A template-free programmable DNA modular synthesis method based on cleavable auxiliary pairing structure

Abstract

The application provides a template-free programmable DNA modular synthesis method based on a cleavable auxiliary pairing structure. Specifically, the method splits a DNA chain to be synthesized into multiple target fragments, connects the multiple target fragments under a template-free condition by designing a starting chain and a to-be-connected chain containing a target sequence, and obtains a target long-chain DNA; wherein the starting chain comprises a fixed region, a spacer region, an auxiliary region, and a single-stranded extension region; the to-be-connected chain comprises a target fragment and a complementary auxiliary region that is complementary to the auxiliary region; the method makes the single-stranded extension region and the target fragment close to each other in space through the complementary pairing of the auxiliary region and the complementary auxiliary region, thereby connecting to form a stem-loop structure, and then removing the complementary auxiliary region through directional cleavage, thereby connecting the target fragment to the single-stranded extension region of the starting chain to complete one cycle; then, other target fragments are sequentially connected through multiple cycles of reaction to obtain the target long-chain DNA.

Description

Technical Field

[0001] This invention belongs to the field of DNA synthesis technology, and more specifically, this invention relates to a template-free programmable modular DNA synthesis method based on cleavable auxiliary pairing structures. Background Technology

[0002] DNA synthesis plays a crucial role as a core technology in synthetic biology, genetic engineering, biomedical research and development, and DNA information storage. Current mainstream DNA synthesis and assembly methods mainly include phosphorous acid chemical synthesis, enzymatic single-base synthesis, and template-based DNA fragment splicing and assembly technology. Each method has its own emphasis in terms of synthesis length, cost, environmental friendliness, and sequence universality.

[0003] The phosphoramide chemical synthesis method is currently the most widely used de novo DNA synthesis technique. This method involves adding single nucleotides one by one to a solid-phase support in an anhydrous organic solvent through a cycle of deprotection, coupling, blocking, and oxidation. It is highly automated and widely used for primer, probe, and short-chain gene synthesis. However, it is essentially a one-dimensional, single-nucleotide addition process, extending only one base per cycle. Furthermore, the small loss in coupling efficiency at each step is exponentially amplified during synthesis. This not only limits the length of directly synthesized DNA but also causes environmental pollution due to the large-scale use of toxic reagents such as trichloroacetic acid and acetonitrile, hindering the development of large-scale green synthesis.

[0004] Enzymatic single-base DNA synthesis utilizes polymerases to catalyze nucleotide elongation in an aqueous environment, enabling template-free de novo synthesis. Compared to chemical methods, it offers advantages such as being environmentally friendly and having mild reaction conditions. However, to achieve precise sequence control, this method requires reversible termination modifications to the 3' end or bases of the nucleotide substrate. These modifications increase synthesis costs, and steric hindrance can reduce enzymatic coupling efficiency. Furthermore, this technology still suffers from the efficiency bottleneck of adding single nucleotides one by one, resulting in time-consuming synthesis of long-chain DNA, which fails to meet the demands for high-throughput and rapid synthesis.

[0005] Traditional long-chain DNA assembly techniques are based on pre-synthesized short-chain oligonucleotides, using polymerases or ligases to assemble long fragments. While this method can obtain long-chain DNA, its operation is essentially fragment splicing rather than de novo synthesis. The assembly process strictly relies on pre-designed homologous overlapping regions or complementary templates, has stringent requirements on template concentration and annealing temperature, and is easily limited by sequence complexity such as high GC content and repetitive sequences. Furthermore, mutations and truncated byproducts in short-chain precursors are easily amplified during assembly, leading to high costs for subsequent clone screening and sequencing verification.

[0006] Existing exploratory studies have attempted to synthesize DNA without template assistance. For example, existing literature has disclosed de novo synthesis strategies based on the synergistic mediation of DNA polymerase and endonuclease. This strategy utilizes the single nucleotide polymerization capability of DNA polymerase under template-free conditions and achieves DNA synthesis with the synergistic cleavage of endonucleases. However, this technology still faces limitations in achieving customized DNA synthesis. This enzymatic de novo synthesis is essentially still a stepwise addition of single bases, failing to overcome the speed bottleneck of single nucleotide elongation. Furthermore, this template-free synthesis based on polymerase and endonuclease exhibits randomness, generating a large number of repetitive sequences or random byproducts containing endonuclease recognition sites. Because this system depends on specific endonuclease recognition sequences, it is difficult to achieve precise directional splicing of arbitrary target sequences, reducing sequence versatility and design freedom.

[0007] In summary, current technologies for DNA synthesis still face certain bottlenecks. While traditional chemical or enzymatic de novo synthesis methods can achieve template-free directional extension, they are limited by single-base cycling patterns, resulting in upper limits on synthesis efficiency and product length. Existing multiple sequence splicing technologies, although capable of multi-base assembly, heavily rely on complementary templates, sacrificing the sequence freedom inherent in de novo synthesis and making them ill-suited for complex sequences. The rapid, customized synthesis of long-chain DNA faces the dual bottlenecks of the time cost of slow single-base stacking and the sequence design limitations imposed by template dependence, both hindering the industrialization efficiency of related fields.

[0008] Therefore, there is an urgent need in this field to develop a DNA synthesis technology that is highly efficient and not limited by complementary template dependence. Summary of the Invention

[0009] The purpose of this invention is to provide a template-free DNA synthesis method.

[0010] The template-free DNA synthesis method of the present invention can realize the direct directional cascade extension of multi-base fragments, breaking through the efficiency of existing single nucleotide synthesis, and providing a template-free de novo DNA synthesis method using multi-base oligonucleotides as basic synthetic units.

[0011] In a first aspect of the present invention, a method for synthesizing nucleic acids is provided, the method comprising: 1) Divide the target nucleic acid into several target fragments to be synthesized, design the starter strand as shown in Formula I and several ligation strands as shown in Formula II, each ligation strand containing one target fragment. The target nucleic acid is either DNA or RNA; X is selected from modified or unmodified nucleotides A, T, G, C, and U; a, b, c, d, e, and f represent the fixed region, the interval region, the auxiliary region, the single-strand extension region, the complementary auxiliary region, and the target fragment, respectively. n represents the quantity; 2) In the reaction system, the auxiliary region and the complementary auxiliary region are complementary to each other, so that the single-chain extension region and the first target fragment in the first chain to be connected are spatially close to each other and then connected. 3) Cut and remove the complementary auxiliary region, thereby connecting the first target fragment to the single-strand extension region of the starting chain; obtain a first single chain containing the first target fragment; and 4) Repeat steps 2) and 3) to sequentially ligate the target fragments from the other strands to be ligated onto the starting strand, thereby obtaining a single strand containing the target nucleic acid.

[0012] In another preferred embodiment, the target nucleic acid is single-stranded DNA or single-stranded RNA.

[0013] In another preferred example, X a1 This indicates the first nucleotide in the fixed region, and so on.

[0014] In another preferred embodiment, the starting chain is fixed to the surface of the magnetic bead via a fixing region.

[0015] In another preferred embodiment, the end of the fixation region is modified with a biotin group, thereby fixing it to the surface of the streptavidin magnetic beads.

[0016] In another preferred embodiment, the fixation region is complementary to the biotin chain fixed on the surface of the streptavidin magnetic beads, thereby fixing it to the surface of the magnetic beads.

[0017] In another preferred embodiment, the fixation region contains 18-23 nucleotides.

[0018] In another preferred embodiment, the fixation region comprises 18, 19, 20, 21, 22, or 23 nucleotides.

[0019] In another preferred embodiment, the interval region is used to provide a flexible connection between the fixed region and the auxiliary region.

[0020] In another preferred embodiment, the spacer region is a polyT sequence.

[0021] In another preferred embodiment, the spacer region contains 4-10 nucleotides.

[0022] In another preferred embodiment, the spacer region comprises 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0023] In another preferred embodiment, the single-strand extension region comprises 3-15 nucleotides.

[0024] In another preferred embodiment, the single-strand extension region comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.

[0025] In another preferred embodiment, the single-chain extension region is used to provide a suspended single chain connected to the target fragment.

[0026] In another preferred embodiment, the target fragment comprises 2-24 nucleotides, preferably 3-16, more preferably 4-8.

[0027] In another preferred embodiment, the target fragment comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.

[0028] In another preferred embodiment, the auxiliary region comprises 6, 7, 8, 9, 10, 11, or 12 nucleotides.

[0029] In another preferred embodiment, the complementary auxiliary region comprises 6, 7, 8, 9, 10, 11, or 12 nucleotides.

[0030] In another preferred embodiment, the complementary auxiliary region contains a specific enzyme cleavage site.

[0031] In another preferred embodiment, the complementary auxiliary region includes a nicking enzyme cleavage site or a USER cleavage site.

[0032] In another preferred embodiment, the complementary auxiliary region comprises a photosensitive group.

[0033] In another preferred embodiment, in step 2), the starting chain and the chain to be connected are paired through an auxiliary region and a complementary auxiliary region to form a stem-loop structure as shown in Formula III. In another preferred embodiment, in step 2), the reaction system is an aqueous phase reaction system.

[0034] In another preferred embodiment, in step 2), the reaction system further includes a cyclase.

[0035] In another preferred embodiment, in step 3), the complementary auxiliary region is cut by means of: nicking enzyme-specific cutting, USER enzyme cleavage, and photosensitive cleavage.

[0036] In another preferred embodiment, in step 3), the cleavage site is located at the last nucleotide of the complementary auxiliary region.

[0037] In another preferred embodiment, the nicking enzyme is selected from: Nt.BspQI, Nb.BbvCI, and Nt.CviPII.

[0038] In another preferred embodiment, after one cycle of steps 2) and 3), a target fragment can be connected to the starting chain, i.e., connected to the single-chain extension region.

[0039] In another preferred embodiment, the method further includes using an assist chain for pulling the target segment in the starting chain and the target segment in the chain to be connected closer to each other in space, thereby connecting them.

[0040] In another preferred embodiment, some nucleotides of the helper chain are complementary to the target fragment in the starting chain, and some nucleotides are complementary to the target fragment in the chain to be joined.

[0041] In another preferred embodiment, the synthesis method can be used for DNA information storage, DNA nanostructure construction, or molecular diagnostics.

[0042] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0043] Figure 1 This diagram illustrates the principle of modular single-stranded DNA synthesis using the first method of starting strand fixation.

[0044] Figure 2 This diagram illustrates the principle of modular single-stranded DNA synthesis using the second method of starting strand fixation.

[0045] Figure 3 The results show a comparison between direct ligation and auxiliary stem ligation in DNA synthesis methods.

[0046] Figure 4 The diagram shows the schematic and characterization results of the two-round "connect-cut" cycle in Example 3 to achieve module-by-module extension.

[0047] Figure 5 The diagram shows the USER enzyme and the photosensitive cleavage method for DNA synthesis, along with the characterization results.

[0048] Figure 6 This demonstrates a DNA information storage application based on a template-free DNA synthesis method. Detailed Implementation

[0049] Through extensive and in-depth research, the inventors have developed a modular DNA synthesis method. This method involves splitting the DNA strand to be synthesized into multiple target fragments, designing a starter strand and a ligation strand containing the target fragments, and then ligating these fragments under template-free conditions. The method forms a stem-loop structure through complementary pairing of the auxiliary region in the starter strand and the complementary auxiliary region in the ligation strand. The complementary auxiliary region is then directionally cleaved to remove the complementary auxiliary region, thereby ligating the target fragment to the starter strand. Subsequently, other target fragments are ligated sequentially through multiple rounds of cyclic reactions to obtain the target long-chain DNA. This invention is based on this method.

[0050] the term To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0051] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0052] Nucleic acid (DNA / RNA) synthesis methods This invention provides a template-free modular nucleic acid synthesis method.

[0053] In one specific embodiment, a template-free modular DNA synthesis method is provided, the method comprising the following steps: 1) Design the starter strand and the ligation strand based on the target DNA sequence to be synthesized. The starter strand includes a fixed region, a spacer region, an auxiliary region, and a single-strand extension region. The ligation strand includes the target fragment and a complementary auxiliary region that is complementary to the auxiliary region. 2) Connect the starting chain to the surface of the magnetic bead through the fixing area; 3) Add the target strand and its ligase reaction system to the system containing magnetic beads and the starting strand. The starting strand and the target strand are connected through complementary pairing of the auxiliary region and the complementary auxiliary region, as well as the connection between the target fragment and the single-strand extension region, thereby forming a stem-loop structure fixed to the magnetic beads. 4) Add a specific cleavage enzyme (e.g., Nt.BspQI cleavage enzyme) to the reaction system to specifically remove the complementary auxiliary region in the strand to be ligated, thereby ligating the target fragment to the starting strand; 5) Clean to remove excess components, repeat the above steps to sequentially ligate the remaining target fragments to obtain single strands containing the target DNA.

[0054] According to the DNA / RNA synthesis method of the present invention, the DNA sequences of each functional region in the starter strand and the strand to be ligated are not limited to the specific sequences listed in the embodiments of the present invention, as long as they can realize the function of each functional region, and can be designed according to the length and bases of the target DNA. The core of the method of the present invention lies in the pairing function of the auxiliary region and the complementary auxiliary region, so that the two DNA fragments, the target fragment and the single-stranded extension region in the starter strand, are spatially close to achieve seamless ligation without the need for a DNA template.

[0055] According to the DNA / RNA synthesis method of the present invention, the starting strand is fixed to the magnetic bead by the fixation region. The starting strand can be directly connected to the magnetic bead, or it can be connected to the magnetic bead by complementary pairing with the complementary fixation region connected to the magnetic bead. The DNA sequences of the fixation region and the complementary fixation region can be designed by the user, as long as they do not interfere with the sequences of other functional regions in the system.

[0056] According to the DNA / RNA synthesis method of the present invention, after the starting strand and the strand to be joined are paired and joined to form a stem-loop structure, the complementary auxiliary region in the strand to be joined needs to be specifically cut. The method of achieving specific cutting is not limited to the nicking enzyme cutting, USER enzyme cutting and photosensitive cutting methods listed in the embodiments of the present invention. Other specific cutting methods can be adjusted according to the DNA specific cutting methods familiar to those skilled in the art, as long as the complementary auxiliary region can be removed from the stem-loop structure.

[0057] The DNA / RNA synthesis method of the present invention enables template-free modular precise directional ligation, and can be used in fields such as DNA information storage, gene synthesis, DNA nanostructure construction and molecular diagnostics. By improving writing efficiency and reducing error accumulation, it provides a general technology platform for high-throughput nucleic acid synthesis and information encoding.

[0058] DNA encoding / DNA information storage In DNA data storage, DNA encoding is the core technology that converts binary data (0s and 1s) from a computer into the four base sequences of A, T, C, and G in a DNA molecule. It's not enough to simply "write" information; this information must also be able to be accurately "read" after undergoing error-prone processes such as synthesis, storage, and sequencing. This process determines the efficiency and reliability of the storage.

[0059] Common problems in DNA encoding: Homopolymer length constraint: Limits the length of consecutive identical bases in the sequence (e.g., no more than 3 or 4). This is because excessively long homopolymers (such as "AAAA") are highly susceptible to insertion or deletion errors during synthesis and sequencing.

[0060] GC content balance (GC-Content Constraint): This requires the total percentage of G and C bases in the sequence to be maintained within a preset range (e.g., 40%-60%). Excessively high or low GC content can lead to abnormal DNA melting temperatures, affecting the uniformity of operations such as PCR amplification.

[0061] Secondary structures and dangerous motifs: Sequences that form stable secondary structures (such as hairpin structures) can interfere with amplification and sequencing. Simultaneously, the coding sequence must avoid being identical to PCR primers, address sequences, etc., to prevent non-specific binding.

[0062] The DNA synthesis method of the present invention avoids interference from redundant sequences and DNA mispairing information storage through modular directional ligation, and can accurately store and reflect digital information.

[0063] Cutting enzyme Nickel endonucleases are a type of restriction endonuclease, a special class of nucleases. Their core function is to cleave only one strand of double-stranded DNA at a specific sequence, creating a "nick," rather than cutting both strands as normally as ordinary restriction endonucleases. Unlike many traditional restriction enzymes that recognize palindromic sequences, nick endonucleases recognize non-palindromic sequences. This allows them to distinguish between the sense and antisense strands of DNA, thus achieving single-strand cleavage.

[0064] The cleavage product is not two DNA fragments, but a circular or linear DNA molecule with a single-stranded gap. This gap is a standard 3'-hydroxyl (3'-OH) and 5'-phosphate (5'-P) end, which can serve as the starting point for a variety of subsequent enzymatic reactions, such as DNA synthesis or exonuclease digestion.

[0065] The cutting enzymes described in this invention include not only naturally occurring cutting enzymes, but also their engineered variants.

[0066] USER enzyme USER enzyme (uracil-specific excision reagent) is a hybrid enzyme that combines uracil DNA glycosylase (UDG) and endonuclease VIII, and is mainly used for DNA repair and molecular biology experiments.

[0067] USER enzyme is composed of two main components: 1) Uracil DNA glycosylation enzyme (UDG): This enzyme can recognize and remove uracil bases in DNA to form an abase site (AP site) while maintaining the integrity of the DNA phosphodiester backbone structure.

[0068] 2) Endo VIII: The lysin activity of this enzyme causes the phosphodiester bonds on both sides of the AP site to break, thereby releasing baseless deoxyribose and forming a single nucleotide gap.

[0069] Compared with the prior art, the main advantages of the present invention include: 1. The DNA synthesis method of the present invention can achieve precise directional ligation of target DNA fragments under template-free conditions.

[0070] 2. Ligating oligo-fragments significantly improves DNA synthesis efficiency.

[0071] 3. Achieve zero redundant sequence residue through precise cutting design.

[0072] 4. The DNA synthesis reaction system is carried out under aqueous conditions, which is suitable for high-throughput and green synthesis.

[0073] 5. The DNA synthesis method of the present invention has good modular expansion capability and can be used for customized synthesis of different sequences.

[0074] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0075] Example 1. A DNA synthesis method based on two initiation strand fixation methods There are two methods for fixing the starting chain: Method 1 (Indirect fixation): The starting strand is fixed to the magnetic beads by complementary pairing with the DNA bases on the streptavidin magnetic beads; Method 2 (direct fixation): The starting chain is directly fixed to the surface of streptavidin magnetic beads by modifying the end with a biotin group.

[0076] DNA modular synthesis method based on method one ( Figure 1 ), including the following steps: Step 1: Design the starter strand and the ligation strand based on the target DNA sequence to be synthesized, wherein: a. The starter strand is a single-stranded DNA, and its sequence consists of four functional regions from the 5' end to the 3' end, including: a single-strand extension region; an auxiliary region a, which is used for complementary pairing with the ligation strand, and this pairing region contains a nicking enzyme recognition sequence, and this sequence is adjacent to the single-strand extension region without spacers; a spacer region, which is a poly T sequence used to provide spatial flexibility; and an immobilization region, which is used for complementary pairing with the biotin strand immobilized on the surface of the magnetic beads, thereby achieving the immobilization of the starter strand on the surface of the magnetic beads; b. The ligation strand can hybridize with the auxiliary region a of the starter strand, and its sequence includes: an auxiliary region a', which is complementary to the auxiliary region a of the starter strand, and contains a nicking enzyme recognition sequence, and this sequence is adjacent to the target sequence region without spacers; and a target sequence region, which is the target sequence fragment to be introduced, and its length is 4–6 bases; c. The full-length target DNA sequence is divided into multiple consecutive target sequence regions, each consisting of 4-6 bases, and each region is designed as a target sequence region for multiple strands to be ligated, thus achieving stepwise synthesis; d. The starting strand is immobilized on the magnetic beads by complementary pairing with the Biotin strand immobilized on the surface of the magnetic beads through the immobilization region; after completing all "ligation-cutting" cyclic synthesis processes, the starting strand and its extension products are dissociated from the surface of the magnetic beads and released into the liquid phase system by adding a substitution strand that is completely complementary to the Biotin strand.

[0077] Step 2: Incubate the Biotin chain described in Step 1 with streptavidin magnetic beads at room temperature for 15-20 min to fix the Biotin chain onto the surface of the magnetic beads. After cleaning to remove unbound components, add the starting chain, the chain to be ligated, the cyclase and its reaction system to the system. First, incubate at 70°C for 3-5 min, then place at room temperature for 3-5 min for annealing, and then carry out a constant temperature ligation reaction at 45-50°C for 1-3 h. In this process, by introducing an auxiliary stem structure into the system, the 5' phosphate group at the end of the chain to be ligated and the 3' hydroxyl group of the starting chain are brought close to each other in space through base complementarity, thereby improving the efficiency of the ligation reaction.

[0078] Step 3: After cleaning the reaction system from Step 2 to remove free components, perform the cleavage reaction according to the specific cleavage method to remove the auxiliary region, ensuring that the cleavage site occurs precisely at the boundary between the auxiliary region a' and the target sequence region, releasing and removing the auxiliary region a'. Cleavage Method 1 (Enzymatic Ceiling): The specific cleavage site is a cleavage enzyme recognition sequence (e.g., the Nt.BspQI recognition site). Add the corresponding cleavage enzyme and cleave at 45-50℃ for 0.5-1 h. Cleavage Method 2 (USER Enzyme Cleavage): The specific cleavage site contains deoxyuracil (deoxyU) modification. Add the USER enzyme to specifically recognize and cleave the phosphodiester bond, and cleave at 37℃ for 0.5-1 h. Cleavage Method 3 (Photosensitive Cleavage): The specific cleavage site contains a photosensitive cleavage group (e.g., iPCLink). Irradiate with 365 nm ultraviolet light to cleave the photosensitive group. Through the above cleavage reactions, the target sequence region is seamlessly retained at the 3' end of the starting chain, and its 5' end exposes an active phosphate group, serving as the acceptor for the next round of ligation.

[0079] Step 4: Repeat steps 2 and 3, using a ligation-cutting cycle to sequentially ligate multiple target sequence modules to the starting strand, achieving module-by-module extension synthesis of the target single-stranded DNA. Finally, the synthesized DNA sequence is released from the solid support surface by substituting the strand.

[0080] DNA modular synthesis method based on method two ( Figure 2 ), including the following steps: Step 1: Design the starter strand and the ligation strand based on the target DNA sequence to be synthesized, wherein: a. The starter strand is a single-stranded DNA, and its sequence consists of three functional regions from the 5' end to the 3' end, including: a single-strand extension region; an auxiliary region a, which is used for complementary pairing with the ligation strand, and this pairing region contains a nicking enzyme recognition sequence, and this sequence is adjacent to the single-strand extension region without spacers; a spacer region, which is a partial NGS sequencing primer sequence used to provide spatial flexibility; the 3' end of the starter strand is modified with a biotin group to achieve immobilization of the starter strand on the magnetic bead surface; b. The ligation strand can hybridize with the auxiliary region a of the starter strand, and its sequence includes: an auxiliary region a', which is complementary to the auxiliary region a of the starter strand, and contains a nicking enzyme recognition sequence, and this sequence is adjacent to the target sequence region without spacers; a target sequence region, which is the target sequence fragment to be introduced, and its length is 4–6 bases; c. The full-length target DNA sequence is divided into multiple consecutive target sequence regions, each consisting of 4-6 bases, and each region is designed as a target sequence region for multiple strands to be joined, in order to achieve stepwise synthesis.

[0081] Step 2: Incubate the biotin-modified starting chain described in Step 1 with streptavidin magnetic beads at room temperature for 15-20 min to fix the starting chain on the surface of the magnetic beads; after cleaning to remove unbound components, add the chain to be ligated and its ligase reaction system to the system, incubate at 70℃ for 3-5 min, then place at room temperature for 3-5 min for annealing, and then add the ligase and carry out the isothermal ligation reaction at 45-50℃ for 1-3 h.

[0082] Step 3: After cleaning the reaction system from Step 2 to remove free components, perform a cleavage reaction using a specific cleavage method to remove the auxiliary region, ensuring the cleavage site occurs precisely at the boundary between the auxiliary region a' and the target sequence region, releasing and removing the auxiliary region a'. After the cleavage reaction is complete, the target sequence region is seamlessly retained at the 3' end of the starting chain, and its 5' end exposes an active phosphate group, serving as the acceptor for the next round of ligation.

[0083] Step 4: Repeat steps 2 and 3, using a "ligation-cutting" cycle to sequentially ligate multiple target sequence modules to the starting strand, achieving module-by-module extension synthesis of the target single-stranded DNA. Finally, a denaturing reaction with 95% formamide is performed to dissociate the starting strand and its extension products from the magnetic bead surface and release them into the liquid phase system.

[0084] Example 2. Comparison of template-free DNA synthesis methods with and without auxiliary regions To verify the ligation efficiency of the DNA traceless ligation extension method of the present invention, a comparison was made between stem-loop ligation based on the auxiliary region and direct ligation without the auxiliary region.

[0085] Figure 3 a is a schematic diagram of the design for direct connection and stem auxiliary connection.

[0086] Figure 3 b shows the denaturing gel electrophoresis results under different ligation methods. Lanes 1 and 2 represent direct ligation reactions, with relatively few ligation products. When using the STEM-assisted ligation strategy, a significantly enhanced target product band can be observed. Furthermore, as... Figure 3Lanes 3 to 8 in diagram b illustrate the analysis of stem-assisted ligation reactions under different strand lengths. The products from each lane were subjected to fluorescence imaging followed by grayscale analysis to assess ligation efficiency. Results showed that when the DNA strand to be ligated was 25 nt in length, the ligation efficiency decreased to approximately 90%; when this length increased to 30 nt, the ligation efficiency increased again. Combined with NUPACK analysis of the secondary structure of the starting and ligated strands, it was found that when the 3′ end single-strand length was 30 nt, the single-stranded DNA may form local secondary structures at the stem end, thereby altering the spatial conformation of the terminal single strand, bringing the ligated groups closer together, and leading to a rebound in ligation efficiency.

[0087] Example 3. Template-free DNA synthesis example (1) Target sequence and oligonucleotide design Taking the target sequence “CAGGATAATGAT (SEQ ID NO. 6)” (12 nt in total) as an example, it is divided into two consecutive target sequence modules: Module 1: CAGGAT; Module 2: AATGATAAT. A starter strand is designed based on the target sequence, including a single-strand extension region, an auxiliary region a, a spacer region, and a fixed region. A ligation strand is designed based on the auxiliary region a in the starter strand and the target sequence modules, where each ligation strand includes an auxiliary region a' and a target sequence region. Specifically, the following are designed: Ligation strand 1: AGGCTCTTCGAATGAT, Ligation strand 2: AGGCTCTTCGCAGGAT. The underlined sequence is the Nt.BspQI nickase recognition site, and the base following the recognition site is the cleavage site. GCTCTTC G | AATGAT.

[0088] In DNA synthesis methods based on indirect fixation, the specific sequences of the starting strand, the strand to be joined, the substituent strand, and the biotin strand are shown in Table 1.

[0089] Table 1. Sequences of the starting chain, the chain to be joined, the replacement chain, and the biotin chain. (2) Magnetic bead fixation and hybridization Biotin chains were incubated with streptavidin magnetic beads at room temperature for about 15 minutes to immobilize the biotin chains on the surface of the magnetic beads. After washing to remove unbound components, the biotin chains were used for subsequent reactions.

[0090] (3) First round of connection reaction The starting chain, the chain to be joined 1, and the cyclase reaction system were added to the system. The mixture was denatured at 70°C for about 5 min, and then annealed at room temperature for about 5 min. After annealing, the cyclase was added to the reaction system, and the mixture was incubated at 50°C for 3 h to carry out the ligation reaction. During this process, the formation of the auxiliary stem structure makes the 5' phosphate group of the chain to be joined and the 3' hydroxyl group of the starting chain spatially close, thereby improving the ligation efficiency.

[0091] (4) First round of cutting reaction After the ligation reaction is completed, the system is cleaned, and then a specific nicking enzyme and its reaction system are added. Here, Nt.BspQI nicking enzyme is used as an example. The reaction is carried out at 50℃ for 1 h. By cutting specific sites in the auxiliary stem structure, the auxiliary structure is removed, and only the target sequence module is retained, achieving traceless extension. A 5' phosphate group is generated at the end of the product for the next round of ligation reaction.

[0092] (5) Second round of cyclic reaction Repeat the above steps of ligation reaction (3) and cleavage reaction (4), add the chain to be ligated 2 to the system, complete the ligation and cleavage of the second target sequence module, and thus achieve the stepwise extension synthesis of the target sequence.

[0093] (6) Product release and detection After completing all connection-cutting cycles, a substituent chain that is completely complementary to the biotin chain is added to the system, causing the starting chain and its extended products immobilized on the surface of the magnetic beads to dissociate and be released into the liquid phase system; the supernatant is collected and the products are characterized and analyzed by gel electrophoresis.

[0094] (7) Results Explanation Figure 4 Electrophoresis results showed that DNA bands of the target length were successfully synthesized. Denaturing polyacrylamide gel electrophoresis results showed that the left lane contained the initiating strand (iDNA), and the right lane contained the products of two rounds of "ligation-cutting" reactions. After two rounds of reactions, the product was mainly the target length band, with a yield of 82.8%. A small number of intermediate bands corresponding to a single round of reactions were also observed, indicating that the ligation-cutting cycle was highly efficient and could achieve seamless extension. This demonstrates that this method can achieve efficient and seamless ligation of multi-base modules under template-free conditions, validating the feasibility of the method.

[0095] Example 4. A template-free DNA synthesis method based on USER enzyme or photosensitive cleavage group. This embodiment verifies that, without relying on specific nicking enzymes, the removal of auxiliary regions and the extension of target DNA sequences can also be achieved by introducing specific chemical modifications at specific sites on the strand to be ligated. The specific steps of this technical solution are as follows: (1) Oligonucleotide structure design To extend the target sequence, a cleavable modification is introduced at the junction of the auxiliary region of the strand to be ligated and the target sequence region to construct a specific cleavage site. In one embodiment, deoxyuracil is introduced at this location. After hybridization between the starting strand and the strand to be ligated, a 10 bp auxiliary double-stranded structure is formed, with the ligation sequence being AGGCTCTTCG / ideoxyU / ACAT. In another embodiment, a photosensitive cleavage group is introduced at this location. After hybridization, an approximately 10 bp auxiliary double-stranded structure is also formed, with the ligation sequence being AGGCTCTTCG / iPCLink / ACAT.

[0096] (2) Connection reaction The starting strand and the modified strand to be ligated were added to the reaction system and treated at 70 °C for 5 min, then cooled to room temperature and held for 5 min to allow them to form a local double-stranded structure through complementary pairing in the auxiliary region. Then, ligase and its buffer system were added, and the reaction was carried out at 50 °C for 3 h to achieve covalent ligation between the strand to be ligated and the starting strand.

[0097] (3) Auxiliary area resection response After ligation, the resulting extended intermediate underwent helper region removal. For the deoxyuridine modification system, USER enzyme was added, and the reaction was carried out for 1 h. This enzyme recognizes the uracil site in the double-stranded structure and induces phosphodiester bond cleavage, thereby removing the helper region and retaining the target sequence. For the photosensitive cleavage system, the reaction product was irradiated under 365 nm ultraviolet light to break the photosensitive group and release the helper region. After the above treatment, the resulting product formed an active end with a 5' phosphate at the target sequence end, which can be used for subsequent ligation reactions.

[0098] (4) Result characterization The reaction products were analyzed by denaturing polyacrylamide gel electrophoresis. Figure 5 The results of a-5b showed that the migration rate of the product after the ligation reaction was reduced compared with that of the unreacted starting chain, indicating that the chain to be ligated had been successfully ligated.

[0099] like Figure 5 As shown in Figure a, the left side illustrates the USER enzyme cleavage, where specific cleavage occurs at the dU modification site. The middle gel electrophoresis results show that the control group contains intact full-length DNA before cleavage, which, after USER enzyme treatment, produces cleavage products, with the corresponding bands marked by black triangles. The right gel image further demonstrates the product characterization results after one round of "ligation-USER enzyme cleavage" reaction, with the target product band clearly visible (shown by black triangles), indicating that the USER enzyme can be effectively used for the cleavage step in this system.

[0100] like Figure 5 As shown in b, the left side illustrates UV cleavage, where UV light can be used to break DNA fragments at the PC-linker site. The middle gel image confirms the feasibility of UV cleavage; no complete full-length DNA bands were observed after cleavage, and the products were mainly short fragments (marked with black triangles). The right gel image shows a round of "ligation-cleavage" reaction based on UV cleavage. The second lane contains the ligation product, showing high ligation efficiency with almost no byproducts; the third lane contains the product after "ligation-UV cleavage," indicating that UV cleavage can also achieve effective cleavage and is suitable for this cycle system.

[0101] The above results demonstrate that by introducing deoxyuracil or photosensitive cleavage groups, the auxiliary region can be effectively removed in a single ligation and cleavage process, generating a reactive end structure. This indicates that the method is versatile in its cleavage mechanism, and the removal of the auxiliary region can be achieved by selecting either enzymatic or photoresponsive methods as needed.

[0102] Example 5. Application of template-free DNA synthesis method To verify the feasibility of applying the multi-round "ligation-cutting" cyclic synthesis strategy in DNA information storage, this embodiment provides a DNA information writing and reading process based on this method. Figure 6 (a-6b), the relevant sequences are shown in Table 2.

[0103] First, the digital information to be stored is converted into a corresponding DNA sequence according to a preset encoding rule. This encoding rule maps different characters to specific oligonucleotide module sequences. Then, several DNA sequences corresponding to characters from the encoding rule are selected as target information fragments. During DNA synthesis, the starting strand immobilized on a solid support undergoes multiple rounds of ligation-cutting cycles. In each cycle, a pre-designed oligonucleotide module is ligated to the end of the extension strand via cyclase-mediated ligation, and auxiliary structures are removed through specific cleavage to achieve seamless extension. Each completed cycle represents the writing of one information module. In this embodiment, three rounds of ligation-cutting cycles are performed, corresponding to the step-by-step writing of three information modules, thereby obtaining the target DNA sequence. After the extension reaction is complete, a termination strand is ligated to the end of the DNA strand. Subsequently, the synthesized product is subjected to PCR amplification for subsequent analysis.

[0104] The amplified products were detected by agarose gel electrophoresis, and the results showed that the multi-round modular synthesis process had good construction efficiency. Finally, the amplified products were sequenced, and the sequencing results were decoded according to the encoding rules, successfully restoring the original input digital information, proving that this method can achieve high-fidelity writing and reading of DNA information. This embodiment demonstrates that the multi-round modular synthesis strategy can achieve step-by-step precise assembly of information modules, with good sequence accuracy and scalability, and can be applied to the field of DNA data storage.

[0105] Table 2. Relevant sequences for DNA data storage All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for synthesizing nucleic acids, characterized in that, The method includes: 1) Divide the target nucleic acid into several target fragments to be synthesized, design the starter strand as shown in Formula I and several ligation strands as shown in Formula II, each ligation strand containing one target fragment. The target nucleic acid is either DNA or RNA; X is selected from modified or unmodified nucleotides A, T, G, C, and U; a, b, c, d, e, and f represent the fixed region, the interval region, the auxiliary region, the single-strand extension region, the complementary auxiliary region, and the target fragment, respectively. n represents the quantity; 2) In the reaction system, the auxiliary region and the complementary auxiliary region are complementary to each other, so that the single-chain extension region and the first target fragment in the first chain to be connected are spatially close to each other and then connected. 3) Cut and remove the complementary auxiliary region to connect the first target fragment to the single-strand extension region of the starting chain; obtain a first single chain containing the first target fragment; and 4) Repeat steps 2) and 3) to sequentially ligate the target fragments from the other strands to be ligated onto the starting strand, thereby obtaining a single strand containing the target nucleic acid.

2. The synthesis method according to claim 1, characterized in that, The starting chain is fixed to the surface of the magnetic bead through a fixing zone.

3. The synthesis method as described in claim 1, characterized in that, The target fragment contains 2-24 nucleotides, preferably 3-16, more preferably 4-8.

4. The synthesis method according to claim 1, characterized in that, The auxiliary region contains 6, 7, 8, 9, 10, 11, or 12 nucleotides.

5. The synthesis method according to claim 1, characterized in that, The complementary auxiliary region contains 6, 7, 8, 9, 10, 11, or 12 nucleotides.

6. The synthesis method according to claim 1, characterized in that, The complementary auxiliary region contains specific enzyme cleavage sites.

7. The synthesis method according to claim 1, characterized in that, The complementary auxiliary region contains a photosensitive group.

8. The synthesis method according to claim 1, characterized in that, In step 2), after the starting chain and the chain to be connected are paired through the auxiliary region and the complementary auxiliary region, a stem-loop structure as shown in Formula III is formed. 。 9. The synthesis method according to claim 1, characterized in that, In step 2), the reaction system is an aqueous reaction system.

10. The synthesis method according to claim 1, characterized in that, In step 3), the complementary auxiliary region is cut in the following ways: nicking enzyme-specific cleavage, USER enzyme cleavage, and photosensitive cleavage.

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