Compositions and methods for phosphoramidite-free enzymatic synthesis of nucleic acids - Patents.com

Abstract

Described herein are compositions and methods for synthesizing any nucleic acid (NA) sequence using entirely natural nucleic acid sources without the need for large-scale phosphoramidite-mediated chemical synthesis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 179,828, filed April 26, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This application has been submitted in ASCII format via EFS-Web and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on April 26, 2022, is named "DNWR-009_001WO_SeqList.txt" and is approximately 17,420 bytes in size. [Background technology]

[0003] There is a need in the art for the synthesis of nucleic acids that significantly reduces the error rate and organic waste generation associated with phosphoramidite-mediated chemical synthesis.In addition, there is a need in the art for the synthesis of nucleic acids at significantly reduced costs.Described herein are compositions and methods for synthesizing any nucleic acid (NA) sequence using completely natural nucleic acid sources without the need for large-scale phosphoramidite-mediated chemical synthesis. Summary of the Invention

[0004] The present disclosure provides one or more addamers, each with a payload of at least 3 base pairs (bp). The full set of 3-mer donor or acceptor addamer designs includes all 64 3-mer bp possibilities as payloads. Other embodiments have similar hairpin designs with payloads of increased length. When the payload is 3, there are 64 possible addamers. With a 4 bp payload, there are 256 possible addamers in a given library. Similarly, for a 5-mer payload, there are 1,024 elements, and for a 6-mer payload, there are 4,096 elements. Because the addamers are double-stranded, it is possible, by placement of various design features, to use less than all possible N-mer maximums for a given library and still achieve complete sequence coverage.

[0005] The adamer can contain a hairpin turn of several bases and a short stretch of sequence that can be used to guide the cleavage of the payload from the hairpin region with an endonuclease. In addition, to facilitate binding to a solid support, the hairpin region can contain any of several possible specific structural sequences, such as an aptamer sequence for affinity purification or binding, an enzyme sequence (DNAzyme) for controlled self-cleavage, a nested endonuclease recognition site for binding to a solid support by sticky end ligation, or an unpaired single-stranded region for binding by hybridization to a solid support-binding anchor sequence.

[0006] Adamers can contain various offset-cleavage type II S restriction endonuclease sites to allow for various overhang lengths or to allow a given entire construct to be cleaved to a specific shorter length. The difference between the left and right endonuclease sites allows for the generation of specific "donor" vs. "acceptor" adamers intermediates when longer NA sequences are generated. As will be understood by those skilled in the art, hundreds of type II S restriction endonucleases (IISREs) have been identified. They are characterized by the fact that the double-stranded DNA recognition site is separated from the cleavage site. Because the method for synthesizing oligonucleotides from adamers relies on ligation, 5' overhangs are preferred, and various IISREs exist that result in 1, 2, 3, 4, and 5 base 5' overhangs as well as blunt ends.

[0007] In one embodiment, an array of unique acceptor and donor initiated payloads is generated by sequential ligation of 3-mer adamers. First, in the loading step, separate solid surfaces preloaded with "ligation studs" with double-stranded multiple cloning sites (MCS) are digested with an appropriate restriction endonuclease (RE), such as BamHI. Similarly, the first 3-mer adamer is RE digested and ligated to the "ligation stud". After exonuclease treatment and rinsing, the ligated adamer construct is digested with an appropriate blunt-cutting IISRE to obtain either a ligated adamer (acceptor) terminated with a blunt, exposed 3-mer sequence, or a free blunt-ended adamer (donor) terminated with an exposed 3-mer. The donor adamer-containing solution is transferred to the desired acceptor adamer-containing well, and these two adamers are blunt-ligated together using human DNA ligase III (hLig3), which has a high efficiency of over 60% for blunt ligation. After ligation, exonuclease is applied to the well to remove unreacted sites and rinse the well. At this point, the well contains solid support-bound adamers with hexameric payloads (see FIG. 8). The hexameric payloads can then be combined by application of the appropriate IISRE to generate acceptor and donor versions. The donor solution is then applied to the acceptor well and ligated via the sticky ends to obtain an extended payload, which is then treated with exonuclease and rinsed to obtain the desired intermediate payload. This reaction cycle is repeated to increase the payload length until the desired NA sequence payload is obtained (see FIG. 8).

[0008] The present disclosure provides an adamer library containing 64 possible 3-mer payloads, 256 possible 4-mer payloads, 1,024 possible 5-mer payloads, or 4,096 possible 6-mer payloads with the following features: a) a double-stranded duplex hairpin overall structure, and b) unique left and right type II S restriction endonuclease (RE) sites that allow for the generation of 0, 1, 2, 3, 4, or 5 bp overhangs.

[0009] In some embodiments, the hairpin region can include a structural feature, such as an aptamer, which can be used to immobilize the adamer to a solid support by molecular affinity for an aptamer ligand in some embodiments, and the hairpin region can include a restriction endonuclease site for restriction endonuclease cleavage to allow adamer ligation to a nucleic acid previously immobilized to a solid support. In some embodiments, the hairpin region can include a lambda phage cos site that can be cleaved by lambda terminase to allow adamer ligation to a nucleic acid previously bound to a solid support. In some embodiments, the hairpin region can include a single-stranded region that can hybridize to a nucleic acid previously bound to a solid support.

[0010] The present disclosure provides nucleic acid sequences derived from the combination of the adamers following restriction endonuclease cleavage and subsequent ligation.

[0011] The present disclosure provides double-stranded DNA anchor sequences with 5'-end modifications for attachment to solid supports, including multiple cloning sites, hairpin structures, and modified exonuclease-resistant 3'-ends.

[0012] The present disclosure provides single-stranded DNA anchor sequences having a 5' end modification for attachment to a solid support, including sequences complementary to a suitable adamer sequence and a modified exonuclease-resistant 3' end.

[0013] The present disclosure provides a method for generating an adammer library, comprising: a) designing library elements, including hairpin sequence and configuration, a type II S restriction endonuclease site, a payload, other restriction endonuclease sites, and target sites for insertion excision; b) cloning each of the different adamer elements of the library into a high copy number plasmid as either a single or multiple copy insert; c) purifying each plasmid or bacteriophage DNA; d) excising the insert using at least one of i) a nickase, ii) a Cas9 nickase with an appropriate guide RNA, and iii) a trans-acting or cis-acting DNAzyme; e) ligating the insert to generate an adamer structure; and f) optionally processing to purify the adamers.

[0014] The present disclosure provides a method for generating an adamer library, the method comprising: a) designing library elements including a hairpin sequence and configuration, a type II S restriction endonuclease site, a payload, other restriction endonuclease sites, and target sites for insertion and removal; b) performing phosphoramidite synthesis of separate top and bottom strands of specific adamers; c) optionally treating the pre-adamer duplex with MutS or a similar error-correcting enzyme; d) contacting the top and bottom strands of the specific adamers with a ligase enzyme to generate an adamer structure; and e) treating the product of step (d) with an exonuclease to purify functional adamers.

[0015] The present disclosure provides a method for attaching one or more adamers to a solid support, the method comprising: a) loading a double-stranded anchor sequence containing a 5' modification onto a solid surface; b) performing bacteriophage lambda terminase digestion of the anchor sequence; c) performing bacteriophage lambda terminase digestion of the one or more adamers; d) incubating the digested one or more adamers with the digested anchor sequence; and e) ligating the digested one or more adamers and the digested anchor sequence.

[0016] The present disclosure provides a method for attaching one or more adamers to a solid support, the method comprising: a) loading a double-stranded anchor sequence containing a 5' modification onto a solid surface; b) performing a restriction endonuclease digestion of the anchor sequence; c) preforming a restriction endonuclease digestion of the one or more adamers; d) incubating the digested one or more adamer sequences and the digested anchor sequence; and ligating the digested one or more adamers and the digested anchor sequence.

[0017] The present disclosure provides a method for binding one or more adamers comprising an aptamer sequence to a solid support, the method comprising: a) binding at least one chemical ligand to the solid support, where the at least one chemical ligand binds to the aptamer sequence; and b) incubating the one or more adamers and the solid support, thereby binding the one or more adamers to the solid support.

[0018] The present disclosure provides a method of synthesizing nucleic acids comprising a) attaching one or more adamers to at least one solid support, b) performing independent restriction enzyme digestion of the acceptor and donor adamers with an appropriate IISRE, c) washing the acceptor adamer reaction volume, d) incubating the donor adamer solution with the acceptor adamer reaction volume, e) ligating the digested donor and digested acceptor adamers, f) performing exonuclease digestion, and g) optionally washing the product of step (f). The resulting product can be used as either the acceptor or donor adamer in subsequent steps, such that a-g are repeated until the desired final product is produced.

[0019] The present disclosure provides a double-stranded adamer, the adamer comprising a) a first type II S restriction endonuclease (IISRE) sequence, b) an N-mer sequence, and c) at least a second IISRE sequence, and at least one end of the adamer comprises a hairpin structure. In some embodiments, the adamer can comprise hairpin structures at both ends of the adamer. In some embodiments, the adamer can comprise a) a first IISRE sequence, b) a second IISRE sequence, c) an N-mer sequence, and d) at least a third IISRE sequence. In some embodiments, the adamer can comprise a) a first IISRE sequence, b) a second IISRE sequence, c) an N-mer sequence, d) a third IISRE sequence, and e) at least a fourth IISRE sequence. In some embodiments, the adamer can further comprise a multiple cloning site (MCS) sequence, which comprises one or more restriction endonuclease sequences.

[0020] In some aspects, the IISRE sequence can be selected from a MlyI sequence, a NgoAVII sequence, a SspD5I sequence, an AlwI sequence, a BccI sequence, a BcefI sequence, a PleI sequence, a BceAI sequence, a BceSIV sequence, a BscAI sequence, a BspD6I sequence, a FauI sequence, an EarI sequence, a BspQI sequence, a BfuAI sequence, a PaqCI sequence, an Esp3I sequence, a BbsI sequence, a BbvI sequence, a BtgZI sequence, a FokI sequence, a BsmFI sequence, a BsaI sequence, a BcoDI sequence, and a HgaI sequence.

[0021] In some embodiments, the hairpin structure can include an aptamer sequence. In some embodiments, the aptamer sequence can be selected from a pL1 aptamer sequence, a thrombin 29-mer aptamer sequence, an S2.2 aptamer sequence, an ART1172 aptamer sequence, an R12.45 aptamer sequence, an Rb008 aptamer sequence, and a 38NT SELEX aptamer sequence.

[0022] The present disclosure provides compositions comprising an adamer of the present disclosure immobilized to a solid support. In some aspects, the solid support can be a bead. In some aspects, the bead can comprise polyacrylamide, polystyrene, agarose, or any combination thereof. In some aspects, the solid support can be a surface of a well or chamber. In some aspects, the well or chamber can be part of a multi-well plate.

[0023] In some embodiments in which the adamer is immobilized on a solid support, the adamer comprises a hairpin structure comprising at least one aptamer sequence, the solid surface comprises at least one ligand that binds to the aptamer sequence, and the adamer is immobilized on the solid surface via binding of the at least one aptamer sequence to the at least one ligand.

[0024] In some embodiments in which the adamer is immobilized on a solid support, the adamer comprises at least one 5' overhang or 3' overhang, the solid surface comprises at least one single-stranded or partially double-stranded nucleic acid molecule having a single-stranded portion complementary to the at least one 5' overhang or 3' overhang, and the adamer is immobilized to the solid surface by hybridizing the at least one 5' overhang or 3' overhang to at least one single-stranded or partially double-stranded nucleic acid on the solid surface.

[0025] In some embodiments in which the adamer is immobilized on a solid support, the adamer comprises at least one 5' overhang or 3' overhang, the solid surface comprises at least one single-stranded or partially double-stranded nucleic acid molecule having a single-stranded portion complementary to the at least one 5' overhang or 3' overhang, and the adamer is immobilized on the solid surface by hybridizing the at least one 5' overhang or 3' overhang to at least one single-stranded or partially double-stranded nucleic acid on the solid surface and ligating the adamer and the at least one single-stranded or partially double-stranded nucleic acid.

[0026] The present disclosure provides a method of producing an adamer of the present disclosure, the method comprising: a) chemically synthesizing a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule, wherein the sequence of the first single-stranded nucleic acid molecule and the sequence of the second single-stranded nucleic acid molecule comprise a portion of the adamer to be produced, the first single-stranded nucleic acid molecule comprising a first region complementary to a second region on the second single-stranded nucleic acid molecule and a second region that is self-complementary, and the second single-stranded nucleic acid molecule comprising the first region that is self-complementary and the second region that is complementary to the first region on the first single-stranded nucleic acid molecule; b) hybridizing the first single-stranded nucleic acid and the second single-stranded nucleic acid to produce a double-stranded nucleic acid molecule; and c) contacting the double-stranded nucleic acid molecule with a ligase enzyme to form a double-stranded adamer structure capped at both ends with a hairpin. In some embodiments, the methods can further comprise treating the product of step (c) with an exonuclease, thereby purifying the properly ligated adamers. In some embodiments, the methods can further comprise contacting the partially double-stranded nucleic acid molecule with a MutS enzyme after step (b) and before step (c).

[0027] The disclosure provides a method of producing an adamer of the disclosure, the method comprising: a) cloning an adamer sequence into a phagemid such that the adamer sequence is flanked on both sides by one or more DNAzymes that can be selectively activated; b) converting the phagemid using a helper phage into a packaged bacteriophage, where the packaged bacteriophage produces a single-stranded DNA comprising the adamer sequence flanked on both sides by one or more DNAzymes; c) purifying the single-stranded DNA produced by the packaged bacteriophage; d) allowing the purified single-stranded DNA to fold back to produce one or more DNAzyme structures and a majority of the double-stranded adamer sequence; e) activating the one or more DNAzymes, thereby excising the adamer sequence from the single-stranded DNA produced by the packaged bacteriophage; and f) contacting the excised adamer with a ligase enzyme. The above method may further comprise treating the product of step (f) with an exonuclease, thereby purifying the properly ligated adamers.

[0028] The present disclosure provides a method of producing an adamer of the present disclosure, the method comprising: a) cloning an adamer sequence into a plasmid; b) propagating the plasmid in a suitable host organism; c) purifying the plasmid from the host organism; d) treating the purified plasmid with one or more of a nickase enzyme and a restriction endonuclease enzyme, or simply one or more nickase enzymes, to excise the adamer sequence from the plasmid; and e) contacting the excised adamer sequence with a ligase to generate a double-stranded adamer structure capped at both ends with a hairpin structure.

[0029] The present disclosure provides a method of synthesizing a nucleic acid molecule comprising a target nucleic acid sequence, comprising: a) providing a first adammer of the present disclosure immobilized to a solid support, the first adammer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adammer of any one of the present disclosures immobilized to a solid support, the second adammer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; and c) contacting the first adammer with an IISRE that cleaves the second IISRE sequence located within the first adammer, thereby generating a first cleaved product that is immobilized to the solid support and comprises the first IISRE sequence, the first N-mer sequence, and a 3' overhang, a 5' overhang, or a blunt end. d) contacting the second adamer with an IISRE that cleaves a third IISRE sequence located in the second adamer, thereby generating a second cleaved product that is released into solution and includes a second N-mer sequence, a fourth IISRE sequence, a 3' overhang, a 5' overhang, or a blunt end, wherein the second cleaved product is capped at one end by a hairpin structure; e) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; f) treating the product of step (e) with an exonuclease, thereby removing unligated first cleaved product and / or second cleaved product; and g) repeating steps (a)-(f) until a nucleic acid molecule comprising the target nucleic acid sequence is synthesized.

[0030] In some embodiments, the ligase enzyme can be human DNA ligase III (hLig3). In some embodiments, the ligase enzyme can be T4 DNA ligase.

[0031] In some embodiments, the target nucleic acid sequence can be at least about 100, or at least about 500, or at least about 1000, or at least about 2000, or at least about 3000, or at least about 4000, or at least about 5000 nucleotides in length.

[0032] In some embodiments, a nucleic acid molecule comprising a target nucleic acid sequence synthesized by the methods of the present disclosure can have a purity of at least 80% or at least 90%. [Brief description of the drawings]

[0033] The above and further features will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings.

[0034] [Figure 1] FIG. 1 is an exemplary schematic diagram of an adamer, a double-stranded nucleic acid molecule containing hairpins at both ends. [Diagram 2] 1 shows an exemplary schematic diagram of various adamer designs of the present disclosure. Several examples of adamer designs are shown, each adamer type having a payload with flanking IISRE sites, a means of attachment to a solid support and a double hairpin to promote exonuclease resistance. The legend shows several possible IISRE and RE sites, as well as a thrombin aptamer hairpin. [Diagram 3] 3 shows sequences of six non-limiting examples of adamers of the present disclosure. The adamer designs include a simple hairpin, multiple cloning sites, and paired IISRE binding sites. In this case, each has one blunt-cutting IISRE, MlyI, which leaves the end of the 3-mer payload, NNN, exposed for blunt ligation. On the left side of each design is a 5' 4-base overhang or 3-base overhang IISRE site for downstream payload extension reactions. The nucleotide sequences of the 10 or more nucleotides presented in FIG. 3 correspond to those set forth in SEQ ID NOs: 1-12. [Figure 4]Exemplary nested type II S restriction endonuclease (IISRE) sequences (hereinafter "IISRE sequences") for use in the adamers of the present disclosure are shown. The nucleotide sequences of 10 or more nucleotides presented in FIG. 4 correspond to those set forth in SEQ ID NOs: 13-28. [Diagram 5] 5 is an exemplary schematic diagram of a method for generating adamers of the present disclosure. In this method, two separate oligonucleotides are synthesized together and hybridized. Treatment with MutS, which binds to mismatched bases and exposes the DNA to exonuclease digestion, is combined with ligation, followed by T7 exonuclease digestion. This process leaves substantially pure adamers. These adamers can be evaluated in oligonucleotide synthesis reactions and then serve as templates for adamer cloning. The nucleotide sequences of 10 or more nucleotides presented in FIG. 5 correspond to those set forth in SEQ ID NOs: 29-32. [Figure 6] Schematic diagram of a phagemid for use in generating adamers of the present disclosure. This phagemid construct contains information for generating bacteriophage from double-stranded DNA, as well as a DNAzyme for efficient excision of pure adamers of clonal origin. From chemically synthesized adamers of any payload length, inserts are generated using For and Rev primers and ligated into the phagemid vector. [Figure 7A]FIG. 1 is an exemplary schematic of a method for generating an adammer of the present disclosure using phagemids and DNAzymes. A schematic of a particular adammer 3-mer payload GCC in an adammer design that includes nested IISREs (blunt cut sites nested with 4-base overhang sites and blunt sites nested with 4-base sites) and paired MCSs (MSC left and MCS right). The design also includes forward and reverse amplification primer sites (For and Rev) and a DNAzyme scar sequence. The adjacent DNAzyme pair is also shown. Single-stranded DNA generated by bacteriophage propagation in E. coli is purified and allowed to fold to generate the majority of the DNAzyme structure and double-stranded adammer sequence. After activation by treatment with Zn+, ligation, and T7 exonuclease treatment, a pure clonal adammer is generated. [Figure 7B] FIG. 1 is an exemplary schematic diagram of a method for generating the adamers of the present disclosure using bimolecular trans-cleavage of an adamer sequence. [Figure 8] 1 is an exemplary schematic diagram of a method of synthesizing nucleic acids of the present disclosure, including the use of the disclosed adamers. First, a first adamer and a second adamer are ligated to a binding stud with an MCS already loaded on a solid support using DNA ligation. A donor construct and an acceptor construct are generated in separate volumes. The donor construct and the acceptor construct are treated with separate IISREs to generate ligatable ends. In this figure, the acceptor is generated by digestion with R1 IISRE, and the released ends and enzyme are discarded by rinsing. A donor construct is generated by digestion with purple L2 enzyme. The donor construct solution (with L2 enzyme) is transferred to the acceptor well and ligated using T4 DNA ligase, which has a high efficiency of over 80% for 2-, 3-, or 4-base sticky end ligation. The well is treated with exonuclease and rinsed. The resulting adamer construct is then ready for the subsequent extension cycle. [Figure 9A]9A and 9B are exemplary schematic diagrams of a method of synthesizing nucleic acids of the present disclosure, including the use of an adamer of the present disclosure to synthesize a target nucleic acid molecule 27 nucleotides in length. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9A corresponds to that set forth in SEQ ID NO: 33. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9E corresponds to that set forth in SEQ ID NO: 34-35. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9G corresponds to that set forth in SEQ ID NO: 36-47. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9H corresponds to that set forth in SEQ ID NO: 48-58. [Figure 9B] 9A and 9B are exemplary schematic diagrams of a method of synthesizing nucleic acids of the present disclosure, including the use of an adamer of the present disclosure to synthesize a target nucleic acid molecule 27 nucleotides in length. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9A corresponds to that set forth in SEQ ID NO: 33. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9E corresponds to that set forth in SEQ ID NO: 34-35. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9G corresponds to that set forth in SEQ ID NO: 36-47. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9H corresponds to that set forth in SEQ ID NO: 48-58. [Figure 9C] 9A and 9B are exemplary schematic diagrams of a method of synthesizing nucleic acids of the present disclosure, including the use of an adamer of the present disclosure to synthesize a target nucleic acid molecule 27 nucleotides in length. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9A corresponds to that set forth in SEQ ID NO: 33. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9E corresponds to that set forth in SEQ ID NO: 34-35. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9G corresponds to that set forth in SEQ ID NO: 36-47. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9H corresponds to that set forth in SEQ ID NO: 48-58. [Figure 9D]9A and 9B are exemplary schematic diagrams of a method of synthesizing nucleic acids of the present disclosure, including the use of an adamer of the present disclosure to synthesize a target nucleic acid molecule 27 nucleotides in length. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9A corresponds to that set forth in SEQ ID NO: 33. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9E corresponds to that set forth in SEQ ID NO: 34-35. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9G corresponds to that set forth in SEQ ID NO: 36-47. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9H corresponds to that set forth in SEQ ID NO: 48-58. [Figure 9E] 9A and 9B are exemplary schematic diagrams of a method of synthesizing nucleic acids of the present disclosure, including the use of an adamer of the present disclosure to synthesize a target nucleic acid molecule 27 nucleotides in length. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9A corresponds to that set forth in SEQ ID NO: 33. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9E corresponds to that set forth in SEQ ID NO: 34-35. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9G corresponds to that set forth in SEQ ID NO: 36-47. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9H corresponds to that set forth in SEQ ID NO: 48-58. [Figure 9F] 9A and 9B are exemplary schematic diagrams of a method of synthesizing nucleic acids of the present disclosure, including the use of an adamer of the present disclosure to synthesize a target nucleic acid molecule 27 nucleotides in length. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9A corresponds to that set forth in SEQ ID NO: 33. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9E corresponds to that set forth in SEQ ID NO: 34-35. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9G corresponds to that set forth in SEQ ID NO: 36-47. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9H corresponds to that set forth in SEQ ID NO: 48-58. [Figure 9G]9A and 9B are exemplary schematic diagrams of a method of synthesizing nucleic acids of the present disclosure, including the use of an adamer of the present disclosure to synthesize a target nucleic acid molecule 27 nucleotides in length. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9A corresponds to that set forth in SEQ ID NO: 33. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9E corresponds to that set forth in SEQ ID NO: 34-35. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9G corresponds to that set forth in SEQ ID NO: 36-47. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9H corresponds to that set forth in SEQ ID NO: 48-58. [Figure 9H] 9A and 9B are exemplary schematic diagrams of a method of synthesizing nucleic acids of the present disclosure, including the use of an adamer of the present disclosure to synthesize a target nucleic acid molecule 27 nucleotides in length. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9A corresponds to that set forth in SEQ ID NO: 33. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9E corresponds to that set forth in SEQ ID NO: 34-35. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9G corresponds to that set forth in SEQ ID NO: 36-47. The nucleotide sequence of 10 or more nucleotides presented in FIG. 9H corresponds to that set forth in SEQ ID NO: 48-58. [Figure 10] 10 is a schematic diagram of a set of restriction enzyme digestion and ligation reactions performed using an adammer of the present disclosure to synthesize a target nucleic acid. The nucleotide sequences of 10 or more nucleotides presented in FIG. 10 correspond to those set forth in SEQ ID NOs: 59-67. [Figure 11] 1 shows the results of gel electrophoresis analysis of enzymatic digestion and ligation reactions performed using the adamer of the present disclosure, as outlined in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present disclosure is directed to compositions and methods for synthesizing nucleic acid molecules that contain specific target nucleic acid sequences. The compositions can include adamers as described in more detail herein. The methods can include the use of these adamers in sequential restriction enzyme cleavage and ligation reactions to generate nucleic acid molecules that contain the target nucleic acid sequence. These methods are described in more detail herein.

[0036] Importantly, unlike existing nucleic acid synthesis methods, the compositions and methods of the present disclosure do not require large-scale phosphoramidite synthesis.As a result, the compositions and methods of the present disclosure are cheaper and faster than existing nucleic acid synthesis methods.In addition, the compositions and methods of the present disclosure generate less toxic organic waste than existing nucleic acid synthesis methods, and are therefore more environmentally conscious.

[0037] Adamar The present disclosure provides a composition comprising at least one adamer. As used herein, the term adamer is used to describe a double-stranded nucleic acid molecule that comprises a hairpin structure at both ends. In some embodiments in which the adamer is immobilized on a solid surface, the adamer may comprise a single hairpin located at the end of the molecule that is not bound to the solid surface. An exemplary schematic diagram of an adamer and two adamers immobilized on a solid surface is shown in FIG. 1. The adamer may comprise one or more features described herein.

[0038] In some embodiments, an adamer can comprise, consist essentially of, or consist of DNA.

[0039] In some embodiments, an adamer can include one or more multiple cloning site (MCS) sequences. In some embodiments, the MCS sequences can include one or more restriction endonuclease (RE) sequences that can be cleaved with a corresponding restriction endonuclease to generate a 3' overhang, a 5' overhang, or a blunt end.

[0040] As will be understood by one of skill in the art, "blunt end" is used to describe the end of a DNA fragment that is free of unpaired nucleotides.

[0041] As will be understood by one of skill in the art, the term 5' overhang is used to refer to a single-stranded portion of a partially double-stranded nucleic acid molecule that is located at the 5' end of one of the strands.

[0042] As will be understood by one of skill in the art, the term 3' overhang is used to refer to a single-stranded portion of a partially double-stranded nucleic acid molecule located at the 3' end of one of the strands.

[0043] In some embodiments, an adamer can include at least one offset-cleaving type II S restriction endonuclease (IISRE) sequence (hereinafter IISRE sequence) that can be cleaved with a corresponding type II S restriction endonuclease (hereinafter IISRE). In some embodiments, an adamer can include at least one IISRE sequence. In some embodiments, an adamer can include at least three IISRE sequences. In some embodiments, an adamer can include at least four IISRE sequences.

[0044] In some embodiments, the IISRE sequence is such that cleavage at the corresponding IISRE results in the creation of a "blunt end."

[0045] In some embodiments, the IISRE sequence is such that cleavage with the corresponding IISRE results in the creation of a 5' overhang that is 1 nucleotide long. In some embodiments, the IISRE sequence is such that cleavage with the corresponding IISRE results in the creation of a 5' overhang that is 2 nucleotides long. In some embodiments, the IISRE sequence is such that cleavage with the corresponding IISRE results in the creation of a 5' overhang that is 3 nucleotides long. In some embodiments, the IISRE sequence is such that cleavage with the corresponding IISRE results in the creation of a 5' overhang that is 4 nucleotides long. In some embodiments, the IISRE sequence is such that cleavage with the corresponding IISRE results in the creation of a 5' overhang that is 5 nucleotides long. In some embodiments, the IISRE sequence is such that cleavage with the corresponding IISRE results in the creation of a 5' overhang that is about 1 nucleotide to about 5 nucleotides long.

[0046] Non-limiting examples of IISRE sequences, along with their corresponding IISREs, and descriptions of the overhangs / blunt ends created by cleavage of the IISRE sequences and corresponding IISREs, are shown in Table 1. Thus, an adamer can include one or more of the IISRE sequences set forth in Table 1. [Table 1-1] [Table 1-2]

[0047] In some embodiments, the hairpin structure or hairpin (used interchangeably) located at the end of the adamer has a length of at least about 1, or at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, or at least about 16, or at least about 17, or at least about 18, or at least about 19, or at least about 20, or at least about 21, or at least about 22, or at least about 23, or at least about 24, or at least about 25, or at least about 26, or at least about 27, or at least about 28, or at least about 29, or at least about 30, or at least about 31, or at least about 32, or at least about 33, or at least about 34, or at least about 35, or at least about 36, or at least about 37, or at least about 38, or at least about 39, or at least about 40, or at least about 41, or at least about 42, or at least about 43, or at least about 44, or at least about 45, or at least about 46, or at least about 47, or at least about 48, or at least about 49, or at least about 50 nucleotides.

[0048] As mentioned above, the adamers are capped at either end by a hairpin structure. The hairpin structure serves several purposes. First, the hairpin provides protection against exonuclease digestion of the adamers. This allows for the removal of unreacted intermediates from a given reaction in the disclosed method, thereby providing purity to both the developing adamers and the product after adamer extension. Second, the hairpin structure provides a means for attaching the adamers to solid supports. These attachments are generated directly by binding of an aptamer that binds to a specific solid support binding ligand, by ligation with an MCS after digestion with a conventional RE such as BamHI, by ligation with a lambda phage cos site, or by hybridization to a single-stranded solid support binding anchor NA. Finally, the hairpins described herein allow for attachment of the adamers to solid supports (e.g., beads) without the need for non-natural modifications such as biotin. Thus, the adamers of the present disclosure can be synthesized using entirely natural means, obviating the need for small-scale and / or large-scale phosphoramidite synthesis. Thus, the disclosed adamers and methods allow for faster and cheaper synthesis of nucleic acid molecules and can generate less toxic waste products.

[0049] In some embodiments, the hairpin located at the end of the adamer can include a structural sequence that allows for affinity purification of the adamer and / or binding of the adamer to a solid support (eg, a bead).

[0050] In some embodiments, the hairpin located at the end of the adamer can contain an enzymatic sequence (eg, a DNAzyme sequence) that allows for controlled self-cleavage.

[0051] In some embodiments, the hairpin located at the end of the adamer can contain one or more restriction enzyme sites. Without wishing to be bound by theory, the one or more restriction enzyme sites in the hairpin can be cleaved with a corresponding restriction enzyme to generate at least one single-stranded overhang, which can then be used to hybridize and / or ligate the cleaved adamer to a solid support (e.g., a bead) that contains a nucleic acid complementary to the at least one single-stranded overhang.

[0052] In some embodiments, the hairpin at the end of the adamer can include an aptamer sequence. Without wishing to be bound by theory, the aptamer sequence can be used for affinity purification and / or binding to a solid support (e.g., beads). Non-limiting examples of aptamer sequences are shown in Table 2. [Table 2]

[0053] In some embodiments, the adamers can include lambda phage cos sites.

[0054] In some embodiments, an adamer can comprise an "N-mer sequence" that comprises a fragment of a nucleic acid synthesized using one of the methods described herein. The terms "N-mer sequence," "payload," and "N-mer payload" are used interchangeably herein.

[0055] In some embodiments, the N-mer sequence can be about 3 nucleotides in length. In some embodiments, the N-mer sequence is about 3 nucleotides in length. An N-mer sequence that is 3 nucleotides in length is referred to herein as a 3-mer.

[0056] In some embodiments, the N-mer sequence can be about 4 nucleotides in length. In some embodiments, the N-mer sequence is about 4 nucleotides in length. An N-mer sequence that is 4 nucleotides in length is referred to herein as a 4-mer.

[0057] In some embodiments, the N-mer sequence can be about 5 nucleotides in length. In some embodiments, the N-mer sequence is about 5 nucleotides in length. An N-mer sequence that is 5 nucleotides in length is referred to herein as a 5-mer.

[0058] In some embodiments, the N-mer sequence can be about 6 nucleotides in length. In some embodiments, the N-mer sequence is about 6 nucleotides in length. An N-mer sequence that is 6 nucleotides in length is referred to herein as a 6-mer.

[0059] In some embodiments, an adamer can include an MCS sequence, a first IISRE sequence, an N-mer sequence, and at least a second IISRE sequence. In some embodiments, an adamer can include an MCS sequence, followed by a first IISRE sequence, followed by an N-mer sequence, followed by at least a second IISRE sequence. Exemplary schematics of the aforementioned adamers are shown in FIG. 2 as adamer design numbers 1-4. In the non-limiting examples of adamer design numbers 1-3 shown in FIG. 2, the first IISRE sequence is an IISRE sequence that creates a 4 nucleotide long 5' overhang when cleaved, the N-mer sequence is a 3-mer sequence, and at least a second IISRE sequence is an IISRE sequence that creates a blunt end when cleaved. In a non-limiting example of adamer design number 4 shown in FIG. 2, the first IISRE sequence is an IISRE sequence that creates a blunt end when cleaved, the N-mer sequence is a 3-mer sequence, and at least the second IISRE sequence is an IISRE sequence that creates a 3 nucleotide long 5' overhang when cleaved.

[0060] In some embodiments, the adamer can include an MCS sequence, a first IISRE sequence, a second IISRE sequence, an N-mer sequence, a third IISRE sequence, and at least a fourth IISRE sequence. In some embodiments, the adamer can include an MCS sequence, followed by a first IISRE sequence, followed by a second IISRE sequence, followed by an N-mer sequence, followed by a third IISRE sequence, followed by at least a fourth IISRE sequence. An exemplary schematic of the adamer described above is shown in FIG. 2 as adamer design number 5. In a non-limiting example of adamer design number 5 shown in FIG. 2, the first IISRE sequence is an IISRE sequence that creates a 4-nucleotide 5' overhang when cleaved, the second IISRE sequence is an IISRE sequence that creates a 4-nucleotide 5' overhang when cleaved, the N-mer sequence is a 3-mer sequence, the third IISRE sequence is an IISRE sequence that creates a 4-nucleotide 5' overhang when cleaved, and at least the fourth IISRE sequence is an IISRE sequence that creates a blunt end when cleaved.

[0061] In some embodiments, an adamer can include a first MCS sequence, a first IISRE sequence, an N-mer sequence, at least a second IISRE sequence, and at least a second MCS sequence. In some embodiments, an adamer can include a first MCS sequence, followed by a first IISRE sequence, followed by an N-mer sequence, followed by at least a second IISRE sequence, followed by at least a second MCS sequence. An exemplary schematic of the aforementioned adamer is shown in FIG. 2 as adamer design number 6. In a non-limiting example of adamer design number 6 shown in FIG. 2, the first IISRE sequence is an IISRE sequence that creates a blunt end when cleaved, the N-mer sequence is a 3-mer sequence, and the at least a second IISRE sequence is an IISRE sequence that creates a 5-overhang of 4 nucleotides when cleaved.

[0062] In some embodiments, the adamer can include a first MCS sequence, a first IISRE sequence, a second IISRE sequence, an N-mer sequence, a third IISRE sequence, at least a fourth IISRE sequence, and at least a second MCS sequence. In some embodiments, the adamer can include a first MCS sequence, followed by a first IISRE sequence, followed by a second IISRE sequence, followed by an N-mer sequence, followed by a third IISRE sequence, followed by at least a fourth IISRE sequence, followed by at least a second MCS sequence. An exemplary schematic of the aforementioned adamer is shown in FIG. 2 as adamer design number 7. In a non-limiting example of adamer design number 7 shown in FIG. 2, the first IISRE sequence is an IISRE sequence that creates a blunt end when cleaved, the second IISRE sequence is an IISRE sequence that creates a 4-nucleotide long 5' overhang when cleaved, the N-mer sequence is a 3-mer sequence, the third IISRE sequence is an IISRE sequence that creates a 4-nucleotide long 5' overhang when cleaved, and at least the fourth IISRE sequence is an IISRE sequence that creates a blunt end when cleaved.

[0063] In some embodiments, an adamer can include a hairpin that includes an aptamer sequence, a first IISRE sequence, an N-mer sequence, at least a second IISRE sequence, and an MCS sequence. In some embodiments, an adamer can include a hairpin that includes an aptamer sequence, followed by a first IISRE sequence, followed by an N-mer sequence, followed by at least a second IISRE sequence, followed by an MCS sequence. An exemplary schematic of the aforementioned adamer is shown in FIG. 2 as adamer design number 7. In a non-limiting example of adamer design number 7 shown in FIG. 2, the aptamer sequence is a thrombin aptamer sequence, the first IISRE sequence is an IISRE sequence that creates a 4 nucleotide long 5' overhang when cleaved, the N-mer sequence is a 3-mer sequence, and the at least a second IISRE sequence is an IISRE that creates a blunt end when cleaved.

[0064] Figure 3 shows sequences of six non-limiting examples of adamers. Each of the adamers shown in Figure 3 is capped with a simple hairpin turn. Each of the adamers shown in Figure 3 includes an MCS sequence, a first IISRE sequence, an N-mer sequence (designated "N"), and a second IISRE sequence. Specifically, the N-mer sequences shown in the adamers in Figure 3 are 3-mer sequences, and the IISRE sequence is selected from BbsI, MlyI, BtgZI, BfuAI, PaqCI, FokI, and EarI.

[0065] When two IISRE sequences are included adjacent to each other within an adamer, these IISRE sequences can be referred to as "nested IISRE sequences" or "nested IISRE sites." In some embodiments, a nested IISRE sequence can include two IISRE sequences that are directly adjacent to each other. In some embodiments, a nested IISRE sequence can include two IISRE sequences that are adjacent to each other but separated by about 1 to about 10 nucleotides.

[0066] Without wishing to be bound by theory, it is possible to nest IISRE sites, since some IISRE sites have cleavage sites that are far enough away from their recognition sites to fit into other IISRE sites between the first site and the payload (see FIG. 4). Thus, in some embodiments, the adamer can include a unique blunt cleavage site and a 4-base overhang site on either side of the payload (N-mer sequence). Without wishing to be bound by theory, this significantly reduces the number of adamer reagents required to perform routine nucleic acid production.

[0067] Without wishing to be bound by theory, the inclusion of nested IISRE sequences in the adamer provides several options for cleaving at the same position with two separate sites in the methods of the present disclosure. The option of cleaving at the same position with two separate sites can reduce the number of separate adamers required in a library (see below) for general nucleic acid synthesis. Figure 4 shows non-limiting examples of nested IISRE sites, including nested BbvI and BbsI sites. Figure 4 also shows three non-limiting examples of aptamer sequences that can be included in the hairpins of the adamers described herein.

[0068] In some aspects, an adumer can include any element known in the art to facilitate cloning, including, but not limited to, cognate sequences of an amplification primer. Without wishing to be bound by theory, including the cognate sequences of an amplification primer in an adumer may allow recovery of a particular adumer design for clonal expansion.

[0069] In some embodiments, an adamer can include any element known in the art to facilitate large-scale production of an adamer by fermentation in a plasmid or bacteriophage, including, but not limited to, a sequence corresponding to a DNAzyme scar and / or a sequence that facilitates smooth folding of the adamer following excision using a particular DNAzyme (see, e.g., Praetorius et al., Nature, 2017, 552, 84-87, which is incorporated by reference in its entirety).

[0070] Adumar Library The present disclosure provides an adamer library that includes a plurality of adamers, the plurality of adamers including one or more different adamer species (ie, adamers having unique sequences).

[0071] The present disclosure provides a 3-mer adamer library comprising a plurality of adamers, the plurality of adamers comprising at least 64 different adamer species, each of which comprises one of 64 possible 3-mer sequences that can be made with adenine, cytosine, guanine, and thymine (4 x 4 x 4 = 64).

[0072] The present disclosure provides a 4-mer adamer library comprising a plurality of adamers, the plurality of adamers comprising at least 256 different adamer species, each of which comprises one of 256 possible 4-mer sequences that can be made of adenine, cytosine, guanine, and thymine (4 x 4 x 4 x 4 = 256).

[0073] The present disclosure provides a 5-mer adamer library comprising a plurality of adamers, the plurality of adamers comprising at least 1,024 different adamer species, each of which comprises one of 1,024 possible 5-mer sequences that can be made of adenine, cytosine, guanine, and thymine (4 x 4 x 4 x 4 = 1,024).

[0074] The present disclosure provides a 6-mer adamer library comprising a plurality of adamers, the plurality of adamers comprising at least 256 different adamer species, each of which comprises one of 256 possible 6-mer sequences that can be made with adenine, cytosine, guanine, and thymine (4 x 4 x 4 x 4 x 4 = 4,096).

[0075] How to Generate Hadamard The adamers described herein can be generated using chemically synthesized nucleic acids in a method as shown generally in FIG. 5. In this method, a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule are chemically synthesized (e.g., using phosphoramidite synthesis). As shown in the top panel of FIG. 5, the first single-stranded nucleic acid molecule comprises a first region that is complementary to a second region on the second single-stranded nucleic acid molecule and a second region that is self-complementary, and the second single-stranded nucleic acid molecule comprises a first region that is self-complementary and a second region that is complementary to the first region on the first single-stranded nucleic acid molecule. The first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule are then hybridized together to generate a partially double-stranded nucleic acid molecule. The partially double-stranded nucleic acid molecule can then be contacted, optionally, with the enzyme MutS, which binds to mismatched bases and exposes the DNA to exonuclease digestion. The partially double-stranded nucleic acid molecule can then be contacted with a ligase enzyme to form a double-stranded adamer structure capped at both ends by a hairpin. After contact with the ligase enzyme, the product can be contacted with T7 exonuclease to purify and concentrate the properly formed adamers.

[0076] Without wishing to be bound by theory, the above-described method of generating aptamers using chemically synthesized single-stranded nucleic acid molecules allows for a rapid turnaround time for testing new adamers designs. Without wishing to be bound by theory, adamers generated using the above-described method can be used as templates for large-scale generation of the above-described adamers using methods that do not require phosphoramidite synthesis, including, but not limited to, cloning of adamers using plasmids or bacteriophage.

[0077] Accordingly, the present disclosure provides a method of generating an adamer as described herein, comprising: a) providing a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule, wherein the sequence of the first single-stranded nucleic acid molecule and the sequence of the second single-stranded nucleic acid molecule comprise a portion of the adamer to be generated, the first single-stranded nucleic acid molecule comprising a first region complementary to a second region on the second single-stranded nucleic acid molecule and a second region that is self-complementary, and the second single-stranded nucleic acid molecule comprising the first region that is self-complementary and the second region that is complementary to the first region on the first single-stranded nucleic acid molecule; b) hybridizing the first single-stranded nucleic acid and the second single-stranded nucleic acid to generate a partially double-stranded nucleic acid molecule; and c) contacting the partially double-stranded nucleic acid molecule with a ligase enzyme to form a double-stranded adamer structure capped at both ends by a hairpin.

[0078] Accordingly, the present disclosure provides a method of producing an adamer as described herein, comprising: a) chemically synthesizing a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule, wherein the sequence of the first single-stranded nucleic acid molecule and the sequence of the second single-stranded nucleic acid molecule comprise a portion of the adamer to be produced, the first single-stranded nucleic acid molecule comprising a first region complementary to a second region on the second single-stranded nucleic acid molecule and a second region that is self-complementary, and the second single-stranded nucleic acid molecule comprising the first region that is self-complementary and the second region that is complementary to the first region on the first single-stranded nucleic acid molecule; b) hybridizing the first single-stranded nucleic acid and the second single-stranded nucleic acid to produce a partially double-stranded nucleic acid molecule; and c) contacting the partially double-stranded nucleic acid molecule with a ligase enzyme to form a double-stranded adamer structure capped at both ends by a hairpin.

[0079] In some embodiments, the above method may further comprise treating the product of step (c) with an exonuclease, thereby purifying the properly ligated adamers.

[0080] In some embodiments, the above method can further comprise contacting the partially double-stranded nucleic acid molecule with a MutS enzyme after step (b) and before step (c).

[0081] Adamers described herein can be generated using a phagemid-based method as shown generally in Figures 6 and 7A (see also Praetorius et al., Nature, 2017, 552, 84-87, and U.S. Patent Application Publication No. US20190203242A1, each of which is incorporated by reference in its entirety for all purposes). In this method, the adamer sequence is cloned into a phagemid (e.g., pBluescript or any other phagemid known in the art) as shown in Figure 6. The adamer sequence is then cloned into a phagemid (e.g., pBluescript or any other phagemid known in the art) as shown in the top panel of Figures 6 and 7A (e.g., DNAzyme Zn2+). 2+ The phagemid is then converted into an M13 packaging bacteriophage using a helper phage by methods well known to those of skill in the art. The M13 packaging bacteriophage produces single-stranded DNA containing the adammer sequence and the flanking DNAzyme, as shown in the top panel of FIG. 7A. The single-stranded DNA produced by the bacteriophage is purified and allowed to fold back to produce the DNAzyme structure and the majority of the double-stranded adammer sequence, as shown in the second panel from the top of FIG. 7A. The DNAzyme is then transferred to a phagemid (e.g., by contacting the DNAzyme with Zn 2+The DNAzyme is activated (by contact with a ligase) and excises the adamer sequence from the single-stranded DNA produced by the bacteriophage. After DNAzyme cleavage, the adamer is allowed to fold and then contacted with a ligase to close the adamer, as shown in the bottom panel of FIG. 7A. Optionally, unligated or improperly formed adamers can be removed by treating the product of the ligation reaction with T7 exonuclease. The exemplary adamer shown in FIG. 7A includes a first MCS sequence, followed by a first IISRE sequence, followed by a second IISRE sequence, followed by an N-mer sequence, followed by a third IISRE sequence, followed by a fourth IISRE sequence, followed by a second MCS sequence. The apostrophe in FIG. 7A denotes the reverse complement of the sequence such that the adamer is self-complementary and forms a double-stranded structure capped at both ends by hairpin domains.

[0082] Alternatively, a similar bacteriophage DNA preparation can be treated with a trans-acting DNAzyme element hybridized to M13 DNA activated by Zn2+, followed by excision of the adamers in a non-self-cleaving manner. As will be appreciated by those skilled in the art, DNAzymes have been shown to act as bimolecular reagents (see Gu, et al., Journal of the American Chemical Society, 2013, 135, 24, 9121-9129, the contents of which are incorporated herein by reference in their entirety). Thus, in some embodiments, adamers are excised by providing a DNAzyme in trans, hybridized to a bacteriophage preparation, and reacting with Zn2+. 2+ This can be achieved using a hemi-DNAzyme sequence that is activated by (see FIG. 7B).

[0083] The disclosure provides a method of producing an adamer as described herein, the method comprising: a) cloning an adamer sequence into a phagemid such that the adamer sequence is flanked on both sides by one or more DNAzymes that can be selectively activated; b) converting the phagemid using a helper phage into a packaged bacteriophage, where the packaged bacteriophage produces a single-stranded DNA comprising the adamer sequence flanked on both sides by one or more DNAzymes; c) purifying the single-stranded DNA produced by the packaged bacteriophage; d) allowing the purified single-stranded DNA to fold back to produce the structure of one or more DNAzymes and a majority of the double-stranded adamer sequence; e) activating one or more DNAzymes, thereby excising the adamer sequence from the single-stranded DNA produced by the packaged bacteriophage; and f) contacting the excised adamer with a ligase enzyme.

[0084] The disclosure provides a method of producing an adamer as described herein, comprising: a) cloning an adamer sequence into a phagemid such that the adamer sequence is flanked on both sides by one or more first portions of DNAzymes that can be selectively activated; b) converting the phagemid into a packaged bacteriophage using a helper phage, where the packaged bacteriophage produces single-stranded DNA comprising the adamer sequence flanked on both sides by one or more DNAzymes; c) purifying the single-stranded DNA produced by the packaged bacteriophage; and d) allowing the purified single-stranded DNA to fold. to generate one or more DNAzyme structures and a majority of the double-stranded adamer sequence; e) contacting the purified single-stranded DNA with one or more oligonucleotides comprising a second portion of a DNAzyme, where the one or more oligonucleotides hybridize to the purified single-stranded DNA, thereby forming one or more complete DNAzymes; f) activating the one or more DNAzymes, thereby excising the adamer sequence from the single-stranded DNA generated by the packaged bacteriophage; and g) contacting the excised adamer with a ligase enzyme.

[0085] The disclosure provides a method of producing an adamer as described herein, comprising: a) cloning an adamer sequence into a phagemid such that the adamer sequence is flanked on both sides by one or more first portions of selectively activated DNAzymes; b) converting the phagemid into a packaged bacteriophage using a helper phage, wherein the packaged bacteriophage produces single-stranded DNA comprising the adamer sequence flanked on both sides by one or more DNAzymes; c) purifying the single-stranded DNA produced by the packaged bacteriophage; and d) converting the purified single-stranded DNA into a phagemid. contacting A with one or more oligonucleotides comprising a second portion of a DNAzyme, where the one or more oligonucleotides hybridize to the purified single-stranded DNA, thereby forming one or more complete DNAzymes; e) allowing the product of step (d) to fold back to produce a majority of the double-stranded adamer sequence; f) activating the one or more DNAzymes, thereby excising the adamer sequence from the single-stranded DNA produced by the packaged bacteriophage; and g) contacting the excised adamer with a ligase enzyme.

[0086] In some embodiments, the methods described above can further comprise treating the excised adumers with a ligase enzyme, followed by treating the product with at least one exonuclease, thereby purifying the properly ligated adumers.

[0087] The adamers described herein can be generated using a plasmid-based method. In the plasmid-based method, the adamers are cloned into a plasmid (e.g., bacteria and / or yeast) that can be propagated and purified on a large scale. The purified plasmid containing the adamer sequence can then be treated with one or more nicase enzymes (e.g., Cas9 nicase with an appropriate guide RNA) and / or restriction endonuclease enzymes to excise the adamer sequence from the plasmid to obtain a double-stranded adamer with a DNA flap at each end. The double-stranded adamers with DNA flaps can then be treated with one or more ligase enzymes to generate a double-stranded adamer capped at both ends by a hairpin structure.

[0088] Accordingly, the disclosure provides a method of producing an adamer as described herein, comprising: a) cloning an adamer sequence into a plasmid; b) propagating the plasmid in a suitable host organism; c) purifying the plasmid from the host organism; d) treating the purified plasmid with one or more of a nickase enzyme and a restriction endonuclease enzyme to excise the adamer sequence from the plasmid; and e) contacting the excised adamer sequence with a ligase to generate a double-stranded adamer structure capped at both ends by a hairpin structure.

[0089] The methods for generating adumers described herein can be used to generate adumer libraries, including, but not limited to, 3-mer adumer libraries, 4-mer adumer libraries, 5-mer adumer libraries, and 6-mer adumer libraries.

[0090] In some embodiments, a 6-mer adumer library can be generated from a 3-mer adumer library. Although not wishing to be bound by theory, the total number of possible hexamers is large, the hexamer payload is a very useful adumer type for oligonucleotide synthesis. In addition, this allows for periodic switching between the IISRE sites encoded by the left and right trimer elements. In a non-limiting example, an initial library is first generated using conventional phosphoramidite synthesis, with a library of 64 element trimer adumers either individually or as a complete set in a DNA microarray pool. The pool of adumer trimer libraries is cloned together into bacteriophage or amplified in vivo. The left and right adumer trimer libraries are prepared and digested to generate blunt ends at the payload sites. The right and left libraries are then ligated to form a large pool of adumers. The unligated material is digested by an exonuclease. The remaining intact adamers are then used as templates to amplify specific hexameric adamers by PCR. Each independent hexameric PCR product can then be cloned into bacteriophage for large-scale production or can be stored for future use.

[0091] Nucleic acid synthesis method using the disclosed adamer The adamers described herein can be used in the methods described herein to synthesize nucleic acid molecules containing any target nucleic acid sequence.

[0092] In some embodiments, the target nucleic acid sequence can be at least about 100, or at least about 200, or at least about 300, or at least about 500, or at least about 600, or at least about 700, or at least about 800, or at least about 900, or at least about 1000, or at least about 1500, or at least about 2000, or at least about 2500, or at least about 3000, or at least about 3500, or at least about 4000, or at least about 4500, or at least about 5000 nucleotides in length. In some embodiments, the target double-stranded nucleic acid can comprise at least one homopolymer sequence.

[0093] In some embodiments, the target nucleic acid sequence can include at least one homopolymer sequence. As used herein, the term homopolymer sequence is used to refer to any type of repetitive nucleic acid sequence, including, but not limited to, single base repeats or small motif repeats. In some embodiments, the homopolymer sequence can be at least about 10 nucleotides, or at least about 20 nucleotides, or at least about 30 nucleotides, or at least about 40 nucleotides, or at least about 50 nucleotides, or at least about 60 nucleotides, or at least about 70 nucleotides, or at least about 80 nucleotides, or at least about 90 nucleotides, or at least about 100 nucleotides in length.

[0094] In some embodiments, a target nucleic acid sequence can have a GC content of at least about 10%, or at least about 20%, or at least about 50%, or at least about.

[0095] As part of the synthetic methods of the present disclosure, one or more adamers can be immobilized on a solid support. The solid support can be any solid support known in the art, including, but not limited to, at least one bead. In some embodiments, the at least one bead can comprise polyacrylamide, polystyrene, agarose, or any combination thereof. In some embodiments, the at least one bead can be magnetic. In some embodiments, the solid support comprises a well or chamber. In some embodiments, the solid support can comprise a plurality of wells or chambers. In some embodiments, the plurality of wells comprises a multi-well plate. In some embodiments, the solid support can comprise glass. In some embodiments, the solid support can comprise a glass slide. In some embodiments, the solid support can comprise quartz. In some embodiments, the solid support can comprise a quartz slide. In some embodiments, the solid support can comprise polystyrene. In some embodiments, the solid support can comprise a polystyrene slide. In some embodiments, the solid support can comprise a coating, where the coating prevents non-specific binding of undesired proteins, undesired nucleic acids, or other undesired biomolecules. In some embodiments, the coating can include polyethylene glycol (PEG). In some embodiments, the coating can include triethylene glycol (TEG).

[0096] In some embodiments where the adamer comprises a hairpin comprising an aptamer sequence, the adamer can be immobilized to a solid support via binding to the aptamer sequence. That is, the solid support can comprise at least one moiety that binds to the aptamer sequence on the adamer. Thus, in a non-limiting example where the adamer comprises a hairpin comprising one of the aptamer sequences listed in Table 2, the solid support can comprise the corresponding ligand listed in Table 2.

[0097] In some embodiments where the adamers comprise an MCS sequence, the adamers can be immobilized to a solid support by a method comprising: a) contacting the adamers with at least one corresponding restriction endonuclease to cleave the MCS sequence, thereby generating a 5' or 3' overhang, and b) hybridizing the 5' or 3' overhang to a complementary single-stranded nucleic acid molecule on a solid support, thereby immobilizing the adamers to the solid support. The foregoing method can further comprise contacting the adamers hybridized to the complementary single-stranded nucleic acid molecule on the solid support with a ligase, thereby ligating the adamers to the complementary single-stranded nucleic acid molecule on the solid support.

[0098] In some embodiments in which the adammer comprises an MCS sequence, the adammer can be immobilized to a solid support by a method comprising: a) contacting the adammer with at least one corresponding restriction endonuclease to cleave the MCS sequence, thereby generating a blunt end; and b) ligating the blunt end of the adammer to a nucleic acid molecule located on a solid support, thereby immobilizing the adammer to the solid support.

[0099] In some embodiments, an adamer bound to a solid support may be referred to herein as a "binding stud."

[0100] A schematic diagram of the disclosed nucleic acid synthesis method is shown in FIG.

[0101] In the first step of the method, an adammer is provided that is immobilized on a solid support (shown as a bead or surface in FIG. 8). This adammer, referred to herein as a "binding stud," is connected at one end to a solid support using any of the methods described above and is capped at the other end with a hairpin. The binding stud also includes an MCS sequence. In the next step of the method, the binding stud is contacted with a restriction endonuclease that cleaves the MCS sequence, thereby generating a 3' overhang, a 5' overhang, or a blunt end. In the next step of the method, the first adammer, including the MCS sequence, a first IISRE sequence (shown as "L1" in FIG. 8), a first N-mer sequence (shown as "Payload number 1" in FIG. 8), and a second IISRE sequence (shown as "R1" in FIG. 8), is contacted with a restriction endonuclease that cleaves the MCS sequence, thereby generating a 3' overhang, a 5' overhang, or a blunt end.

[0102] In the next step of the method, the cleaved first adamer is ligated to the cleaved binding stud by contacting the cleaved binding stud, the cleaved adamer, and a ligase enzyme, thereby generating a first ligation product that is immobilized on a solid support and includes an MCS sequence, a first IISRE sequence, a payload number 1 sequence, and a second IISRE sequence (see left side of FIG. 8). The first ligation product is then treated with an exonuclease to remove any unligated binding studs and / or the first adamer.

[0103] The above steps are then repeated using another adumer immobilized on a solid support and a second adumer comprising an MCS sequence, a third IISRE sequence (shown as "R2" in Figure 8), a second N-mer sequence (shown as "Payload Number 2" in Figure 8), and a fourth IISRE sequence (shown as "L2" in Figure 8) to generate a second ligation product immobilized on a solid support and comprising an MCS sequence, a third IISRE sequence, a payload number 2 sequence, and a fourth IISRE sequence (see the right side of Figure 8).

[0104] In the next step of the method, the first ligation product is contacted with an IISRE (shown as "R1 enzyme in FIG. 8") that cleaves the second IISRE sequence (R1), thereby generating a 3' overhang, a 5' overhang, or a blunt end, thereby generating a) a first cleaved product that is immobilized on a solid support and includes the MCS sequence, the first IISRE sequence (R1), the payload number 1 sequence, and a 3' overhang, a 5' overhang, or a blunt end, and b) a second cleaved product that includes the second IISRE sequence (R1). The second cleaved product is then discarded by washing.

[0105] In the next step of the method, the second ligation product is contacted with an IISRE (shown as "L2 enzyme" in Figure 8) that cleaves the fourth IISRE sequence (L2), thereby generating a 3' overhang, a 5' overhang, or a blunt end, thereby creating a) a third cleaved product that is released into solution and comprises a hairpin, a third IISRE sequence (R2), a payload number 2 sequence, and a 3' overhang, a 5' overhang, or a blunt end at one end, and b) a fourth cleaved product that is immobilized to a solid support and comprises an MCS sequence and a fourth IISRE sequence (L2).

[0106] In the next step, the first cleaved product and the third cleaved product are ligated together by contacting the first cleaved product, the third cleaved product, and a ligase enzyme (e.g., a solution containing the third cleaved product is transferred to a solution containing the first cleaved product immobilized on a solid surface and a ligase enzyme is added to the solution), thereby generating a third ligation product that is immobilized on a solid surface and includes the MCS sequence, the first IISRE sequence (L1), the payload number 1 sequence, the payload number 2 sequence, and the third IISRE sequence (R2). The ligation reaction is then treated with an exonuclease to remove any unligated first cleaved product and / or the third cleaved product.

[0107] The above steps can be repeated until the target nucleic acid sequence is synthesized.

[0108] A schematic of the synthesis of an exemplary 27 nucleotide long target nucleic acid sequence is shown in Figures 9A-9H. The sequence to be synthesized is shown at the top of Figure 9A. The sequence is subdivided into eleven 6-mer fragments that overlap with either three or four nucleotides that are incorporated into the adamers that are ligated together to synthesize the target nucleic acid sequence. Figure 9B shows an assembly tree of an exemplary target nucleic acid sequence that maps the order in which adamers containing 6-mer fragments are ligated to efficiently synthesize the target nucleic acid sequence. There are several different methods that conflict with the assembly and placement of odd vs. even overhangs, but this assembly order should be determined by the compatibility of the IISRE enzyme site with the sequence to be generated. In Figure 9B, the numbered 6-mers (1)-(11) correspond to the numbered 6-mers in Figures 9C-9H. The numbers at each node of the tree correspond to the payload length at each step of the assembly. The "4" and "3" indicate the length of the overhangs used. The length of the resulting payload sequence is length=a+bn, where "a" and "b" are the lengths of the input payload and "n" is the length of the overhang.

[0109] The first step in the synthesis of a target nucleic acid sequence is shown in Figure 9C, showing the loading of an adammer containing a 3-mer sequence of GAC and an adammer containing a 3-mer sequence of ATC to form an adammer containing a GACATG 6-mer, which is 6-mer number 1 in Figure 9B. To generate the GACATG hexamer, a first binding stud containing the MCS sequence and a first adammer containing the MCS sequence, a first IISRE sequence (shown as "L1" in Figure 9C), a 3-mer sequence containing the sequence GAC, and a second IISRE sequence (shown as "R1" in Figure 9C) are contacted with one or more restriction endonucleases to cleave the MCS sequence, thereby generating a complementary overhang. These complementary overhangs are then hybridized, and the adamer and binding stud are ligated together by contacting the hybridized complex with a ligase enzyme to obtain ligation product number 1, which is immobilized on a solid surface and includes the MCS sequence, a first IISRE sequence (L1), a 3-mer sequence GAC, and a second IISRE sequence (R1). The same process is repeated with a second binding stud including the MCS sequence and a second adamer including the MCS sequence, a third IISRE sequence (shown as "L2" in FIG. 9C), a 3-mer sequence including the sequence ATG, and a fourth IISRE sequence (shown as "R2" in FIG. 9C) to obtain ligation product number 2, which is immobilized on a solid surface and includes the MCS sequence, a third IISRE sequence (L2), a 3-mer sequence ATG, and a fourth IISRE sequence (R2). Ligation product number 1 is then contacted with an IISRE (shown in FIG. 9C as "R1 enzyme") that cleaves the second IISRE sequence (R1), thereby generating cleaved product number 1, which is immobilized on a solid surface and contains the MCS sequence, the first IISRE sequence (L1), and the 3-mer sequence GAC, followed by a blunt end. Similarly, ligation product number 2 is contacted with an IISRE (shown in FIG. 9C as "L2 enzyme") that cleaves the third IISRE sequence (L2), thereby generating cleaved product number 2, which is released into solution and contains the blunt end, the 3-mer sequence ATG, and the fourth IISRE sequence (R2).The cleaved product number 1 and the cleaved product number 2 are then ligated together using a ligase enzyme to obtain ligation product number 3, which is immobilized on a solid support and contains the MCS sequence, the first IISRE sequence (L1), the 6-mer sequence GACATG, and the fourth IISRE sequence (R2). These products can be optionally treated with an exonuclease to remove any unligated cleaved product number 1 and / or cleaved product number 2. The steps described in this paragraph can be repeated with additional adamers containing different 3-mer sequences to generate adamers containing 6-mer SEQ ID NOs: 2-11 as shown in FIG. 9B.

[0110] The method continues in Figure 9D, showing the ligation of an adammer containing a 6-mer SEQ ID NO:1 to an adammer containing a 6-mer SEQ ID NO:2 (see Figure 9B). Adamer number 1 is immobilized on a solid surface and contains an MCS sequence, a first IISRE site (L1) of Figure 9C, a 6-mer sequence GACATG (6-mer SEQ ID NO:1 of Figure 9B), and a fourth IISRE sequence (R2) of Figure 9C. Adamer number 2 is immobilized on a solid surface and contains an MCS sequence, a fifth IISRE site ("L3" in Figure 9D), a 6-mer sequence ATGAGG (6-mer SEQ ID NO:2 of Figure 9B), and a sixth IISRE site (shown as "R3" in Figure 9D). Adamer number 1 contacts an IISRE cleaving the fourth IISRE site (R2) to generate a single-stranded overhang on the N-mer sequence, thereby generating cleaved product number 3, which is immobilized on a solid surface and includes an MCS sequence, a first IISRE sequence (L1), and an N-mer sequence with a single-stranded overhang. Adamer number 2 contacts an IISRE cleaving the fifth IISRE site (L3) to generate a single-stranded overhang on the N-mer sequence, thereby generating cleaved product number 4, which is released into solution and includes an N-mer sequence with a single-stranded overhang and a sixth IISRE sequence (R3). The cleaved product number 3 and the cleaved product number 4 are then ligated together using a ligase enzyme to obtain ligation product number 4, which is immobilized on a solid surface and includes a MCS sequence, a first IISRE sequence (L1), an N-mer sequence GACATGAGG (the first 9 nucleotides in the target nucleic acid sequence to be synthesized), and a sixth IISRE sequence (R3). Ligation product number 4 can be optionally treated with an exonuclease to remove any unligated cleaved product number 1 and / or cleaved product number 2.

[0111] The method continues in Figure 9E, where ligation product number 4 and an adumer containing the 6-mer sequence number 3 (see Figure 9B) are treated with the corresponding IISRE to generate cleaved products which are then ligated together to generate an adumer containing the N-mer sequence GACATGAGGGT (SEQ ID NO: 75), which is immobilized on a solid surface and represents the first 11 nucleotides of the target nucleic acid sequence to be synthesized.

[0112] Successive IISRE digestions and ligations are repeated in Figures 9F-9H according to the assembly map shown in Figure 9B until an adammer containing an N-mer sequence corresponding to the 27 nucleotide long target nucleic acid sequence is synthesized. In a final step, the 27 nucleotide long target nucleic acid sequence can be excised from the final synthetic adammer by treating the final synthetic adammer with an IISRE that cleaves IISRE sequences adjacent to the 27 nucleotide long target nucleic acid sequence.

[0113] The aforementioned method can be described as follows: a) providing a first adamer of the present disclosure immobilized on a solid support, the first adamer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adamer of the present disclosure immobilized on a solid support, the second adamer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; and c) contacting the first adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer, thereby generating a first cleaved product that is immobilized on a solid support and comprises the first IISRE sequence, the first N-mer sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end. d) contacting the second adamer with an IISRE that cleaves a third IISRE sequence located in the second adamer, thereby generating second cleaved products that are released into solution and include a second N-mer sequence, a fourth IISRE sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end, wherein the second cleaved products are capped at one end by a hairpin structure; e) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; f) treating the product of step (e) with an exonuclease, thereby removing unligated first cleaved product and / or second cleaved product; and g) repeating steps (a)-(f) until a nucleic acid molecule containing the target nucleic acid sequence is synthesized.

[0114] The aforementioned method can be described as follows: a) providing a first adamer of the present disclosure immobilized to a solid support, the first adamer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adamer of the present disclosure immobilized to a solid support, the second adamer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; c) contacting the first adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer, thereby generating a first cleaved product that is immobilized to a solid support and comprises the first IISRE sequence, the first N-mer sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end; and d) contacting the second adamer with an IISRE that cleaves a third IISRE sequence located in the second adamer, thereby generating second cleaved products that are released into solution and include a second N-mer sequence, a fourth IISRE sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end, wherein the second cleaved products are capped at one end by a hairpin structure; e) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; f) treating the product of step (e) with an exonuclease, thereby removing unligated first cleaved product and / or second cleaved product; and g) repeating steps (c)-(f) until a nucleic acid molecule comprising the target nucleic acid sequence is synthesized.

[0115] The method can be described as follows: a) providing a first adamer of the present disclosure immobilized on a solid support, the first adamer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adamer of the present disclosure immobilized on a solid support, the second adamer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; c) contacting the first adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer, thereby generating a first cleaved product that is immobilized on a solid support and comprises the first IISRE sequence, the first N-mer sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end; and d) providing a second adamer of the present disclosure immobilized on a solid support, the first IISRE sequence, followed by a first N-mer sequence, followed by a hairpin structure. contacting the second adamer with an IISRE that cleaves a third IISRE sequence located within the second adamer, thereby generating a second cleaved product that is released into solution and includes a second N-mer sequence, a fourth IISRE sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end, wherein the second cleaved product is capped at one end by a hairpin structure; e) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; f) treating the product of step (e) with an exonuclease, thereby removing unligated first cleaved product and / or second cleaved product; and g) repeating steps (a)-(f) with one or more additional adamers until a nucleic acid molecule containing the target nucleic acid sequence is synthesized.

[0116] The aforementioned method can be described as follows: a) providing a first adamer of the present disclosure immobilized on a solid support, the first adamer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adamer of the present disclosure immobilized on a solid support, the second adamer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; c) contacting the first adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer, thereby generating a first cleaved product that is immobilized on a solid support and comprises the first IISRE sequence, the first N-mer sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end; and d) contacting the second adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer. a) contacting a second cleaved product with an IISRE that cleaves a third IISRE sequence located within the second adamer, thereby generating second cleaved products that are released into solution and include a second N-mer sequence, a fourth IISRE sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end, wherein the second cleaved product is capped at one end by a hairpin structure; b) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; c) treating the product of step (e) with an exonuclease, thereby removing unligated first cleaved product and / or second cleaved product; and g) repeating steps (c)-(f) with one or more additional adamers until a nucleic acid molecule comprising the target nucleic acid sequence is synthesized.

[0117] The aforementioned method can be described as follows: a) providing a first adamer of the present disclosure immobilized on a solid support, the first adamer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adamer of the present disclosure immobilized on a solid support, the second adamer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; c) contacting the first adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer, thereby generating a first cleaved product that is immobilized on a solid support and comprises the first IISRE sequence, the first N-mer sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end; and d) contacting the second adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer. contacting the first cleaved product with an IISRE that cleaves a third IISRE sequence located within the second adamer, thereby generating second cleaved products that are released into solution and include a second N-mer sequence, a fourth IISRE sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end, wherein the second cleaved product is capped at one end by a hairpin structure; e) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; f) treating the product of step (e) with an exonuclease, thereby removing unligated first cleaved product and / or second cleaved product; and g) repeating steps (a)-(f) using the product of step (f) and one or more additional adamers until a nucleic acid molecule comprising the target nucleic acid sequence is synthesized.

[0118] The aforementioned method can be described as follows: a) providing a first adamer of the present disclosure immobilized on a solid support, the first adamer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adamer of the present disclosure immobilized on a solid support, the second adamer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; c) contacting the first adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer, thereby generating a first cleaved product that is immobilized on a solid support and comprises the first IISRE sequence, the first N-mer sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end; and d) contacting the second adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer. with an IISRE that cleaves a third IISRE sequence located within the target nucleic acid sequence, thereby generating second cleaved products that are released into solution and include a second N-mer sequence, a fourth IISRE sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end, wherein the second cleaved products are capped at one end by a hairpin structure; e) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; f) treating the product of step (e) with an exonuclease, thereby removing unligated first cleaved product and / or second cleaved product; and g) repeating steps (c)-(f) using the product of step (f) and one or more additional adumers until a nucleic acid molecule comprising the target nucleic acid sequence is synthesized.

[0119] The aforementioned method can be described as follows: a) providing a first adamer of the present disclosure immobilized on a solid support, the first adamer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adamer of the present disclosure immobilized on a solid support, the second adamer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; c) contacting the first adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer, thereby generating a first cleaved product that is immobilized on a solid support and comprises the first IISRE sequence, the first N-mer sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end; and d) contacting the second adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer. contacting the first cleaved product with an IISRE that cleaves a third IISRE sequence located within the N-mer, thereby generating second cleaved products that are released into solution and include a second N-mer sequence, a fourth IISRE sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end, wherein the second cleaved product is capped at one end with a hairpin structure; e) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; f) treating the product of step (e) with an exonuclease, thereby removing any unligated first cleaved product and / or the second cleaved product; and g) repeating any combination of steps (a)-(f) using the product of step (f) and / or one or more additional adumers until a nucleic acid molecule comprising the target nucleic acid sequence is synthesized.

[0120] The method can be described as follows: a) providing a first adamer of the present disclosure immobilized on a solid support, the first adamer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adamer of the present disclosure immobilized on a solid support, the second adamer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; c) contacting the first adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer, thereby generating a first cleaved product that is immobilized on a solid support and comprises the first IISRE sequence, the first N-mer sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end; and d) contacting a second adamer with an IISRE that cleaves the second IISRE sequence located within the second adamer. a third IISRE sequence associated with said target nucleic acid sequence with an IISRE that cleaves said third IISRE sequence, thereby generating second cleaved products that are released into solution and include a second N-mer sequence, a fourth IISRE sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end, wherein the second cleaved products are capped at one end by a hairpin structure; e) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; f) treating the product of step (e) with an exonuclease, thereby removing any unligated first cleaved product and / or the second cleaved product; and g) repeating any combination of steps (c)-(f) using the product of step (f) and / or one or more additional adumers until a nucleic acid molecule comprising the target nucleic acid sequence is synthesized.

[0121] In some aspects of the disclosed method, the ligase enzyme can be human DNA ligase III (hLig3). As will be appreciated by those skilled in the art, hLig3 exhibits high blunt-end ligation efficiency (greater than 60%). In some aspects of the disclosed method, the ligase enzyme can be T4 DNA ligase. As will be appreciated by those skilled in the art, T4 DNA ligase exhibits high ligation efficiency (greater than 80%) of nucleic acid fragments containing 3' or 5' overhangs of 2, 3, or 4 nucleotides in length. The ligase enzyme can be any ligase enzyme known in the art.

[0122] In some embodiments of the disclosed methods, the exonuclease can be T7 exonuclease.

[0123] In some aspects of the disclosed methods, the synthesized target nucleic acid sequence has a purity of at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99%.

[0124] In some aspects, the purity of the synthesized target nucleic acid sequence refers to the percentage of the total ligation products formed as part of a single ligation reaction or multiple ligation reactions that correspond to the correct / desired ligation product. Without wishing to be bound by theory, the disclosed method involving ligation of nucleic acid molecules can generate multiple ligation products, some of which correspond to the correct / desired ligation product and some of which are undesired (side reactions, incorrect ligation, etc.). The purity of the ligation product or the synthesized target molecule can be expressed as a percentage that corresponds to the percentage of the total ligation products formed that correspond to the correct / desired ligation product.

[0125] Example 1 - Synthesis of Hadamer Base The following is a non-limiting example of an adumer-based synthesis method of the present disclosure used to synthesize a target nucleic acid molecule. In this example, a series of restriction enzyme digestions followed by ligation were performed as outlined in FIG. 10. The products of each of the ligation reactions were analyzed using gel electrophoresis. The results of this analysis are shown in FIG. 11. The results shown in FIG. 11 demonstrate that the method of the present disclosure, including repeated restriction enzyme digestions followed by ligation reactions using an adumer of the present disclosure, can efficiently synthesize a nucleic acid molecule.

Claims

1. A double-stranded Addamer, a) a first type II S restriction endonuclease (IISRE) sequence; and b) an N-mer sequence; and c) at least a second IISRE sequence, A double-stranded adamer, wherein at least one end of the adamer comprises a hairpin structure.

2. The adamer of claim 1 , wherein the adamer comprises a hairpin structure at both ends of the adamer.

3. The said adamant is a) a first IISRE sequence; and b) a second IISRE sequence; and c) an N-mer sequence; d) at least a third IISRE sequence.

4. The said adamant is a) a first IISRE sequence; and b) a second IISRE sequence; and c) an N-mer sequence; d) a third IISRE sequence; and and e) at least a fourth IISRE sequence.

5. The adammer of claim 1 , further comprising a multiple cloning site (MCS) sequence, the MCS sequence comprising one or more restriction endonuclease sequences.

6. 2. The adammer of claim 1, wherein at least one of the IISRE sequences is selected from the group consisting of MlyI, NgoAVII, SspD5I, AlwI, BccI, BcefI, PleI, BceAI, BceSIV, BscAI, BspD6I, FauI, EarI, BspQI, BfuAI, PaqCI, Esp3I, BbsI, BbvI, BtgZI, FokI, BsmFI, BsaI, BcoDI, and HgaI sequences.

7. The adammer of claim 1 , wherein at least one hairpin structure comprises an aptamer sequence.

8. 8. The adamer of claim 7, wherein the aptamer sequence is selected from a pL1 aptamer sequence, a thrombin 29-mer aptamer sequence, a S2.2 aptamer sequence, an ART1172 aptamer sequence, a R12.45 aptamer sequence, a Rb008 aptamer sequence, and a 38NT SELEX aptamer sequence.

9. A composition comprising the adammer of claim 1 immobilized on a solid support.

10. The composition of claim 9 , wherein the solid support is a bead.

11. The composition of claim 10 , wherein the beads comprise polyacrylamide, polystyrene, agarose, or any combination thereof.

12. The composition of claim 9 , wherein the solid support is the surface of a well or chamber.

13. The composition of claim 12 , wherein the well or chamber is part of a multi-well plate.

14. the adamer comprises a hairpin structure comprising at least one aptamer sequence; the solid surface comprises at least one ligand that binds to the aptamer sequence; The composition of claim 9 , wherein the aptamer is immobilized on the solid surface via binding of the at least one aptamer sequence to the at least one ligand.

15. the adamer comprises at least one 5' or 3' overhang; the solid surface comprises at least one single-stranded or partially double-stranded nucleic acid molecule having a single-stranded portion complementary to the at least one 5' overhang or 3' overhang; 10. The composition of claim 9, wherein the adamer is immobilized to the solid surface by hybridizing the at least one 5' overhang or 3' overhang to the at least one single-stranded or partially double-stranded nucleic acid on the solid surface.

16. the adamer comprises at least one 5' or 3' overhang; the solid surface comprises at least one single-stranded or partially double-stranded nucleic acid molecule having a single-stranded portion complementary to the at least one 5' overhang or 3' overhang; 10. The composition of claim 9, wherein the adamer is immobilized on the solid surface by hybridizing the at least one 5' overhang or 3' overhang to the at least one single-stranded or partially double-stranded nucleic acid on the solid surface and ligating the adamer and the at least one single-stranded or partially double-stranded nucleic acid.

17. 2. A method for producing the hadamar of claim 1, comprising the steps of: a) chemically synthesizing a first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule, the sequence of the first single-stranded nucleic acid molecule and the sequence of the second single-stranded nucleic acid molecule comprising a portion of the adamer that is generated; the first single-stranded nucleic acid molecule comprises a first region that is complementary to a second region on the second single-stranded nucleic acid molecule and a second region that is self-complementary; synthesizing a second single-stranded nucleic acid molecule comprising a first region that is self-complementary and a second region that is complementary to the first region on the first single-stranded nucleic acid molecule; b) hybridizing the first single-stranded nucleic acid and the second single-stranded nucleic acid to generate a partially double-stranded nucleic acid molecule; c) contacting the partially double-stranded nucleic acid molecule with a ligase enzyme to form a double-stranded adamer structure capped at both ends by a hairpin.

18. 18. The method of claim 17, further comprising treating the product of step (c) with an exonuclease, thereby purifying the properly ligated adamers.

19. 19. The method of claim 17 or claim 18, further comprising contacting the partially double-stranded nucleic acid molecule with a MutS enzyme after step (b) and before step (c).

20. 2. A method for producing the hadamar of claim 1, comprising the steps of: a) cloning the adammer sequence into a phagemid such that the adammer sequence is flanked on both sides by one or more DNAzymes that can be selectively activated; b) converting the phagemid into a packaged bacteriophage using a helper phage, wherein the packaged bacteriophage produces a single-stranded DNA comprising the adamer sequence flanked on both sides by one or more DNAzymes; c) purifying the single-stranded DNA produced by the packaged bacteriophage; and d) allowing the purified single stranded DNA to fold to generate the structure of the one or more DNAzymes and the majority of the double stranded adamer sequence; e) activating the one or more DNAzymes, thereby excising the adumer sequence from the single-stranded DNA produced by the packaged bacteriophage; f) contacting the excised adammer with a ligase enzyme.

21. 21. The method of claim 20, further comprising treating the product of step (f) with an exonuclease, thereby purifying the properly ligated adamers.

22. 2. A method for producing the hadamar of claim 1, comprising the steps of: a) cloning the adumer sequence into a plasmid; b) propagating said plasmid in a suitable host organism; c) purifying the plasmid from the host organism; and d) treating the purified plasmid with one or more of a nickase enzyme and a restriction endonuclease enzyme to excise the adamer sequence from the plasmid; e) contacting the excised adamer sequence with a ligase to generate a double-stranded adamer structure capped at both ends by a hairpin structure.

23. 1. A method for synthesizing a nucleic acid molecule comprising a target nucleic acid sequence, comprising: a) providing a first adumer of claim 1 immobilized on a solid support, said first adumer comprising a first IISRE sequence, followed by a first N-mer sequence, followed by a second IISRE sequence, followed by a hairpin structure; b) providing a second adumer of claim 1 immobilized on a solid support, said second adumer comprising a third IISRE sequence, followed by a second N-mer sequence, followed by a fourth IISRE sequence, followed by a hairpin structure; c) contacting the first adamer with an IISRE that cleaves the second IISRE sequence located within the first adamer, thereby generating a first cleaved product that is immobilized on the solid support and includes the first IISRE sequence, the first N-mer sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end; d) contacting the second adamer with an IISRE that cleaves the third IISRE sequence located within the second adamer, thereby generating a second cleaved product that is released into solution and includes the second N-mer sequence, the fourth IISRE sequence, and at least one of a 3' overhang, a 5' overhang, and a blunt end, wherein the second cleaved product is capped at one end by a hairpin structure; e) ligating the first cleaved product and the second cleaved product using a ligase enzyme to generate a first ligation product; f) treating the products of step (e) with an exonuclease, thereby removing unligated first cleaved products and / or second cleaved products; g) repeating any combination of steps (a)-(f) using the product of step (f) and / or one or more additional adumers of claim 1 until the nucleic acid molecule containing the target nucleic acid sequence is synthesized.

24. 24. The method of claim 23, wherein the ligase enzyme is human DNA ligase III (hLig3).

25. 24. The method of claim 23, wherein the ligase enzyme is T4 DNA ligase.

26. 24. The method of claim 23, wherein the target nucleic acid sequence is at least about 100, or at least about 500, or at least about 1000, or at least about 2000, or at least about 3000, or at least about 4000, at least about 5000 nucleotides in length.

27. 24. The method of claim 23, wherein the synthesized nucleic acid molecule comprising the target nucleic acid sequence has a purity of at least 80%.

28. 24. The method of claim 23, wherein the synthesized nucleic acid molecule comprising the target nucleic acid sequence has a purity of at least 90%.

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