oligo, such as is depicted in Figure 1-2.
  • any of the oligonucleotides in a binding set can have degenerate nucleotides in its variable sequence.
  • at least one oligonucleotide in a binding set has degenerate nucleotides.
  • only the anchor strand has a variable sequence with degenerate nucleotides.
  • the anchor strands are depicted as having degenerate oligonucleotides (designated “N”) in the variable sequence.
  • a binding set is a group of oligonucleotides that bind to one another in the methods.
  • O1-O3 is a binding set, as is 04-06.
  • oligos of a binding set can bind substantially to one another (i.e. not merely by a small amount). In another embodiment oligos of a binding set bind to each other without mismatched base pairs. In one embodiment at least one oligo of a binding set binds completely to one or two other oligos of the binding set, i.e. no with no unmatched bases.
  • a “target” oligo is an second oligo that a first oligo is intended to bind to in the methods.
  • One or more anchor strands in a method can have 3 or 4 or 5 or 6 or 7 or 8 or 3-5 or 3-6 or 3-7 or 3-8 or 4-5 or 4-6 or 4-7 or 4-8 or 6-10 or more than 8 or more than 10 or more than 12 degenerate nucleotides in its variable sequence.
  • the one or more degenerate nucleotides in an oligo can be present as one consecutive sequence to comprise a degenerate sequence, or the degenerate nucleotides can be separated singly or in groups of two or more consecutive degenerate nucleotides throughout the oligo (e.g. an anchor strand). In some embodiments degenerate nucleotides are present only within a variable sequence of the oligos, or only within the variable sequence of the anchor strand(s).
  • Degenerate oligonucleotides present at a location in the oligo library have multiple sequences at the location and can be grouped together and considered as one member of the library.
  • a location in the oligo library containing the multiple sequences of degenerate oligonucleotides is termed a degenerate oligo location.
  • degenerate oligonucleotides can be co-located at a single location in the oligo library. While in some embodiments all possible sequences of a degenerate oligonucleotide are provided at the same location (e.g. all 1024 possible sequences of a degenerate oligo having 5 degenerate nucleotides), in other embodiments multiple degenerate oligonucleotides can be located in groups of convenient numbers at multiple different locations in the oligo library. Degenerate nucleotides allow the user to therefore greatly reduce the number of positions in the oligo library.
  • oligos having one or more degenerate nucleotides can be colocated together at a single defined location in the library
  • oligos having variable sequences with no degenerate nucleotides can each have their own defined location in the library, i.e. a separate location for each sequence.
  • An oligonucleotide having one or more degenerate nucleotides can be co-located at a single location with all possible sequences of the oligo for each degenerate position present at the single location.
  • degenerate sequences D1-D4096 can all be present at each of variable locations L1-L256 for 03, with each location having a distinct sequence for the nondegenerate positions on the sequence, and oligos of all possible sequences at the degenerate positions.
  • location LI for the example 03 can be SEQ ID NO: 3 NNNACTCNNN (VI), plus degenerate sequences D1-D4096 having the non-degenerate portion of variable sequence, and all possible sequences for the degenerate nucleotides in each location.
  • degenerate sequences D1-D4096 will all have non-degenerate sequence V2.
  • degenerate sequences D1-D4096 will all have non-degenerate sequence V3, and so on.
  • degenerate sequences D1-D4096 for 03 are all present at locations L1-L256 for 03, with each degenerate sequence having the non-degenerate portion of the variable sequence.
  • All anchor strands will thus contain the same set portion of the sequence, but all will vary in sequence at the degenerate nucleotides.
  • the library can have 256 locations for 03 in this example.
  • the invention also provides methods of synthesizing a product DNA molecule from a library of oligonucleotide members.
  • the library of oligonucleotide members can have fewer than 10,000 or fewer than 5,000 oligonucleotide members, and the oligonucleotide members in the library are sufficient to assemble any possible polynucleotide sequence.
  • the method involves assembling oligonucleotide members from the library to obtain the product DNA molecule.
  • oligonucleotides O1-O6, are members in the library.
  • 01-06 can each have one or more variable sequences.
  • the oligonucleotide library can be comprised on any one or more of a DNA chip, solid support, solid phase, bead, microfluidic surface, plate, etc, or other structure where oligonucleotides can be stored at defined locations and be available for retrieval and use in the methods.
  • the library will contain a distinct location for each of the possible variable sequences of 01- 06.
  • 01-02 and 04-05 have a variable region having 5 variable nucleotides
  • the number of locations to accommodate the possible sequences of the oligos is 4 to the 5th power, thus 4x4x4x4x4 equals 1,024.
  • 01 oligos can have five variable nucleotides and thus 1024 possible sequences, which can be present at 1024 defined locations for 01 with a single defined variable sequence at each location, and similar for 02 and 04-5.
  • each anchor strand has four non-degenerate nucleotides, and six degenerate nucleotides.
  • the library can also have 256 locations for each of 03 and 06 to accommodate the non-degenerate nucleotides, with each location having a distinct sequence for non-degenerate nucleotides.
  • each of the 256 locations can have all possible degenerate sequences, thus 4,096 degenerate oligo sequences are present together at each of the 256 locations for the set nucleotides of the variable sequence.
  • a location in the oligo library can be a well of a plate, a tube, or any other structure that segregates an oligonucleotide member in a distinct location, spatially separated from other members of the library sufficiently for it to be accessed individually and as a species at this distinct location.
  • the oligos can be maintained in their distinct locations as a single molecule (from which a complementary sequence can be synthesized) or as a multiple copies of the same molecule (from which a small volume can be taken and used in synthesis procedures).
  • the distinct locations can be identifiable to a software program that can be configured with a mechanical component or device that retrieves library members from the distinct location for use in a method of the invention where the defined oligonucleotide library member is required.
  • an oligo library can be located in a collection of assay plates or small tubes, each containing a member of the oligo library, and to which instrumentation components can go and retrieve an oligo library member according to software instructions, which can be located on a non-transitory computer-readable medium.
  • the non-transitory computer readable medium can also contain programmed instructions and/or steps for synthesizing a product DNA molecule according to any of the methods disclosed herein, and the programmed instructions and/or steps can be provided to an instrument in communication with the computer-readable medium.
  • the programmed instructions or steps can direct the instrumentation to perform the assembly of a DNA molecule of pre-defined sequence according to any method disclosed herein, or to perform any of the methods provided herein.
  • oligonucleotide library are present at distinct locations, spatially separated from other members of the library.
  • a member of the library can be a specific sequence present at its location (either singly or multiple copies).
  • the member of the library containing degenerate sequences can be all possible degenerate sequences (or in some embodiments a subset of all possible sequence) in view of the number of degenerate nucleotides, and present at a distinct location.
  • the distinct location can be defined by any suitable technique, for example reference points in a microscopic picture or grid of the solid support containing the oligo library.
  • the distinct location can be stored on and/or communicated by a non-transitory computer-readable medium.
  • the product DNA molecules can be assembled if desired into larger product dsDNA molecules.
  • the product dsDNA molecules will be doublestranded blunt end DNA.
  • DNA molecules can be synthesized so that the variable sequences between product dsDNA molecules contain an overlapping sequence.
  • the dsDNA can be digested with a restriction endonuclease that cleaves within the variable sequence of each dsDNA and leaves overhang sequences or "sticky ends” in the dsDNA fragments remaining. These overhang sequences can then be used to assemble the dsDNA fragments into a larger DNA molecule through annealing to complementary 3’ and/or 5’ sequences on additional dsDNA fragments.
  • the product DNA molecule can be synthesized having single-stranded overhang sequences of one or more nucleotides, or of 4 nucleotides or 5 nucleotides or 6 nucleotides or 7 nucleotides or 8 nucleotides or a more than 8 nucleotide single-stranded overhang.
  • Type IIS restriction enzymes cleave DNA at a defined distance from their recognition site and leave a 5’ and 3’ single-stranded overhang.
  • the recognition site can be programmed to lie outside of the variable sequence, and the cleavage site can be programmed to lie within the variable sequence, leaving 3’ and/or 5’ overhangs on the resulting dsDNA fragments.
  • Type IIS restriction endonucleases also find application in the invention for producing additional dsDNA fragments having single-stranded overhangs.
  • the singlestranded overhangs can be present at the 3’ and/or 5’ ends, depending on where in the molecule the dsDNA fragment is to be positioned relative to other fragments.
  • dsDNA molecules can be programmed to have active recognition sites on the 3’ and 5’ sides of the dsDNA molecule and on both sides of the variable sequence.
  • the dsDNA molecules can also be programmed to have cleavage sites towards the 5’ and 3’ ends of the variable sequence.
  • dsDNA fragments can be joined by annealing dsDNA fragments having complementary overhanging 3’ or 5’ sequences and ligating to form a longer DNA molecule. Multiple additional dsDNA fragments having 3’ and 5’ overhangs can be annealed to dsDNA fragments having complementary 3’ or 5’ overhangs.
  • dsDNA fragments at any step can be annealed to a plurality of additional dsDNA fragments to more rapidly advance the size of the variable sequence of the product dsDNA molecule.
  • a dsDNA molecule can be synthesized having a 100 bp variable sequence or larger.
  • the restriction enzyme utilized in the invention can be a Type IIS restriction enzyme.
  • the Type IIS restriction enzyme is one that only cleaves dsDNA.
  • Type IIS restriction sites can be encoded into the conserved flanking sequences, as illustrated in Figure 1. Any Type IIS restriction enzyme can be utilized.
  • the restriction sites can be BsmBI sites, or BsmBi sites, or Ecil sites, or BspMI sites, or Faul sites, etc.
  • BsmBi recognizes the sequence 5'- CGTCTC(N)-3' (SEQ ID NO: 16). The enzyme generally cleaves to the 3' side of N.
  • Bsal is another Type IIS restriction enzyme, which recognizes the sequence 5'-GGTCTC(Nl)-3' (SEQ ID NO: 17) and generally cleaves to the 3' side of N.
  • Persons of ordinary skill with resort to this disclosure will realize many other Type IIS restriction enzymes can be utilized in the invention. Such persons can also easily determine where the enzymes will cut in any particular application.
  • any of the DNA molecules utilized in or produced by the methods can contain one or more Type IIS restriction endonuclease recognition sites.
  • DNA is stable even over periods of thousands of years and even in many extreme environments, giving it great advantages for storing information. Any of the methods disclosed herein can be applied to encoding digital data into DNA.
  • One or more product DNA molecule(s) can have a sequence that comprises an encoded non-genetic message.
  • One or more product DNA molecule(s) can have a sequence that corresponds to bytes of information that encode the non-genetic message. The bytes of information can be decoded with reference to a key that assigns one or more language character(s) to each encoded character or byte of information.
  • a 16 bp DNA molecule can be synthesized and easily accommodates four bytes of information, where each byte is encoded by an assigned sequence of nucleotides.
  • a four nucleotide sequence represents a byte of information, which can correspond to a character (e.g. a letter or numeral).
  • 256 characters can be encoded in each byte of information (4x4x4x4).
  • the alphabet of any language in the world can be easily accommodated within these 256 bytes of information and a sufficient number of numerals and other characters utilized in communication as well.
  • the message can be encoded in a reference language, such as English, French, German, Italian, Spanish, Latin, Japanese, Hindi, Chinese, Russian, or any language.
  • a reference language can also include numbers and special characters, even though not formally part of the reference language. But any information can be encoded in the DNA sequence in any language.
  • the product DNA can also encode a character (e.g. a letter, a word, a number, a punctuation mark, word character, or other characters utilized in communication) indicating where in the sequence the information encoded by that DNA molecule is to be placed.
  • Figure 5 depicts 16 bp product DNA molecules having four bytes of four nucleotides each. The last byte in each product DNA sequence indicates the location in the message where the preceding three bytes are placed; this is conveniently a numeral but can be any character that can be placed into a definable sequence. While a 4 nucleotide byte provides up to 256 identifiers the byte can be any convenient length of nucleotides.
  • bytes can be comprised of 5 nucleotides or 6 nucleotides (allowing for 4,096 identifiers), or 7 or even 8 nucleotides, or more than 8 nucleotides, allowing for many more identifiers to be included.
  • Limited numbers of identifiers can also be expanded by placing DNA molecules in a single well up to the number of identifiers, and then assembling the messages from the DNA in the order of the sequence of wells. Using this method with only a 4 nucleotide identifier even a single 384 well plate can contain over 98,000 DNA molecules (256 molecules x 384 wells), which can be assembled in order to provide almost 300,000 bytes of information (in addition to the identifier).
  • the invention provides methods of storing data in a DNA sequence, which can involve determining a sequence of DNA that encodes a non-genetic message according to a coding scheme that can translate the non-genetic message from a reference language into a DNA sequence and vice versa; synthesizing the sequence of DNA that encodes the non-genetic message according to a method disclosed herein; and thereby store data in a DNA sequence.
  • a coding scheme is a set of codes (e.g. 4 or 3 nucleotide codons, an example of which is shown in Figure 5) that assign a particular character of a reference language to a particular codon.
  • the standard DNA codon table is a coding scheme, but it may be advantageous to use a coding scheme that is not easily transcribable.
  • Other examples of coding schemes are known to persons of ordinary skill in the art.
  • the invention can also be applied to the synthesis of guide RNAs (gRNA) for use in CRISPR-Cas9 methods. Using the methods any sequence of gRNA can be quickly constructed.
  • Guide RNA constructs can also be constructed from oligonucleotides in the oligonucleotide library.
  • a product DNA molecule can be synthesized in the methods having a DNA sequence that encodes an initial guide structure.
  • the initial guide RNA structure can encode a gRNA with the necessary prokaryotic or eukaryotic transcriptional elements for in vitro transcription in proper order, for example any one or more of a promoter, a sequence of gRNA, and a terminator.
  • the gRNA can encode a Cas9-binding hairpin (Cas9 handle).
  • the transcriptional elements include a promoter and/or a terminator.
  • the product DNA molecule can encode 20 bases for the gRNA.
  • Figure 6 depicts one embodiment in which a dsDNA molecule is synthesized into an initial guide structure having the transcriptional elements.
  • the product dsDNA molecule can encode a guide structure or gRNA or other RNA molecule. Since all possible polynucleotide sequences can be assembled from the oligo library, any initial guide structure or gRNA or RNA can be assembled in the methods.
  • the method involves annealing at least two oligonucleotides of about 30-60 nucleotides in length with an anchor strand about 30-70 nucleotides in length according to the methods disclosed herein.
  • the method involves annealing at least two oligonucleotides of about 40-50 nucleotides in length with an anchor strand about 40-50 nucleotides in length according to the methods disclosed herein.
  • the method involves annealing at least two oligonucleotides of about or about 40-50 nucleotides in length with an anchor strand about 40-60 nucleotides in length.
  • the anchor strand can utilize 4-6 or 6 degenerate oligonucleotides.
  • the method involves annealing at least two oligonucleotides of about or about 40-50 nucleotides in length with an anchor strand about 45-55 nucleotides in length.
  • the anchor strand can utilize 4-6 or 6 degenerate oligonucleotides.
  • This example shows the synthesis of a dsDNA molecule of desired sequence having a 100 base pair variable region in a hierarchal method.
  • the “L0” reaction included two oligonucleotides 01 and 02 (each 45 nucleotides), each of which had a variable sequence of 5 nucleotides, a conserved flanking sequence of about 20 nucleotides, and a primer binding site of about 20 nucleotides.
  • the anchor strand 03 was programmed to have a variable sequence of 10 nucleotides and be 50 nucleotides in length.
  • the oligonucleotides were selected so that the sequence produced by the O1-O3 synthesis (LO) would be a portion of the 100 nucleotide variable sequence of the pre-determined total dsDNA molecule, and would have a variable sequence of about 10 nucleotides.
  • the oligonucleotides were also selected to encode a restriction site for Bsal, a Type IIS endonuclease, on the 5’ side of the DNA molecule (for later ligation with a paired dsDNA molecule having an active recognition site on the 3’ side of the DNA molecule).
  • a solution was prepared containing oligonucleotides O1-O2 (two oligonucleotides) and 03 (the anchor strand) (2 ul of pool at 100 pM).
  • the oligonucleotides were placed into wells containing T4 DNA ligase buffer (0.5 ul), water (2.4 ul), and T4 DNA ligase (0.1 ul).
  • the solution was incubated for 1 hour at 16 °C, then for 10 minutes at 65 °C.
  • PCR1 PCR amplification
  • a digestion and ligation step DL1 was then performed. Water (2.3 ul), T4 ligation buffer (0.5 ul), Bsal enzyme (0.1 ul), T4 DNA ligase (0.1 ul), and the PCR1 product were mixed together. The mixture was incubated for 1 minute at 37 °C followed by 1 minute at 16 °C and cycled 10 times. Finally, the mixture was held at 80 °C for 20 minutes. A step of PCR (PCR2) was then performed on the DL1 product in a mixture of water (2 ul), 5’ and 3’ primers (1 uM), the DNA polymerase above (5 ul), and then diluted 150x. PCR cycles and CIP+CE and proteinase K were performed as above. The dsDNA molecule produced had a variable sequence of 16 nucleotides.
  • PCR3 was then performed on the DL2 product in a mixture of water (2 ul), 5’ and 3’ primers (1 uM), the DNA polymerase above (5 ul), and then diluted 150x. PCR cycles and calf intestinal phosphatase (CE) and proteinase K digestions were performed as in step 5 above. Amplification products were verified on a gel showing the presence of 88, 68, 68, and 88 bp products, which had a variable sequence of 28 nucleotides. The 28mer containing dsDNA molecule was also produced so that it would have a Type IIS restriction site on one side of the molecule.
  • a digestion reaction was performed and the resulting dsDNA fragment was combined with dsDNA fragments from parallel reactions, one which was a dsDNA fragment that was all variable sequence and derived from a digestion of a dsDNA molecule with recognition sites on both sides of the dsDNA, while maintaining the conserved flanking sequences from the 3’ and 5’ ends to allow for efficient ligation and to enable universal primers to be used in downstream PCR (for example illustrated in Figure IB).
  • a ligation step was performed on the dsDNA fragments (DL3) using 16.5 ul water, lOx T4 ligation buffer (2.5 ul), Bsal (0.5 ul), T4 DNA ligase (0.5 ul), and 5 ul of the pooled PCR3 product. The mixture was incubated for 1 minute at 37 C followed by 1 minute at 16 °C and cycled 25 times. Finally the mixture was held at 80 °C for 20 minutes.
  • a step of PCR was then performed on the product in a mixture of water (6 ul), 5’ and 3’ primers (2 ul of 1 uM), the DNA polymerase above (10 ul), and 2 ul of the digestion and ligation product. PCR cycles and CE and proteinase K were performed as in step 5 above. Amplification products were verified on a gel showing the presence of a 180 bp product, which had a variable sequence of 100 nucleotides. The molecule was sequenced and found to have the correct sequence with no errors.
  • This example shows construction of a universal oligonucleotide library. Considerations in selecting a library included whether flanking sequences that would serve as robust universal priming sequences and ensure that 5’ and 3’ flanking sequences were distinct enough so that PCR primer sequences would not cross-react in the PCR steps. A common feature in all the flanks was a Type IIS site and this was held constant within the flanking sequence and designed around. These sequences were generated by computational design but can also be generated manually.
  • flanking sequences were empirically selected by ordering approximately eight sequences from a commercial supplier and testing them directly in PCR. The best flanking sequence set was then selected. The “flank set” was tested with the 5’ and 3’ primer pair, the 5’ only, and the 3’ only to ensure that the expected PCR product would be generated.
  • variable sequences were added to the sequences. Note that all possible permutations of the variable bases were needed to be able to construct any DNA sequence. For example, if 5 variable bases were added to the 3’ end of 01, there was 4 to the 5 th power or 1,024 different 01 sequences in separate microtiter wells where 4 is the number of DNA bases available and 5 is the number of variable bases utilized in the 01 oligo. These variable sequences were generated by available computational design programs but can also be generated manually. [0100] In the case of 01, the variable bases were added to the 3’ end (e.g., 5 variable bases). In the case of 02, the variable bases were added to the 5’ end (e.g., 5 variable bases).
  • variable sequence non-degenerate bases were added to the central part of the oligo (e.g., 4 non-degenerate bases) to support the ligation of 01 and 02 at their abutting interfaces and then surrounded by degenerate N bases as these bases prevent the unnecessary expansion of the library.
  • the degenerate N bases were synthesized on the oligo synthesizer by combining all four DNA bases for the N position, thus a 03 anchor oligo was a mixture of sequences. For example, if a single 03 anchor had a total of six N positions there would be a total 4 to the 6 th power or 4,096 different molecules within a single library well. Not all the molecules in this library well were viable 03 anchors for the ligation of 01 + 02, but only a fraction of the 4,096 molecules were needed to support a robust LO ligation.
  • the oligos that made up the library were then synthesized in microtiter plate format in such a way that all oligo members had a discrete well location within the library.
  • the wells were in single micro-tubes or microtiter plate formats of 96 and 384-wells, but they can be any format that allows for the physical separation of library oligo members.
  • the location of each member was precisely known and could be accessed when the oligo components were pooled together, either manually or by laboratory liquid handling automation.
  • oligos (01, 02 & 03) were pooled into a single well and these oligos corresponded to the first 10 bp (bases 1 to 10) of the lOObp sequence in this example.
  • oligos were then pooled (i.e., the next set of 01, 02 & 03) into an adjacent well. These oligos constituted another lObp but overlapped the first lObp in “a.” above by 4bp, which constituted bases 6-14 of the lOObp sequence in this example.
  • Table 1 This table shows the number of oligo members in an entire library set that were needed to build any 10 -> 16 — > 28 — > lOObp DNA fragment. The total number of oligo members needed was 9216.
  • Table 2 This table shows the nucleotide lengths for each of the oligo members within the entire library set. The length of the non-degenerate nucleotides of the variable sequence is shown in parenthesis.
  • SEQ ID NO: 1 DNA, artificial sequence AGGGA
  • SEQ ID NO: 2 DNA, artificial sequence CGTTG
  • SEQ ID NO: 3 DNA, artificial sequence NNNACTCNNN
  • SEQ ID NO: 4 DNA, artificial sequence TTGCG
  • SEQ ID NO: 14 DNA, artificial sequence
  • SEQ ID NO: 16 DNA, BsmBI recognition site, Bacillus stearothermophilus CGTCTC(N)

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Abstract

L'invention concerne des procédés de synthèse d'une molécule d'ADN de produit de toute séquence d'ADN possible à partir d'une banque universelle d'oligonucléotides chevauchants. Le procédé consiste à combiner une pluralité d'oligonucléotides chevauchants dans un pool de réaction, où les séquences de la pluralité d'oligonucléotides comprennent au moins une sous-séquence de la molécule d'ADN du produit. Le procédé comprend également le recuit de la pluralité d'oligonucléotides, la réalisation d'une étape de ligature, et d'une étape d'amplification pour ainsi synthétiser une sous-séquence de la molécule d'ADN de produit. L'invention peut être utilisée pour synthétiser une molécule d'ADN de toute séquence possible à partir de la banque universelle, ce qui peut être réalisé grâce à une procédure d'assemblage hiérarchique. Dans un mode de réalisation, la banque universelle comprend moins de 10 000 oligonucléotides préfabriqués pouvant être synthétisés en toute séquence d'ADN possible. Dans n'Importe quel mode de réalisation, la molécule d'ADN de produit a un taux d'erreur inférieur à 1 erreur pour 2 000 nucléotides.
PCT/US2021/059422 2021-11-15 2021-11-15 Procédés de synthèse de molécules d'acide nucléique WO2023086109A1 (fr)

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Publication number Priority date Publication date Assignee Title
US20130309725A1 (en) * 2010-05-28 2013-11-21 Gen9, Inc. Methods and Devices for In Situ Nucleic Acid Synthesis
US20210277446A1 (en) * 2020-03-03 2021-09-09 Codex Dna, Inc. Methods for assembling nucleic acids

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130309725A1 (en) * 2010-05-28 2013-11-21 Gen9, Inc. Methods and Devices for In Situ Nucleic Acid Synthesis
US20210277446A1 (en) * 2020-03-03 2021-09-09 Codex Dna, Inc. Methods for assembling nucleic acids

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