A cell-free mRNA template high-throughput preparation method and system based on continuous multi-enzyme reaction and online quality control

Abstract

The present disclosure belongs to the technical field of mRNA drug production and manufacturing, and relates to a cell-free mRNA template high-throughput preparation method and system based on continuous multi-enzyme reaction and online quality control. The method comprises the following steps: providing a target mRNA coding region fragment, a polyA coding sequence and a circularization linker; generating a single-stranded circular DNA through a first directional enzyme cutting-linking reaction; obtaining a single-stranded DNA concatemer through rolling circle replication amplification; and connecting the polyA coding sequence through a second directional enzyme cutting-linking reaction after fragmenting the single-stranded DNA concatemer to generate a linear DNA template for in vitro transcription. The present application realizes cell-free, high-throughput and automated template preparation, and is especially suitable for personalized mRNA therapy.

Description

Technical Field

[0001] This disclosure belongs to the field of mRNA drug manufacturing technology, specifically relating to a high-throughput method and system for preparing cell-free mRNA templates based on continuous multi-enzyme reactions and online quality control, providing a high-throughput, automated cell-free DNA template preparation process suitable for personalized mRNA therapies. Background Technology

[0002] mRNA drugs, especially personalized cancer vaccines based on neoantigens, present revolutionary demands on manufacturing processes due to their clinical application models: high personalization—each patient requires a specific mRNA sequence tailored to their unique mutation profile; microscale production—single doses are typically in the microgram to milligram range; and extremely short production and delivery cycles—usually requiring the entire process from tumor tissue sequencing to formulation release to be completed within weeks. In vitro transcription using T7 RNA polymerase is a common core technology for preparing active mRNA molecules. However, the method of preparing the DNA template, the starting template for the in vitro transcription reaction, directly determines the flexibility, speed, and product quality of the entire manufacturing process.

[0003] Currently, mainstream large-scale in vitro transcription DNA templates heavily rely on recombinant plasmids amplified through fermentation using *E. coli* as the host. While this paradigm is mature, it has several inherent limitations. First, bacterial genomic fragments, endotoxins, and host proteins may remain in the plasmid template, posing additional challenges and introducing potential safety risks for downstream purification. Second, when cloning and amplifying plasmids containing long homopolymer sequences (such as A / T sequences encoding poly(A) tails of mRNA) in vivo, homologous recombination can easily lead to deletions or heterogeneity in the length of this region, affecting the stability and translation efficiency of the final mRNA product. Furthermore, to meet Good Manufacturing Practices (GMP) requirements for pharmaceuticals, a large master cell bank and working cell bank system needs to be established, maintained, and validated, which is time-consuming and costly.

[0004] To overcome the inherent risks of plasmid systems, the industry is exploring cell-free DNA template preparation technologies. However, current mainstream cell-free methods (such as linear PCR amplification) still face new challenges: it is difficult to precisely control the length and uniformity of the poly(A) tail while ensuring sequence fidelity; their discrete, multi-step operation mode is difficult to meet the requirements of high-throughput, automation, and continuous production, and they are insufficient in preventing cross-contamination and online quality control. Although some advanced technologies have made progress, they have not completely broken away from the biological source of "specialized plasmids" and have not achieved a completely plasmid-free process from the source.

[0005] In summary, existing technologies suffer from systemic deficiencies in addressing the personalized, high-throughput, and highly compliant clinical production needs. At the paradigm level, they heavily rely on bacterial fermentation, failing to eliminate the risk of biological impurities and dependence on GMP-compliant strain libraries. Furthermore, the process is coupled with sequences, lacking flexibility and structurally contradicting the "multi-sequence, small-batch, rapid-turnover" model of personalized vaccines. At the quality control level, there is a lack of precise control over the length and uniformity of poly(A) tails. Long homopolymeric sequences are unstable in plasmids, leading to length heterogeneity and affecting translation efficiency and half-life. Cloning and amplification of complex templates (such as those containing repetitive sequences or special linkers) results in low efficiency or even failure. At the system adaptation level, multi-step discrete operations require frequent capping and intermediate purification, resulting in low throughput and strong reliance on manual processes, making them unsuitable for automated, closed-loop continuous production. The lack of online quality control and anti-contamination design makes rolling circle amplification products prone to cross-contamination, affecting environmental stability and detection reliability.

[0006] Therefore, there is an urgent need in this field to develop a new method for preparing cell-free mRNA templates that combines high fidelity, high throughput, high integration, and suitability for industrial quality control and compliant production, so as to promote the translation of personalized mRNA therapies from laboratory research to widespread clinical application. Summary of the Invention

[0007] Existing research in this field mainly focuses on the ligation efficiency of the cyclization step itself, while the impact of the cyclization method on the fragmentation efficiency of subsequent rolling circle replication products has not been addressed as an independent technical issue. The inventors of this disclosure have experimentally discovered that the annealing efficiency and accessibility of restriction enzyme sites in rolling circle replication products generated by the traditional head-to-tail self-ligation method are not ideal.

[0008] To address the above technical problems, this disclosure provides a method for preparing a cell-free mRNA template, comprising the following steps (a)-(e): (a) Provide a first nucleic acid fragment containing the target mRNA coding region, a second nucleic acid fragment containing a polyA coding sequence, and a circular adapter; The ring-shaped joint has a stem-ring structure; (b) The first nucleic acid fragment is directionally assembled and circularized with the circular adapter through a first directional enzyme digestion-ligation reaction to generate a single-stranded circular DNA template; In this process, stem-loop structures of the circular adapter are connected to both ends of the first nucleic acid fragment to form a single-stranded circular DNA with a dumbbell-shaped structure. (c) Obtain single-stranded DNA polymers containing multiple copies of the target sequence by rolling circle replication amplification; The single-stranded circular DNA template enables the amplified single-stranded DNA multiplexes to self-fold through intramolecular annealing, forming multiple double-stranded stem regions. (d) Fragmenting the single-stranded DNA multiplex; (e) The second nucleic acid fragment is ligated to the product of the fragmentation treatment by a second directional enzyme digestion-ligation reaction to generate a linear DNA template for in vitro transcription.

[0009] In some embodiments, the enzyme digestion efficiency of the single-stranded DNA multiplex in the fragmentation process is ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0010] In some embodiments, the first directional enzyme digestion-ligation reaction and / or the second directional enzyme digestion-ligation reaction are performed using restriction endonucleases and ligases.

[0011] In some implementations, the rolling circle replication amplification is performed using DNA polymerase.

[0012] In some embodiments, the restriction endonuclease is a Type IIS restriction endonuclease, and the ligase is a T4 DNA ligase.

[0013] In some embodiments, the DNA polymerase is phi29 DNA polymerase.

[0014] In some implementations, all steps of the method are carried out continuously in a single reaction vessel.

[0015] In some implementations, the first nucleic acid fragment is derived from a gene synthesis fragment, plasmid, or PCR amplification product.

[0016] In some embodiments, the first nucleic acid fragment comprises, from the 5' end to the 3' end, the following in sequence: a first E1 site, a T7 promoter sequence, a target RNA coding region, an E2 site, an optional E3 site, and a reversed second E1 site; wherein the E1 site is the restriction enzyme site of the first directional restriction enzyme digestion-ligation reaction, and the E2 and / or E3 sites are the restriction enzyme sites of the fragmentation treatment and / or the second directional restriction enzyme digestion-ligation reaction.

[0017] In some embodiments, the fragmentation process and / or the second directional enzyme digestion-ligation reaction are performed via a one-step directional enzyme digestion-ligation reaction at the E2 site.

[0018] In some implementations, the E1 site is located inside the PCR primer.

[0019] In some implementations, all steps of the method are performed sequentially in a single reaction vessel by continuously diluting the reaction buffer, without any intermediate purification steps.

[0020] In some implementations, steps (d) and (e) are performed via a one-step directional enzyme digestion-ligation reaction.

[0021] In some implementations, the sequential dilution includes a 1.5-3 fold dilution during step (b).

[0022] In some implementations, the sequential dilution includes diluting by 25-50 times during step (c).

[0023] In some implementations, the sequential dilution includes a 1.5-3 fold dilution during steps (d) and (e).

[0024] In some embodiments, the sequential dilution includes: (1) diluting by 2 times during step (b); (2) diluting by 50 times during step (c); and / or (3) diluting by 2 times during steps (d) and (e).

[0025] In some embodiments, the polyA coding sequence includes a sticky end at the 5' end that is complementary to the target site of the single-stranded DNA multiply.

[0026] In some implementations, the viscous ends are formed in one of the following ways: (1) Direct chemical synthesis of sequences with protruding ends, formed by double-strand annealing; (2) Introduce a stem-loop structure containing restriction enzyme sites into the polyA coding sequence, exposing sticky ends after enzyme digestion; or (3) A partial double-chain structure is formed by double-chain annealing, with one end retaining a sticky end.

[0027] In some embodiments, the method further includes online quality control of the reaction process throughout the preparation process.

[0028] In some implementations, the online quality control utilizes FRET technology; the FRET technology employs two oligonucleotide probes labeled with a first fluorescent dye and a second fluorescent dye, and when a directional enzyme digestion-ligation reaction occurs, the two dyes move spatially close to each other to generate a FRET signal.

[0029] In some embodiments, the fluorescent dye is labeled at the 5' end or middle of the oligonucleotide.

[0030] In some embodiments, the first fluorescent dye and the second fluorescent dye are selected from AF488 and Cy3, Cy3 and Cy5, FAM and TAMRA, or Alexa Fluor 488 and Alexa Fluor 594.

[0031] In some embodiments, the first fluorescent dye is AF488 and the second fluorescent dye is Cy3.

[0032] In some embodiments, the first fluorescent dye is Cy3 and the second fluorescent dye is Cy5.

[0033] In another aspect, this disclosure provides a cell-free mRNA template high-throughput preparation system, said system for use in the method described above, comprising: (1) A modular nucleic acid component comprising a first nucleic acid fragment, a second nucleic acid fragment, and a circular adapter as described above; (2) A continuous multi-enzyme reaction unit configured to perform steps (a)-(e) sequentially in a single reaction vessel and having a built-in dilution control module; (3) Online quality control unit, which is configured to monitor the reaction process in real time using FRET technology, including a fluorescence detection module and a FRET signal analysis module.

[0034] In some implementations, the sequential multi-enzyme reaction unit is configured to process multiple different target mRNA sequences in parallel.

[0035] In some implementations, the continuous multi-enzyme reaction unit is an automated workstation.

[0036] In some implementations, the single reaction vessel is a 96-well plate.

[0037] In some implementations, the dilution control module is configured to dilute by 1.5-3 times during step (b), by 25-50 times during step (c), and by 1.5-3 times during steps (d) and (e).

[0038] In some implementations, the dilution control module is configured to dilute by 2 times in step (b), by 50 times in step (c), and by 2 times in steps (d) and (e).

[0039] In another respect, this disclosure provides a cell-free mRNA template preparation kit for performing the aforementioned methods or for assembling the aforementioned systems.

[0040] This disclosure provides a complete, modular, and continuously operating high-throughput system for preparing cell-free mRNA templates, particularly suitable for the rapid production of personalized mRNA drugs. Its beneficial effects include, but are not limited to: (1) Through innovative design of the rolling circle replication template topology, a single-stranded circular template is generated by cyclization of the stem-loop linker, replacing the traditional double-stranded circular template. Its rolling circle product forms a uniform double-stranded unit through self-folding, which significantly improves the annealing efficiency and the accessibility of the enzyme cleavage site, thereby greatly improving the fragmentation efficiency and product uniformity.

[0041] (2) It integrates a variety of optional cyclization strategies, RCA conditions, polyA design schemes, dilution optimization strategies, FRET online quality control methods and anti-pollution design; (3) Includes optimized reaction systems, connectors, and polyA element designs to improve efficiency; (4) It can flexibly select combinations of different technical features according to specific needs to prepare linear DNA templates for in vitro transcription in an efficient, flexible and precise manner. Attached Figure Description

[0042] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this specification and, together with the description, serve to explain the principles of this specification.

[0043] Figure 1 shows a schematic diagram of the preparation process of a novel mRNA in vitro transcription (IVT) template.

[0044] Figure 2 shows schematic diagrams of the structures of key molecules in the preparation of the novel mRNA template, in which... Figure 2 'a' represents the sequence structure of the starting molecule. Figure 2 b represents the structure of a cyclized molecule. Figure 2 c represents the structure of the rolling ring replication cascade sequence. Figure 2 d is a schematic diagram of the fragmentation process of the rolling ring replication product. Figure 2 e represents the synthesized polyA fragment. Figure 2 f is a schematic diagram showing the connection results between the fragmented RCA product and the polyA sequence.

[0045] Figure 3 shows the gel electrophoresis verification results of each intermediate product in the preparation of the novel mRNA template.

[0046] Figure 4 shows a comparison of the biological activities of mRNA prepared by the method of the present invention and mRNA prepared by the conventional method in 293T cells.

[0047] Figure 5 shows the effect of different dilution factors on the amount of rolling ring replication (RCA) product.

[0048] Figure 6 shows a schematic diagram comparing the structures of the two cyclization strategies.

[0049] Figure 7 shows gel electrophoresis diagrams comparing the fragmentation efficiency of RCA products; where A is a comparison of the efficiency of two cyclization strategies, and B is a comparison of the efficiency before and after introducing mismatch.

[0050] Figure 8 shows a schematic diagram of the structure of three polyA encoded sequences.

[0051] Figure 9 shows a detailed structural diagram of the B-type stem-ring polyA sequence.

[0052] Figure 10 shows a gel plot verifying the ligation efficiency of the RCA fragmentation product with the B-type polyA sequence.

[0053] Figure 11 shows a schematic diagram of the principle of FRET monitoring of unidirectional enzyme digestion and ligation reactions.

[0054] Figure 12 shows the FRET signal efficiency detection graph after the enzymatic ligation reaction of AF488 and Cy3-labeled oligos. Detailed Implementation

[0055] I. Definition In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0056] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. In this disclosure, unless otherwise stated, the scientific and technical terms used have meanings commonly understood by those skilled in the art. It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, and variations thereof are certainly possible. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used in this disclosure are all widely used terms and routine procedures in their respective fields. It should also be understood that the terminology used in this disclosure is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0057] In this disclosure, the conjunction term "and / or" between multiple elements means that it includes both the meaning of "and" and "or".

[0058] In this disclosure, the terms “comprising,” “including,” “having,” and “containing,” and any variations thereof, are intended to cover non-exclusive inclusion. The term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term “comprising” includes the more restrictive terms “consisting of” and “substantially consisting of”. In this disclosure, the term “containing” indicates that various ingredients may be used together in mixtures or compositions of this disclosure.

[0059] In this disclosure, the terms “about” or “approximately” applied to one or more target values ​​refer to values ​​similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the terms “approximately” or “about” refer to a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0060] As used in this article, the term "mRNA in vitro transcription (mRNA in vitro transcription)" is used to refer to the transcription of m in vitro In vitro transcription (IVT) refers to the process of chemically synthesizing IVT mRNA molecules in a cell-free system using components such as DNA templates, ribonucleotides, and RNA polymerase. It is a key production step in the preparation of mRNA vaccines and therapies.

[0061] As used herein, the term "Type IIS restriction endonuclease" refers to a subclass of type II restriction endonucleases. Type II restriction endonucleases (RE II) are a class of endonucleases that recognize specific short nucleotide sequences (usually palindromic sequences) and cleave the DNA double strand within or at a fixed position within that sequence. In this patent, unless otherwise specified, the recognition site and the cleavage site are the same. The core feature of Type IIS restriction endonucleases is that the recognition site and the cleavage site are mutually separate, allowing for directed cleavage and ligation reactions to achieve seamless, directed assembly of DNA fragments.

[0062] As used in this article, the term "T7 promoter sequence" refers to a DNA sequence that can be highly specifically recognized and bound by phage-derived T7 RNA polymerase. Its core conserved sequence is TAATACGACTCACTATA, which can guide the transcription of T7 RNA polymerase into its downstream initiating RNA. It is the most commonly used promoter for in vitro transcription and synthesis of mRNA.

[0063] As used in this article, the term "IVT mRNA" refers to a single-stranded RNA molecule synthesized through an in vitro transcription reaction using linearized DNA as a template. Its sequence ranges from the precise transcription start site (+1) to the 3' end of the linearized DNA template. A fully functional IVT mRNA typically contains a 5' cap structure, a 5' untranslated region, an open reading frame of the target gene, a 3' untranslated region, and a 3' polyadenylated tail.

[0064] As used in this article, the term "linearization" refers to the process of precisely cutting circular plasmids or linear template DNA into linear DNA downstream of the transcription unit using restriction endonucleases. It is a key step in the in vitro transcription preparation of mRNA templates and can control the length and sequence consistency of the RNA product.

[0065] As used herein, the term "polyA sequence" refers to a homopolymer tail chain located at the 3' end of an mRNA molecule, consisting of tandemly linked adenosine (A) nucleotides, typically comprising 100-150 A bases. This sequence interacts with intracellular poly(A)-binding proteins, enhancing mRNA stability and translation efficiency. The uniformity and integrity of its length are crucial for the function and quality of mRNA drugs. In some embodiments of this disclosure, a polyA splicing design is employed—a design that uses a one-way enzymatic digestion-ligation reaction to directionally ligate a chemically synthesized, precisely-length polyA sequence module to a specific location on a DNA template in vitro. This avoids the instability associated with in vivo cloning or in vitro amplification of longer homopolymer sequences, ensuring the accuracy and uniformity of the polyA coding sequence in the transcription template.

[0066] As used in this article, the term "Rolling Cycle Amplification (RCA)" refers to a technique that uses polymerases with high continuous synthesis capacity and strand displacement activity, such as Phi29 DNA polymerase, to perform isothermal nucleic acid amplification using single-stranded circular DNA as a template. This technique can produce ultra-long linear single-stranded DNA products composed of tandemly linked complementary copies of the template sequence.

[0067] As used in this article, the terms "directional restriction enzyme digestion-ligation reaction" or "one-way restriction enzyme digestion-ligation reaction" refer to highly efficient DNA directional assembly technology represented by GoldenGate assembly. It utilizes the synergistic effect of Type IIS restriction endonuclease and T4 DNA ligase in the same reaction system. Through sequence design, DNA fragments are digested to produce unique non-palindromic complementary sticky ends, thereby forcing the fragments to be seamlessly and irreversibly ligated in a preset order and direction.

[0068] As used herein, the term "circular molecule" refers to a specialized adaptor molecule for preparing single-stranded circular DNA templates for rolling circle amplification. In some embodiments of this disclosure, the circular molecule is formed by intramolecular base complementarity folding of a specially designed single-stranded DNA to form a stem-loop (hairpin) structure, with complementary sticky ends at its 5' and 3' ends. It can be directionally ligated to a target DNA fragment via a one-way enzyme digestion-ligation reaction to achieve intramolecular circularization, significantly improving circularization efficiency and direction specificity.

[0069] As used herein, the term "first nucleic acid fragment" refers to a nucleic acid molecule containing the core sequence of the target mRNA coding region. In some embodiments of this disclosure, the first nucleic acid fragment is a linear double-stranded or single-stranded DNA, which, from its 5' end to its 3' end, sequentially comprises: a first restriction enzyme site (E1 site) for a first directional restriction enzyme digestion-ligation reaction, a T7 promoter sequence, the target RNA coding region, a second restriction enzyme site (E2 site) and an optional third restriction enzyme site (E3 site) for fragmentation processing and / or a second directional restriction enzyme digestion-ligation reaction, and a second E1 site with the opposite orientation to the first restriction enzyme site. The first nucleic acid fragment may be derived from gene synthesis, plasmid digestion, or PCR amplification products.

[0070] As used herein, the term "second nucleic acid fragment" refers to a nucleic acid molecule containing a polyA coding sequence. In some embodiments of this disclosure, the second nucleic acid fragment is a chemically synthesized, precisely-length DNA sequence having a sticky end at its 5' end capable of complementary pairing with fragmented single-stranded DNA ligands for integration into the 3' end of a linear DNA template via a second directional enzyme digestion-ligation reaction.

[0071] As used herein, the terms "stem-loop structure" or "hairpin structure" are used interchangeably, referring to a nucleic acid structure in which two complementary or substantially complementary sequences within the same nucleic acid molecule anneal to form a partially double-stranded region, containing a single-stranded loop. An exemplary structure is shown below. Figure 1 As shown in b.

[0072] As used herein, the term "dumbbell-shaped structure" refers to a nucleic acid molecule with one or more hairpin structures that is structurally linear but topologically circular, suitable for in vitro replication or amplification. When this molecule denatures or substantially denatures, it exists as a single-stranded circular nucleic acid molecule. An exemplary structure is shown below. Figure 1 As shown in b.

[0073] As used herein, the term "double-stranded stem region" refers to a double-stranded segment formed by intramolecular annealing of a single-stranded nucleic acid molecule through Watson-Crick base complementarity pairing. When there are two or more complementary segments in a single-stranded nucleic acid molecule, a periodic fold-back structure can be formed through self-folding. This structure is a multi-stem-loop structure containing multiple parallel double-stranded nucleic acid segments (i.e., the double-stranded stem regions). Adjacent double-stranded stem regions are sequentially connected by U-shaped single-stranded linking loops formed by single-stranded nucleic acids, forming a periodic, serpentine arrangement. An exemplary structure is shown below. Figure 6 As shown in a.

[0074] As used herein, the term "single-stranded circular DNA" refers to a closed single-stranded circular DNA molecule formed by the directional ligation and circularization of the first nucleic acid fragment with a circularization adapter through a first directional enzyme digestion-ligation reaction. This single-stranded circular DNA serves as a template for subsequent rolling circle replication amplification.

[0075] As used herein, the term "single-stranded DNA polyp" refers to a linear single-stranded DNA product generated by rolling circle replication amplification using single-stranded circular DNA as a template, consisting of multiple unit-length complementary copies of the template sequence tandemly. This polyp product is then fragmented into unit-length linear fragments.

[0076] As used herein, the term "fragmentation" refers to the process of cutting single-stranded DNA multiples generated by rolling circle replication into unit-length linear DNA fragments. In a preferred embodiment of this disclosure, the fragmentation is achieved by specific digestion at predetermined E2 and / or E3 sites using Type IIS restriction endonucleases, and this digestion step can be completed in one step in the same reaction system as a second directional digestion-ligation reaction.

[0077] As used herein, the terms "E1 site," "E2 site," and "E3 site" refer to different restriction endonuclease recognition and cleavage site sequences artificially designed and introduced into the first nucleic acid fragment. In this disclosure, the sites are preferably Type IIS restriction endonuclease recognition sites. The E1 site is used for the first directional restriction enzyme digestion-ligation reaction, mediating the directional ligation and circularization of the first nucleic acid fragment with the circularization adapter; the E2 site and optionally the E3 site are used for fragmentation processing, cutting single-stranded DNA multiplexes into unit-length fragments, and simultaneously or subsequently mediating a second directional restriction enzyme digestion-ligation reaction with the second nucleic acid fragment. In some embodiments of this disclosure, through low carry-over design (LCD), the RCA template structure is modified to place the circularization site (E1 restriction site) inside the PCR primer, so that the circularized RCA template lacks a complete PCR primer binding site, thereby preventing cross-contamination of subsequent PCR processes after RCA product leakage.

[0078] As used herein, the term “single reaction vessel” refers to a process in which all or the main enzyme reactions in steps (a) through (e) are carried out continuously in the same physical vessel (e.g., but not limited to, a PCR tube, centrifuge tube, or a single well of a 96-well plate) without involving any transfer of the reaction solution to another vessel.

[0079] As used herein, the terms "serial dilution" or "serial dilution reaction buffer" refer to a technique used when performing multiple enzymatic reactions sequentially in a single reaction vessel. This involves adding appropriate amounts of dilution buffer or water to the reaction system to adjust conditions such as ionic strength, pH, or enzyme cofactor concentration, enabling subsequent reactions to proceed smoothly without intermediate purification steps. In some embodiments of this disclosure, the serial dilution includes dilutions of specific folds after the first directional enzyme digestion-ligation reaction, after rolling circle replication amplification, and before the second directional enzyme digestion-ligation reaction.

[0080] As used herein, the term "sticky end" refers to a single-stranded protruding sequence at the end of a double-stranded DNA molecule, produced by restriction endonuclease cleavage or chemical synthesis. In this disclosure, the sticky end is preferably a non-palindromic sequence produced by Type IIS restriction endonuclease cleavage, which can undergo sequence-specific annealing with the end of another DNA fragment through a designed complementary sequence, thereby achieving efficient, directional, and seamless assembly of the fragment in a directional enzyme digestion-ligation reaction.

[0081] As used herein, the term "online quality control" refers to a quality control method that monitors the reaction process in real time, continuously, or at intervals during preparation, without sampling the reaction system or pausing the reaction. In some embodiments of this disclosure, the online quality control utilizes FRET technology to indicate the occurrence and completion of the directed enzyme digestion-ligation reaction by detecting changes in specific fluorescence signals in the reaction system.

[0082] As used herein, the term "modular nucleic acid assembly" refers to a combination of nucleic acid elements prepared or designed in advance to implement the methods described in this disclosure, comprising a circular adapter, a first nucleic acid fragment, and a second nucleic acid fragment. "Modular" means that each nucleic acid element has a standardized interface sequence (e.g., compatible sticky ends generated after Type IIS digestion), allowing template preparation to be completed under the same reaction system and process by simply replacing the target RNA coding region in the first nucleic acid fragment for different target mRNA sequences.

[0083] As used herein, the term "continuous multi-enzyme reaction unit" refers to an integrated functional module configured to sequentially perform a first directed enzyme digestion-ligation reaction, rolling circle replication-amplification, fragmentation, and a second directed enzyme digestion-ligation reaction within a single reaction vessel. This unit includes a built-in dilution control module for automatically adding dilution buffer to the reaction system according to a preset program to adjust the conditions required for each reaction step.

[0084] As used herein, the term "phi29 DNA polymerase" refers to a DNA polymerase derived from Bacillus subtilis phage phi29, which possesses high continuous synthesis capacity and strong strand displacement activity. In this disclosure, it is preferably used for isothermal rolling circle replication amplification using single-stranded circular DNA as a template.

[0085] As used herein, the term "T4 DNA ligase" refers to an ATP-dependent DNA ligase derived from T4 bacteriophage that catalyzes the formation of phosphodiester bonds between adjacent 5'-phosphate and 3'-hydroxyl groups in double-stranded DNA and can ligate single-stranded DNA nicks under certain conditions. In this disclosure, it is preferably used in synergy with Type IIS restriction endonucleases to achieve efficient ligation of DNA fragments in directed enzyme digestion-ligation reactions.

[0086] As used herein, the term "Golden Gate assembly" refers to a DNA-directed assembly technique that utilizes the synergistic action of Type IIS restriction endonucleases and DNA ligases in the same reaction system. Through sequence design, each DNA fragment is digested to produce unique non-palindromic complementary sticky ends, thereby forcing the fragments to be seamlessly and irreversibly joined in a predetermined order and orientation. The "unidirectional enzyme digestion-ligation reaction" or "directed enzyme digestion-ligation reaction" in this disclosure encompasses technical solutions based on this principle.

[0087] As used in this article, the term "high-throughput" refers to an industrial process design that reconstructs the traditional multi-step, discrete template preparation process into an integrated, closed, and programmable automated production system to meet the needs of large-scale and compliant production of mRNA personalized drugs. This system enables key steps such as enzyme digestion, ligation, and amplification to be performed continuously in the same reaction vessel without intermediate purification. It also incorporates online quality control nodes to achieve automated tracking of samples and data.

[0088] As used in this article, the term "FRET (fluorescence resonance energy transfer)" refers to the nonradiative energy transfer process between two fluorescent molecules (donor and acceptor). When the donor molecule is excited, it can transfer energy to the acceptor molecule which is closer (usually <10nm), causing the acceptor molecule to emit fluorescence. This can be used to detect the progress of a one-way enzymatic ligation reaction in real time.

[0089] This article provides a novel method for preparing mRNA templates. Through optimized steps such as one-way enzyme digestion and ligation, rolling circle amplification, fragmentation, and polyA ligation, it achieves efficient and continuous preparation of mRNA templates. At the same time, it provides clear definitions of relevant terms to clearly explain the technical solution disclosed herein.

[0090] II. Detailed Implementation Plan In one aspect, this disclosure provides a method and system for high-throughput preparation of cell-free mRNA templates based on continuous multi-enzyme reactions and online quality control. The overall process of this method is as follows: using starting molecules, a linear DNA template for in vitro transcription (IVT) is synthesized through a series of continuous enzymatic reactions.

[0091] 1. Sequence Design In some embodiments, the starting molecule is selected from gene synthesis products, plasmids, or linear DNA from other sources.

[0092] In some embodiments, the starting molecule is selected from a PCR-amplified linear DNA fragment.

[0093] In some specific implementations, the sequence structure of the starting molecule is as follows: Figure 2 As shown in Figure a, from the 5' end to the 3' end, the sequence includes: upstream primer, E1 site, T7 promoter sequence, RNA sequence consisting of 5'UTR-CDS-3'UTR, E2 / E3 site, reverse E1 site, and downstream primer. The E1 site is used for directional restriction enzyme digestion and ligation-circularization, the T7 promoter is required for IVT, and the E2 / E3 sites are used for directional restriction enzyme digestion and ligation of the polyA tail.

[0094] In some specific implementations, the E1 site can be designed inside the PCR primer so that the circularized PCR primer sequence is not included in the circular molecule, thereby preventing cross-contamination of the RCA product to the subsequent PCR process.

[0095] In some implementations, the initiating molecule is a gene synthesis fragment containing E1 sites at its 5' and 3' ends, respectively.

[0096] In some specific implementations, the starting molecule is chemically synthesized linear DNA that does not contain any PCR primer sequences, but only contains the E1 site, T7 promoter, target RNA coding region, and E2 site.

[0097] In some implementations, the starting molecule is a plasmid that is linearized by a restriction endonuclease and contains E1 sites at both ends.

[0098] 2. Continuous multi-enzyme reaction process In some implementations, mRNA is synthesized through the following steps: Step 1, a one-way enzyme digestion-ligation reaction targeting the E1 site to form a cyclized molecule; Step 2, RCA amplification of the cyclized product; Step 3, fragmentation of the RCA product; Step 4, ligation with a polyA sequence. Figure 1 ) In some specific embodiments, the unidirectional enzymatic digestion-ligation-circularization includes: directionally assembling and circularizing the hairpin adapter sequence and linearization initiator molecule under the action of restriction endonuclease and ligase. Specifically, the circularization adapter is a nucleic acid molecule with a stem-loop structure, with sticky ends at both ends that can complement the ends of the first nucleic acid fragment. Under the combined action of Type IIS restriction endonuclease and ligase, the upper 5' end of one end of the first nucleic acid fragment is connected to one end of the circularization adapter, and the lower 3' end, complementary to the upper strand, is connected to the other end of the circularization adapter; the other end of the first nucleic acid fragment is connected to the circularization adapter in the same manner. Thus, the first nucleic acid fragment and the circularization adapter together constitute a molecule that is topologically circular and structurally dumbbell-shaped. The two ends of this dumbbell-shaped structure are the stem loop formed by the circularization adapter (equivalent to the two spherical ends of the dumbbell), and the middle double-stranded segment is the first nucleic acid fragment (equivalent to the handle of the dumbbell). The dumbbell-shaped structure, after denaturation, exists as a single-stranded circular DNA molecule. Its circular topology ensures that it can serve as a template for continuous reading by polymerase during the subsequent rolling circle replication step, thereby efficiently synthesizing single-stranded DNA polynucleotides containing multiple copies of the target sequence. Figure 1 b).

[0099] In some specific implementations, mRNA is synthesized through the following steps: Step 1, a one-way enzyme digestion-ligation reaction is performed at the E1 site to ligate the double-stranded digestion product with a hairpin-fit sequence, forming a hairpin-shaped circular molecule; Step 2, the circular product is subjected to RCA amplification; Step 3, the RCA product is fragmented; Step 4, ligation with a polyA sequence. Figure 1 ) In some specific implementations, the one-way restriction ligation-circularization includes: one-way restriction ligation mediated by Type IIS restriction endonuclease.

[0100] In some specific embodiments, the one-way enzymatic cleavage ligation-cyclization includes: directly cleaving the E1 sites at both ends of the starting molecule, and then using a ligase to connect the starting molecules end to end to form a cyclic molecule.

[0101] In some specific implementations, the one-way enzymatic ligation-circularization includes: directly catalyzing the circularization of single-stranded DNA using a circulase.

[0102] The cyclization linker described in this disclosure functions to: (i) directionally connect to the target fragment via sticky ends; and (ii) provide a single-stranded loop region as a template for continuous loop reading by RCA polymerase via a stem-loop structure. The realization of these functions does not depend on a specific nucleotide sequence of the linker. Specifically, the design of the cyclization linker only needs to satisfy the following: its sticky ends are complementary to the overhangs at both ends of the target fragment, and the sequence between the two sticky ends can form a stem-loop structure through intramolecular base complementarity. The stem region needs to have sufficient length to ensure a stable double-stranded state at the reaction temperature; this is a conventional design that can be determined based on base pairing thermodynamics (e.g., it can be quickly calculated using Tm (°C) = 2 × (A + T) + 4 × (G + C)). Provided this stability requirement is met, the specific base sequences, lengths, and stem-loop ratios of the stem and loop regions can be arbitrarily chosen by those skilled in the art based on synthetic convenience or preference, without affecting the cyclization efficiency or subsequent RCA yield. Therefore, the specific sequence and structural parameters of the ring-shaped connector are not necessary for realizing the present invention. Those skilled in the art can design and obtain ring-shaped connectors adapted to the method system of the present invention based on ordinary technical knowledge.

[0103] In some specific implementations, the RCA amplification includes: using a circularized molecule as a template, performing rolling circle replication under the action of DNA polymerase to obtain a single-stranded DNA multiplex containing multiple copies of the target sequence.

[0104] In some specific embodiments, the DNA polymerase is phi29 DNA polymerase.

[0105] In some specific embodiments, the fragmentation includes digesting the RCA product with a restriction endonuclease that recognizes the E2 site to obtain fragmented DNA of uniform length.

[0106] In some specific implementations, the fragmentation includes: digesting the RCA product with a Type IIS restriction endonuclease, and generating sticky ends after digestion for direct use in subsequent ligation.

[0107] In some specific implementations, the fragmentation includes digesting the RCA product with a conventional type II restriction endonuclease to produce sticky ends or blunt ends.

[0108] In some preferred embodiments, the fragmentation efficiency is characterized by the ratio of the amount of enzyme digestion product to the amount of RCA product.

[0109] In some preferred embodiments, the fragmentation efficiency is ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0110] In some specific embodiments, the ligation with the polyA sequence includes: performing a second directional restriction enzyme digestion and ligation of the fragmented product and the chemically synthesized polyA sequence under the action of restriction endonuclease and ligase to generate a complete IVT linear DNA template.

[0111] In some specific embodiments, the ligase is T4 DNA ligase.

[0112] In some specific implementations, processes 3 and 4 can be carried out by a one-step directional enzymatic ligation reaction.

[0113] In some specific implementations, the obtained IVT DNA template is purified using magnetic beads, and then IVT is used to prepare mRNA.

[0114] 3. Continuous reaction system In some specific implementations, multiple enzyme reactions in the aforementioned processes 1-4 can be carried out continuously, and the intermediate reaction can eliminate the influence of its buffer solution on the next enzyme reaction by reasonable dilution, without the need for intermediate purification steps.

[0115] In some specific implementations, the aforementioned method further includes: diluting the sample 1.5-3 times after the one-way enzyme digestion-ligation reaction in process 1.

[0116] In some specific implementations, the aforementioned method further includes performing a first dilution after RCA amplification in process 2.

[0117] In some specific implementations, the first dilution is a dilution factor of 25-50 times.

[0118] In some specific implementations, the aforementioned method further includes a second dilution following the fragmentation and polyA sequence ligation of the RCA products from processes 3 and 4.

[0119] In some specific implementations, the second dilution is a dilution factor of 1.5 to 3 times.

[0120] In some specific implementation schemes, the dilution factor is as follows: 1.5-3 times dilution after directional enzyme digestion, ligation, and circularization reaction; a third dilution after circularization amplification, directional fragmentation, and polyA ligation.

[0121] In some specific implementations, the third dilution is a dilution factor of 1.5 to 3 times.

[0122] In some preferred embodiments, the first dilution is a dilution factor of 2, the second dilution is a dilution factor of 50, and the third dilution is a dilution factor of 2.

[0123] In some specific implementations, multiple enzyme reactions in processes 1-4 described above are carried out continuously in a single reaction vessel.

[0124] In some specific implementations, multiple enzyme reactions in processes 1-4 described above are carried out continuously in an automated workstation.

[0125] In some specific implementations, the multiple enzymatic reactions in processes 1-4 described above are carried out continuously in a 96-well plate.

[0126] 4. PolyA Encoding Sequence Design In some implementations, the polyA sequence in the mRNA is formed via IVT using a PolyT single-stranded template.

[0127] In some implementations, the polyA coding sequence is chemically synthesized and then ligated with the RCA fragmentation product via targeted enzymatic digestion.

[0128] In some specific implementations, the structure of the polyA encoding sequence is type A: polyT is a linear single strand, and the 5' end forms a sticky end with a short oligo ( Figure 8 A).

[0129] In some specific implementations, the polyA coding sequence has a B-type structure: the 5' end is a stem-loop structure containing a Type IIS restriction site, which, after digestion, forms a sticky end consistent with type A, and downstream is a single-stranded polyT sequence ( Figure 8 B).

[0130] In some specific implementations, the polyA encoding sequence has a C-type structure: it contains both a long polyA chain and a long polyT chain, with one end forming a sticky terminator consistent with the A-type structure. Figure 8 C).

[0131] In some preferred embodiments, the polyA coding sequence has a B-type structure. The stem-loop structure at the 5' end of the polyA coding sequence can form a stem-loop double strand at the 5' end, which can be linked to the gene sequence after enzyme digestion, facilitating a one-way enzyme digestion reaction.

[0132] In some preferred embodiments, the connection efficiency between the fragmented product and the polyA coding sequence is ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0133] In some implementations, the polyA coding sequence contains a polyT sequence of 100-120 T bases.

[0134] In some implementations, the polyA coding sequence contains a polyT sequence of 50-80 T bases.

[0135] In some implementations, the polyA coding sequence contains a polyT sequence of 150-200 T bases.

[0136] The function of the polyA coding sequence in this disclosure is to provide a DNA template containing a polyT, enabling the mRNA product after in vitro transcription to obtain a polyA tail of precise length. This function requires only that the 5' end of the polyA coding sequence possess a sticky end complementary to the fragmented product for directional ligation, regardless of the specific length of the polyT sequence, the formation method of the sticky 5' end, or the presence or absence of a stem-loop structure. Those skilled in the art will understand that the design of the polyA coding sequence can be accomplished using only the following conventional methods: (a) preparing a polyT sequence of the desired length through chemical synthesis; (b) directly introducing a sticky end at the 5' end through chemical synthesis, or exposing the sticky end through enzymatic digestion of the stem-loop structure, or retaining the sticky end after forming a partial double strand through polyA / polyT annealing (including but not limited to the A, B, and C structures verified in Example 5); (c) selecting the appropriate polyT length (e.g., 50-200 nt) according to the required polyA tail length of the target mRNA. Example 5 has verified that the polyA coding sequences of three different exemplary structural forms can achieve efficient directional enzymatic ligation with the RCA fragmented product. Therefore, the specific structural form of the polyA encoding sequence, the polyT length, and the formation method of the 5' end sticky end can be arbitrarily selected and designed by those skilled in the art as needed, and are not necessary for realizing the present invention.

[0137] 5. FRET detection In some implementations, a method is included for real-time detection of the extent of the one-way enzymatic ligation reaction.

[0138] In some implementations, the method is FRET detection.

[0139] In some embodiments, the method includes employing two oligonucleotide chains labeled with a first fluorescent dye and a second fluorescent dye. When the unidirectional enzymatic ligation reaction proceeds sufficiently, the first and second fluorescent dyes generate a FRET signal due to their spatial proximity.

[0140] In some embodiments, the first and second fluorescent dyes are labeled at the 5' end of the oligonucleotide chain. Figure 11 (Left side).

[0141] In some embodiments, the first and second fluorescent dyes are labeled at the middle position of the oligonucleotide chain. Figure 11 (Right side).

[0142] In some specific embodiments, the first fluorescent dye and the second fluorescent dye are selected from fluorescent pairs that can generate FRET signals on a multifunctional microplate reader.

[0143] In some specific embodiments, the first fluorescent dye and the second fluorescent dye are selected from AF488 and Cy3.

[0144] In some specific embodiments, the first fluorescent dye and the second fluorescent dye are selected from Cy3 and Cy5.

[0145] In some specific embodiments, the first fluorescent dye is AF488 and the second fluorescent dye is Cy3.

[0146] In some specific embodiments, the first fluorescent dye is Cy3 and the second fluorescent dye is Cy5.

[0147] In some implementations, a real-time quantitative PCR instrument or a multi-functional microplate reader is used to detect the FRET signal.

[0148] In some implementations, the FRET detection is performed only once at the reaction endpoint.

[0149] In some implementations, the FRET detection is performed continuously during the reaction process, and the signal change curve is recorded in real time.

[0150] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include embodiments having combinations of all or some of the described features. The enzymes, instruments, and reagents used in the experiments of this disclosure are all conventional types in molecular biology, and appropriate specifications and models can be selected according to actual experimental needs.

[0151] Example Example 1: Preparation of mRNA template The starting material was the initial PCR product (PCR template sequence as shown in SEQ ID NO: 1), which was a linear DNA fragment with the following structure: Figure 2 As shown in SEQ ID NO: 2, the primer contains an upstream PCR primer binding site, an E1 site (containing a BsmBI restriction site), a T7 promoter, a target RNA sequence, an E2 site, a reverse E1 site, and a downstream PCR primer binding site. The concentration is 40 ng / μL, the volume is 25 μL, and the total amount is 1 μg. The upstream and downstream PCR primer sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0152] 1. Directed enzymatic cleavage and cyclization reaction Directed enzyme digestion, ligation, and circularization reactions were performed using a total reaction volume of 50 μL: 25 μL of PCR product (containing 1 μg DNA) at a concentration of 40 ng / μL was taken, and 1 μL of BsmBI Type IIS restriction endonuclease (Yugong Biotechnology, EG22507V), 1 μL of T4 DNA ligase, 5 μL of ligase buffer, and 1 μL of 10 μM hairpin adapter sequence were added. Finally, ddH2O was added to bring the total volume to 50 μL, and the mixture was thoroughly mixed before the reaction was carried out.

[0153] After incubation at room temperature for 2 hours, the total volume after the reaction was still 50 μL, which is equivalent to a 2-fold dilution compared to the 25 μL of PCR product added upstream.

[0154] 2. Rolling circle replication (RCA) amplification Based on the optimization results of continuous enzyme reaction conditions (see Example 3), the amount of RCA product obtained under 50-fold dilution was greater than that under 25-fold and 15-fold dilution, therefore a 50-fold dilution ratio was selected. Rolling circle replication amplification used a reaction system with a total volume of 2.5 mL: 50 μL of the upstream reaction product was taken, and after dilution in step 1, the DNA concentration in the reaction solution was approximately 20 ng / μL; 10 μl of phi29 DNA polymerase, 250 μl of reaction buffer, 10 μl of 100 mM dNTP, and ddH2O were added to bring the total volume to 2.5 mL, mixed well, and then the reaction was carried out.

[0155] Incubate at 37℃ for 2-5 hours. The total volume after the reaction is complete is 2.5 mL, which is equivalent to a 50-fold dilution compared to the 50 μL reaction solution added upstream in step 1. Samples can be taken periodically during the reaction to detect the amount of product.

[0156] The expected concentration of RCA product is approximately 40 ng / μL, and the total yield is approximately 100 μg.

[0157] 3. Fragmentation and polyA join A one-step directional enzymatic digestion and ligation method was adopted to simultaneously complete the fragmentation and polyA ligation process. The total reaction volume was 5.0 mL: 2.5 mL of upstream reaction product (containing RCA product concentration of approximately 40 ng / μL) was taken, and 5-50 μg of polyA sequence was added (depending on the molecular weight of the upstream template, the final molar ratio of polyA fragment to upstream fragment was approximately 1.5:1), 50 μl of DNA T4 ligase (Yugong Biotechnology, EG15205S), 25 μl of BsaI (Yugong Biotechnology, EG15518), and 500 μl of T4 DNA ligase buffer were added. Finally, ddH2O was added to bring the total volume to 5.0 mL, and the mixture was stirred before the simultaneous enzymatic digestion and ligation reaction was carried out.

[0158] The reaction was carried out at room temperature for 2 hours. The total volume after the reaction was completed was 5.0 mL, which is equivalent to a 2-fold dilution compared to the 2.5 mL of reaction solution added upstream in step 2.

[0159] 4. Product purification and validation DNA purification and recovery kit using magnetic beads (Yugong Biotechnology, PEDM-48) was used, and the product was purified according to the manufacturer's instructions. A final yield of 100 μg to 200 μg of IVT DNA template was obtained, with a concentration of 1-2 μg / μl and a total volume of 100 μl. Agarose gel electrophoresis was used to verify the intermediate products of each experimental step and the final IVT DNA template obtained.

[0160] As shown in Figure 3, each intermediate product was observed to have a clear band of the expected size (2.2 KB) without any obvious impurities, indicating that the preparation process was stable.

[0161] Example 2: Validation of the biological activity of the mRNA product 293T cells were cultured to the logarithmic growth phase and seeded at 50,000 cells / well in 96-well plates. Each cell was transfected with 100 ng of conventionally prepared mRNA and mRNA prepared using the method of this invention, with a blank control group included. Three technical replicates were set up for each sample. After transfection, cells were cultured for 16 hours, and the fluorescence intensity of each group was detected using a fluorescence detection kit (Yisheng Biotechnology, 11413ES60).

[0162] mRNA biological activity verification results are as follows: Figure 4 As shown, the mRNA (plasmid) is firefly mRNA prepared using conventional methods. mRNA-1 / 2 is firefly mRNA prepared using the method of this invention. Transfecting 293T cells with the same amount of mRNA resulted in the same biological activity.

[0163] Example 3: Optimization of continuous enzyme reaction conditions Referring to the preparation method in Example 1, the continuous enzyme reaction conditions were optimized, and the optimal dilution factor was finally determined.

[0164] Three identical RCA reaction systems were set up and diluted 50-fold, 25-fold, and 15-fold respectively. Rolling ring replication was performed under the same reaction conditions. After the reaction, the amount of RCA product in each reaction system was quantitatively detected by gel electrophoresis.

[0165] The results are as follows Figure 5 As shown, the RCA product yield was highest at a 50-fold dilution, and continuous enzymatic reactions with optimized dilution ratios can efficiently prepare IVT DNA templates.

[0166] Example 4: Verification of RCA product fragmentation efficiency Hairpin-shaped looping strategy and conventional looping strategy (e.g.) are applied respectively. Figure 6 The cyclization reaction (shown) was performed, and then rolling cyclization was carried out under the same conditions using two cyclized molecules as templates to obtain two RCA products. These products were then fragmented by enzyme digestion. Finally, the banding was observed by gel electrophoresis and the fragmentation efficiency was analyzed. Fragmentation efficiency = fluorescence value of fragmented product / fluorescence value of unfragmented product.

[0167] The conventional circularization strategy involves self-ligating the PCR product (template and primer sequences are the same as in Example 1) from head to tail.

[0168] The hairpin cyclization strategy involves directional enzyme digestion and ligation. The main cyclized molecule is obtained by digesting the PCR product with BsmBI and then ligating it with hairpin aptamers (the sequences of the hairpin molecules are shown in SEQ ID NO: 4 and 5). Hairpin molecules can be formed independently through a standard annealing procedure (naturally decreasing from 95℃ to 22℃, 1℃ / min). The hairpin cyclization reaction system is as follows: 1 μg of PCR product and 100 ng each of the two hairpin aptamers are added to a 50 μl reaction mixture. The reaction is carried out at room temperature (37℃). The resulting product is purified using magnetic beads, and an equal volume is used for the RCA reaction (1 ng cyclized molecule / reaction).

[0169] Experimental results are as follows Figure 7 As shown in Figure A, from left to right, the products are: Marker, rolling circle amplification product of hairpin cyclic molecule, fragmented product of RCA product of hairpin cyclic molecule, RCA product of ordinary cyclic molecule, and fragmented product of RCA product of ordinary cyclic molecule. Analysis shows that the fragmentation efficiency of RCA product of hairpin cyclic molecule (>90%) is significantly better than that of RCA product of ordinary cyclic molecule (<40%).

[0170] To verify that the function of the cyclized adapter depends on the stable pairing of the stem region double strand structure rather than a specific sequence, a stem region mismatch variant (SEQ ID NO: 14) was designed based on the original adapter (15 bp stem region, completely complementary, Tm=42℃) for comparative experiments: by introducing a single mismatched base pair (C34A, replacing GC pairing with GA mismatch), the stability of the stem region double strand was significantly reduced, while the sequence consistency remained basically unchanged. Other conditions were the same as the original adapter, and cyclization and RCA amplification were performed according to the steps in Example 1. The cyclization efficiency and RCA product amount were detected, respectively. The results are as follows: Figure 7 As shown in B, after introducing a single mismatch, the amount of RCA product drops to 63% of that in the perfectly complementary stem region.

[0171] The above results indicate that, compared to sequence consistency, the correct complementary pairing of bases in the stem region is more crucial for the realization of cyclization linker function.

[0172] Example 5: PolyA Sequence Design and Connection Efficiency Verification The three polyA sequences A, B, and C, as well as the stem-loop polyA sequence used in the experiment, were all obtained through chemical synthesis. The RCA fragmentation product was prepared according to the method in Example 1.

[0173] Three different polyA sequence design schemes were validated, as shown in Figure 8. Molecule A forms a sticky end with a complete polyT (containing 100-120 T bases, SEQ ID NO: 7) at its 3' end and a short oligo (SEQ ID NO: 8). It can undergo directional enzymatic ligation with RCA fragmentation products. Molecule B has a stem-loop structure at its 5' end, containing a Type IIS cleavage site. After cleavage, it forms a sticky end consistent with A, suitable for ligation with RCA fragmentation products (…). Figure 9 (SEQ ID NO: 9). Molecule C contains long polyA chains and long polyT chains (SEQ ID NO: 6, 7), forming consistent sticky ends of molecule A.

[0174] Fragmentation and polyA ligation can be performed via directional enzymatic digestion ligation. In a 50 μl reaction system, add 1 μg template, 10-50 ng polyA sequence (molar ratio of main molecule to polyA sequence is about 1:1.5), BsaI restriction enzyme, 5 μl T4 DNA, and 10 μl T4 DNA ligase buffer, and react at room temperature for 2 hours.

[0175] After the reaction, an appropriate amount of the sample to be tested was mixed with the loading buffer and loaded into the wells of a pre-prepared polyacrylamide gel. The polyacrylamide gel had a concentration of 8% and was electrophoresed in 1×TBE buffer at a constant voltage (e.g., 100-150 V) until the indicator dye migrated to the appropriate position on the gel. After electrophoresis, the gel was stained in a nucleic acid fluorescent dye solution (e.g., SYBR Gold or Gel Red) in the dark for 15-30 minutes, and then observed and images were acquired using a gel imaging system. The gray values ​​of each band were quantified using image analysis software such as ImageJ. Figure 10 The middle arrow indicates that the ligation efficiency is calculated as the gray value of fragment A divided by (gray value of fragment A + gray value of fragment B). Experimental results show that all types of polyA sequences can be ligated with RCA fragmentation products through directional enzyme digestion. Among them, the stem-circular polyA sequence of type B has the best ligation efficiency, with approximately 95% or more of the fragmentation products undergoing the ligation reaction, resulting in efficient acquisition of complete DNA IVT templates. The ligation efficiency of type A is over 80%, and that of type C is over 55%.

[0176] Example 6: FRET monitoring of one-way enzymatic digestion ligation reaction The labeled oligos were synthesized by Junji Biotechnology Co., Ltd., and the label could be located at the 5' end or in the middle (Figure 11, left or right). After annealing, they were combined to form molecules A (SEQ ID NO: 10, 11) and B (SEQ ID NO: 12, 13). The FRET monitoring reaction was established as follows: 500 ng of molecule A and 500 ng of molecule B, 10 μl of T4 DNA ligase buffer, 5 μl of T4 DNA ligase, and 1 μl of BsaI restriction enzyme were added to a 100 μl reaction system. The reaction was carried out at room temperature for 1 hour, and the signal changes were monitored in real time using a FRET signal detection instrument to analyze the effect of dye labeling position and reaction conditions on signal efficiency.

[0177] like Figure 12 As shown, oligos with different concentrations of AF488 and Cy3 labeled at the 5' end can all produce obvious FRET signals after enzyme digestion and ligation, and the reaction can be monitored in real time.

[0178] The exemplary sequences used in the disclosed embodiments are shown in Table 1, but it should be understood that the technical solutions of this disclosure are not limited to the specific sequences described. The above description of specific embodiments of this disclosure is for illustration and example only and should not be construed as limiting this disclosure to the precise form disclosed. Based on the teachings of this disclosure, those skilled in the art can make various modifications and variations thereto. The purpose of selecting and describing exemplary embodiments is to elucidate the principles and applicability of this disclosure so that those skilled in the art can implement and utilize various exemplary solutions and variations thereof. The scope of this disclosure is defined by the claims and their equivalents.

[0179] Table 1: Nucleotide Sequences

Claims

1. A method for preparing a cell-free mRNA template, comprising the following steps (a)-(e): (a) Provide a first nucleic acid fragment containing the target mRNA coding region, a second nucleic acid fragment containing a polyA coding sequence, and a circular adapter; in, The circumferential joint has a stem-ring structure; (b) The first nucleic acid fragment is directionally assembled and circularized with the circular adapter through a first directional enzyme digestion-ligation reaction to generate a single-stranded circular DNA template; In this process, stem-loop structures of the circular adapter are connected to both ends of the first nucleic acid fragment to form a single-stranded circular DNA with a dumbbell-shaped structure. (c) Obtain single-stranded DNA polymers containing multiple copies of the target sequence by rolling circle replication amplification; The single-stranded circular DNA template enables the amplified single-stranded DNA multiplexes to self-fold through intramolecular annealing, forming multiple double-stranded stem regions. (d) Fragmenting the single-stranded DNA multiplex; (e) The second nucleic acid fragment is ligated to the product of the fragmentation treatment by a second directional enzyme digestion-ligation reaction to generate a linear DNA template for in vitro transcription.

2. The method according to claim 1, wherein the enzyme digestion efficiency of the single-stranded DNA multiplex in the fragmentation treatment is ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

3. The method according to claim 1 or 2, wherein: The first directional enzyme digestion-ligation reaction and / or the second directional enzyme digestion-ligation reaction are performed using restriction endonucleases and ligases; the rolling circle replication amplification is performed using DNA polymerase. Preferably, the restriction endonuclease is a Type IIS restriction endonuclease, and the ligase is a T4 DNA ligase; Preferably, the DNA polymerase is phi29 DNA polymerase; More preferably, all steps of the method are carried out continuously in a single reaction vessel.

4. The method according to any one of claims 1-3, wherein the first nucleic acid fragment is derived from a gene synthesis fragment, a plasmid, or a PCR amplification product; Preferably, the first nucleic acid fragment comprises, from the 5' end to the 3' end, the following in sequence: a first E1 site, a T7 promoter sequence, a target RNA coding region, an E2 site, an optional E3 site, and a reversed second E1 site; in, The E1 site is the restriction site of the first directional restriction enzyme digestion-ligation reaction, and the E2 and / or E3 sites are the restriction sites of the fragmentation treatment and / or the second directional restriction enzyme digestion-ligation reaction. Preferably, the fragmentation process and / or the second directional enzyme digestion-ligation reaction are performed by a one-step directional enzyme digestion-ligation reaction at the E2 site; Preferably, the first nucleic acid fragment further comprises PCR primers; More preferably, the E1 site is located inside the PCR primer.

5. The method according to any one of claims 1-4, wherein all steps of the method are performed sequentially and continuously in a single reaction vessel by means of continuous dilution of the reaction buffer, without including intermediate purification steps; Preferably, steps (d) and (e) are performed via a one-step directional enzymatic digestion-ligation reaction; Preferably, the sequential dilution comprises one or more of the following (1)-(3): (1) Dilute 1.5-3 times during step (b); (2) Dilute 25-50 times during step (c); (3) Dilute by 1.5-3 times during steps (d) and (e); More preferably, the sequential dilution comprises: (1) Dilute by 2 times during step (b); (2) Dilute by 50 times during step (c); and / or (3) Dilute by 2 times during steps (d) and (e).

6. The method according to any one of claims 1-5, wherein the polyA coding sequence includes a sticky end at the 5' end capable of complementary pairing with the target site of the fragmented single-stranded DNA multiply; Preferably, the viscous ends are formed in one of the following ways: (1) Direct chemical synthesis of sequences with protruding ends, formed by double-strand annealing; (2) Introduce a stem-loop structure containing restriction enzyme sites into the polyA coding sequence, exposing sticky ends after enzyme digestion; or (3) A partial double-chain structure is formed by double-chain annealing, with one end retaining a sticky end.

7. The method according to any one of claims 1-6, wherein the method further comprises online quality control of the reaction process throughout the preparation process; Preferably, the online quality control utilizes FRET technology; the FRET technology employs two oligonucleotide probes labeled with a first fluorescent dye and a second fluorescent dye. When the directional enzyme digestion-ligation reaction occurs, the two dyes move close to each other in space to generate a FRET signal. Preferably, the fluorescent dye is labeled at the 5' end or middle position of the oligonucleotide; More preferably, the first fluorescent dye and the second fluorescent dye are selected from AF488 and Cy3, Cy3 and Cy5, FAM and TAMRA, or Alexa Fluor 488 and Alexa Fluor 594; More preferably, the first fluorescent dye is AF488 and the second fluorescent dye is Cy3; or the first fluorescent dye is Cy3 and the second fluorescent dye is Cy5.

8. A cell-free mRNA template high-throughput preparation system, said system for performing the method of any one of claims 1-7, comprising: (1) A modular nucleic acid component comprising the first nucleic acid fragment, the second nucleic acid fragment, and a circular adapter; (2) A continuous multi-enzyme reaction unit configured to perform steps (a)-(e) sequentially in a single reaction vessel and having a built-in dilution control module; (3) Optional online quality control unit, which is configured to monitor the reaction process in real time using FRET technology, including a fluorescence detection module and a FRET signal analysis module.

9. The system of claim 8, wherein the sequential multi-enzyme reaction unit is configured to process multiple different target mRNA sequences in parallel; Preferably, the continuous multi-enzyme reaction unit is an automated workstation; preferably, the single reaction vessel is a 96-well plate. Preferably, the dilution control module is configured to dilute by 1.5-3 times in step (b), by 25-50 times in step (c), and by 1.5-3 times in steps (d) and (e). More preferably, the dilution control module is configured to dilute by 2 times in step (b), by 50 times in step (c), and by 2 times in steps (d) and (e).

10. A cell-free mRNA template preparation kit for performing the method of any one of claims 1-7 or for assembling the system of claim 8 or 9.

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