Thermostable peptide-DNA oligoconjugates and methods of using same

Abstract

The present invention relates to a composition ("TSP-DNA Oligo") comprising: a thermostable peptide ("TSP"), wherein the peptide comprises: an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, or an amino acid sequence with at least about 92% sequence identity to a sequence as set forth in SEQ ID NO; 1, or SEQ ID NO: 2, or SEQ ID NO: 3 or SEQ ID NO: 4; and a "DNA Oligo" linked to the thermostable peptide through a linker, wherein the linker comprises: an azide group, an alkyne group, an isocyanate group, or a tetrazole group; wherein the "DNA oligo" comprises a promoter sequence for a T7, T3, SP6 RNA polymerase, or a known variant of said promoter sequence, or a sequence complementary to said promoter sequence or said variant.

Description

[0001] THERMOSTABLE PEPTIDE-DNA OLIGOCONJUGATES AND METHODS OF USING SAME

[0002] BACKGROUND OF THE INVENTION

[0003] In recent years, RNA-based therapeutics have emerged as a promising class of drugs for treating a wide range of diseases, including genetic disorders, infectious diseases, and cancer. Unlike traditional small molecule drugs or protein-based therapies, RNA therapeutics, particularly mRNA-based ones, offer several advantages including the ability to target specific proteins, rapid development timelines, and potential for personalized medicine. However, the widespread adoption of RNA therapeutics is contingent upon efficient manufacturing processes that can produce high- quality RNA molecules reproducibly and at scale.

[0004] The manufacturing of RNA molecules, especially mRNA, is a complex and multi-step process involving enzymatic transcription from a suitable DNA template followed by purification and formulation steps. The efficiency and quality of the mRNA synthesis step significantly impacts the overall yield and purity of the final product. Traditionally, in vitro transcription (IVT) using a DNA template with a bacteriophage-derived promoter, an RNA polymerase, tor example, bacteriophage SP6, T3 or T7 RNA polymerase and ribonucleoside triphosphates (NTPs), has been the preferred method for mRNA production due to its scalability and versatility. Currently, IVT is performed as an in-solution process. In solution, after serving as the template for the IVT reaction, the DNA is enzymatically digested before the mRNA product is subsequently purified. Because of this, the DNA can only be used for one round of IVT thus leading to an increased demand and process cost. Additionally, the DNA is typically digested by an enzyme that must be added at posttranscription step, thus adding additional process times and cost to the manufacturing process.

[0005] The template DNA serves as the blueprint for mRNA synthesis during IVT. The quality and integrity of the template DNA directly influence the fidelity and efficiency of the transcription process. Any degradation or instability in the template DNA can lead to reduced transcription efficiency and compromised mRNA quality. Therefore, a method of ensuring stability and robustness of the template DNA would be crucial for achieving high-yield and high-quality mRNA production. Additionally, efficient utilization of resources and reduction of production costs are paramount in the commercial manufacturing of RNA therapeutics. The reuse of immobilized DNA templates for multiple rounds of transcription would be an approach to enhance efficiency.

[0006] SUMMARY OF THE INVENTION

[0007] The present invention addresses the need for efficient and robust methods in molecular biology and biotechnology tor manufacturing mRNA with enhanced yield.

[0008] In one aspect, the present invention provides a streamlined process of template DNA manufacturing. For instance, the invention provides a novel composition comprising a thermostable peptide (“SpyTag”) and a DNA oligonucleotide (“DNA oligo”). This composition facilitates the synthesis of ftill-length linear template DNA, containing the thermostable peptide, from a plasmid construct or synthetic construct. The inclusion of this peptide facilitates the immobilization of the template DNA to a solid support, ensuring its stability and reusability in downstream applications. Moreover, the generation of the template DNA containing the thermostable peptide (i.e., the TSP- DNA Template) is a PCR-based process; and it omits the need for linearization of plasmid using restriction enzymes. It is also a cell free in vitro process, thus mitigating risks of host microbial DNA, proteins, and endotoxins contamination.

[0009] In another aspect, the present invention provides a process of Immobilization to Solid Support. For instance, the invention provides a process for immobilizing a peptide-containing template DNA onto a solid support (“SpyTag-DNA”). This immobilization is achieved via a specific binding partner pair involving a polypeptide associated with a thermostable peptide. This step enables a template DNA to be covalently linked to a solid surface, facilitating subsequent purification and processing steps during transcription reactions. For instance, immobilizing a template DNA onto a solid support allows for its reuse in subsequent transcription reactions, thereby minimizing material waste and streamlining the manufacturing process. Moreover, immobilization offers advantages such as improved reaction kinetics, easier separation of DNA from reaction mixtures, and increased stability of the template DNA during transcription. In a further aspect, the present invention provides in vitro Transcription tor mRNA Prod uction. For instance, the invention provides u tilization of the immobilized template DNA for IVT. This process involves the transcription of the immobilized template DNA into mRNA, a critical step in the manufacture of mRNA-based therapeutics, vaccines, and research tools. The immobilization of the template DNA ensures spatial confinement and stability during the transcription process, leading to the production of mRNA molecules with enhanced yield and purity.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a schematic representation of the functionalization of a solid support for conjugation with a SpyCatcher polypeptide.

[0012] FIG. 2 is an SDS-PAGE gel showing the complex formation between SpyTag-DNA oligo (17.3 kDa) and SpyCatcher (15.7 kDa). Lane 1 shows SpyCatcher only; Lane 2 shows SpyCatcher + SpyTag-DNA oligo; Lane 3 shows SpyCatcher + DNA oligo.

[0013] FIG. 3 is a plasmid map of pUC19 plasmid according to SEQ ID NO: 12, which encodes GFP. Restriction sites and important elements of the pDNA are indicated. T7 promoter, 5’ UTR, GFP, 3’UTR and Poly(A) tail are indicated.

[0014] FIG. 4 is a DNA gel electrophoresis showing SpyTagDNA generated from PCR. PCR products were loaded and analyzed on a 1% agarose gel stained with SYBR green I dye.

[0015] FIG. 5 is an agarose gel electrophoresis. The SpyTagDNA was used as a template for RNA in vitro transcription. In vitro transcribed RNA was analyzed using agarose gel electrophoresis and stained with SYBR green II dye. Lane 1 shows the RNA generated from SpyTagDNA and Lane 2 shows RNA generated from linearized plasmid DNA.

[0016] FIG. 6 is a schematic diagram illustrating the assembly of a solid-phase IVT system for mRNA synthesis. The SpyCatcher protein (B) is coupled onto solid support e.g., magnetic bead (A), and it serves as the binding partner for SpyTagDNA (C) as they are capable of spontaneously forming an isopeptide bond. This enables its use in the immobilization of SpyTagDNA. Immobilized SpyTagDNA can then serve as a template for solid-phase IVT.

[0017] FIG. 7 is a solid-phase IVT from SpyTagDNA immobilized on SpyCatcher-magnetic beads. The immobilized SpyTagDNA was used as a template for RNA IVT. In vitro transcribed RNA was analyzed using agarose gel electrophoresis and stained with SYBR green II dye. Lane 1 shows the RNA generated from immobilized SpyTagDNA and Lane 2 shows RNA generated from linearized plasmid DNA.

[0018] FIG. 8A is an RNA gel electrophoresis showing RNA generated from 6 sequential IVT reactions. The lane numbers represent the IVT cycle number.

[0019] FIG. 8B is a histogram showing the RNA yield from the 6 sequential cycles.

[0020] FIG. 9 is an HPLC chromatograms of purified in vitro transcript showing that RNA synthesized in the 6 IVT cycles, showing nearly identical integrity of the synthesized RNA. The analytic HPLC profile of the produced RNA samples from linearized plasmid DNA and immobilized SpyTagDNA for successive IVT reactions.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] To address the need for efficient processes to manufacture RNA reproducibly and at scale, the methods of the present invention utilize immobilized template DNA for in vitro transcription. In order to achieve this, the inventors generated a thermostable peptide-DNA oligonucleotide conjugate that can be used to synthesize full length linear template DNA containing the peptide, which in turn can be used to in the manufacture of mRNA of a gene of interest.

[0023] Thermostable Peptide-DNA Oligo (“TSP-DNA Oligo”)

[0024] In one aspect, the present invention provides a thennostable peptide (TSP) which can withstand temperatures during a polymerase chain reaction (PCR) (e.g., up to 98°C). In one embodiment, the said peptide comprises 1) an amino acid sequence as set forth in SEQ ID NO: 1, or ii) an amino acid sequence with at least about 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence as set forth in SEQ ID NO: 1, or iii) an amino acid sequence as set forth in SEQ ID NO: 2, or iv) an amino acid sequence with at least about 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence as set forth in SEQ ID NO: 2, or v) SEQ ID NO: 3, or vi) an amino acid sequence with at least about 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence as set forth in SEQ ID NO: 3, or vii) SEQ ID NO: 4, or viii) an amino acid sequence with at least about 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence as set forth in SEQ ID NO: 4. Within tire scope of the present invention, not only the wild-type peptide sequence can be used, but also functional variants thereof. Variants of the peptide have a sequence which differs from that of the wild-type peptide by one or more amino acid substitutions, deletions, or additions, resulting in a sequence identity to the wildtype peptide of at least about 92%.

[0025] The presence of the following amino acids in the peptide typically contribute to its thermostability: a) Arginine (R) and Lysine (K): These amino acids can form strong hydrogen bonds and salt bridges, which help stabilize the peptide structure. b) Valine (V), Isoleucine (I), and Methionine (M): These non-polar amino acids contribute to the hydrophobic core, enhancing stability. c) Proline (P): Adds rigidity to the peptide structure. d) Glycine (G): Its small size allows for tight packing and flexibility in the peptide structure.

[0026] The thermostable peptide is conjugated to a DNA oligonucleotide (“DNA oligo") to form a “TSP-DNA Oligo.” (In some places of this specification, the “TSP-DNA Oligo” is referred to as the “Spy Tag DNA oligo.”) In one embodiment, the “DNA oligo" comprises spacer nucleotides (“spacer”) and a promoter. The “spacer” can comprise about 20-150 bases on the end proximate to the TSP (i.e., 5" end).

[0027] In some embodiments, the promoter sequence can be a promoter for a T7, T3, SP6 RNA polymerase or a known variant thereof, including sequences which are complementary to the promoter sequences or their variants. Examples of suitable promoter sequences are SEQ ID NO: 15 (T7 RNA polymerase), SEQ ID NO: 16 (T3 RNA polymerase) and SEQ ID NO: 17 (SP6 RNA polymerase).

[0028] An example of a “DNA oligo” is a DNA sequence as set forth in SEQ ID NO: 5, or a sequence with at least about 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence as set forth in SEQ ID NO: 5. In SEQ ID NO: 5, the first about 30 bases of the oligonucleotide (i.e., 5’ end) function as a spacer. The last 18 bases of the oligonucleotide correspond to the T7 promoter sequence. An example of a “TSP-DNA oligo” is a sequence as set forth in SEQ ID NO: 11, or a sequence with at least about 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence as set forth in SEQ ID NO: 11.

[0029] In some embodiments, the TSP is conjugated with the “DNA Oligo” through a linker. The linker can comprise different chemical moieties attached to each of the TSP and the “DNA oligo,” preferably chemical moieties involved in click chemistry, for example: a) Azides (Ns): Azides are highly reactive and can participate in click reactions, particularly the copper-catalyzed azide-alkyne cycloaddition (CuAAC), where they react with alkynes to form 1 ,2,3-triazoles; b) Alkynes (C≡C): Alkynes are unsaturated hydrocarbons containing a triple bond (C≡C). In click chemistry , alkynes are often used as substrates in reactions with azides, leading to the formation of 1 ,2,3-triazoles; c) Isocyanates (N=C=O): Isocyanates are organic compounds containing the isocyanate functional group (-N=C=O). Isocyanates can participate in click reactions, particularly Ute copper- free strain-promoted azide-alkyne cycloaddition (SPAAC), where they react with azides to form 1 ,4- disubstituted 1 ,2,3-triazoles; d) Tetrazoles (N4): Tetrazoles are heterocyclic compounds containing a five-membered ring with four nitrogen atoms. They are used as reagents in click chemistry, particularly in copper-free click reactions, where they react with alkynes to fomi triazoles.

[0030] In a preferred embodiment, the TSP is modified with the azide or thiol group, and the “DNA Oligo” is modified with dibenzocyclooctyne (DBCO) or maleimide group. The click chemistry reactions between the said chemical moieties will generate the “TSP-DNA Oligo” conjugate. In one embodiment, the “TSP-DNA Oligo” is immobilized onto a solid support via a binding partner pair involving a polypeptide. The solid support can comprise magnetic or nonmagnetic particulate materials, including porous or non-porous beads. The beads can be formed from a variety of crosslinked or non-crosslinked polymers of natural or synthetic origin. The particle size of the solid support can range from approximately 1 micron to approximately 100 microns. Examples of suitable solid supports include agarose, sepharose, magnetic beads, and glass beads. The solid support is functionalized with a binding polypeptide to facilitate covalent bonding with the thermostable peptide (“TSP”). Examples of such binding polypeptides include peptides having at least about 95% sequence identity to any one of SEQ ID NOs: 6 to 9. In particular, the immobilization is achieved through a SpyTag / SpyCatcher system, wherein the SpyTag is part of the thermostable peptide and the SpyCatcher is part of the polypeptide immobilized on the solid support via reactive functional groups. The reactive functional group may comprise straight or branched hydrocarbon chains, which may be saturated or unsaturated and contain from three (C3) to twenty-four (C24) carbon atoms. Chemically, the reactive functional group may be selected from, but is not limited to, epoxides, thioesters, maleimides, or other saturated or unsaturated hydrocarbon-based moieties. The thermostable peptide retains its function in binding to its binding partner pair when subjected to temperatures (up to 98°C) used in a standard PCR.

[0031] In another aspect, the invention relates to the process for immobilization of the “TSP-DNA Oligo” to a solid support via its binding partner pair (polypeptide). To facilitate a specific and stable immobilization of the “TSP-DNA Oligo”, the inventors utilize a two-part linker comprising a peptide tag (peptide) and a polypeptide (protein) that is capable of spontaneously forming an isopeptid e bond. The peptide tag / binding partner pair, termed SpyTag / SpyCatcher, has been described and used in diverse applications, including:

[0032] 1. Protein Engineering and Bioconjugation: SpyTag / SpyCatcher system has been widely used for site-specific protein labeling and bioconjugation due to its rapid and irreversible conjugation kinetics (Zakeri et al. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences, 109(12), E690-E697). 2. Vaccine Development: SpyTag / SpyCatcher system has been employed for the modular decoration of virus-like particles (VLPs) with antigens, facilitating the development of multivalent vaccines (Brune et al. (2016) Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Scientific Reports, 6, 19234).

[0033] 3. Cell Surface Engineering: SpyTag / SpyCatcher system has been utilized for the site-specific immobilization of proteins onto cell surfaces, enabling the study of cell-matrix interactions with high precision (Khalil et al. (2012) Single Molecule Characterization of the Interactions between Extracellular Matrix Proteins and the Cell Surface Using Nanoscale Smart Probes. PLoSONE, 7(10), e46088).

[0034] 4. Bioactive Surface Coating: SpyTag / SpyCatcher system has been applied for the functionalization of surfaces with bioactive proteins, offering a versatile approach for surface modification in various biomedical and biotechnological applications (Fairhead, M., & Howarth, M. (2015). Site-specific biotinylation of purified proteins using BirA. Methods in Molecular Biology, 1266, 171-184).

[0035] 5. Material Science and Biofabrication: SpyTag / SpyCatcher system has been integrated into smart polymers and hydrogels for the development of stimuli-responsive materials and biofabrication techniques, such as 3D printing of protein-based structures (Roy, P., & Damera, S. (2020). Smart Polymers and Their Biomedical Applications. Polymers in Advanced Technologies, 31(10), 2247-2263).

[0036] The said polypeptide (protein) comprises i) an amino acid sequence as set forth in SEQ ID No: 6, or ii) an amino acid sequence with at least about 80% sequence identity to a sequence as set forth in SEQ ID NO: 6. The said protein can also comprise i) an amino acid sequence as set forth in SEQ ID NOS: 7-9, or ii) an amino acid sequence with at least about 80% sequence identity to a sequence as set forth in SEQ ID NOS: 7-9. The said two-part linker is with reference to a pending patent application number U.S.S.N. 17 / 435,742. However, the application of the two-part linker related to the present invention is novel and unrelated to the pending patent.

[0037] The polypeptide (protein) may further comprise an amino acid sequence facilitating purification, such as an oligohistidine tag. Preferably, the oligohistidine tag comprises six histidine residues (SEQ ID NO: 10). The invention also relates to a solid support to which the polypeptide as defined above is immobilized by covalent bonding, preferably by a thioether bond via thiol-ene reaction. The polypeptide may be bound to the thiol-activated support material via a cysteine residue, preferably the cysteine residue has been introduced by substituting an amino acid within the wild-type polypeptide with cysteine (SEQ ID NO: 6). Covalent immobilization is typically favored due to the strong binding affinity between the protein and the support material, aiming to minimize the likelihood of proteins detaching from the support, commonly known as leaching.

[0038] In some embodiments, the solid support comprises bead-like particles, which may be magnetic or non-magnetic. The beads may be porous or non-porous, and may be formed from polymers that are either crosslinked or non-crosslinked, and of natural or synthetic origin. The particle size of the solid support may range from approximately 1 micron to approximately 100 microns.

[0039] In certain embodiments, the solid support is functionalized with a reactive functional moiety, designated as "R" in FIG. 1. The R group may be linear or branched, and may include saturated or unsaturated hydrocarbon chains containing from three (C3) to twenty-four (C24) carbon atoms. The R group may be selected from, but is not limited to, epoxides, thioesters, maleimides, or other saturated or unsaturated hydrocarbon-based functional groups.

[0040] The covalent interaction between the “TSP-DNA Oligo” and its binding polypeptide partner further stabilizes the immobilization of the to-be-formed DNA template to the support. This may allow a more controlled and efficient way of immobilization.

[0041] Manufacture of TSP-Linear DNA Template

[0042] In one aspect of the invention, the “TSP-DNA Oligo” is used as a primer in a polymerase chain reaction (PCR) using a plasmid construct or synthetic DNA construct as an “initial DNA template.” The generated product of the PCR is TSP-containing linear template DNA (“TSP-DNA Template”) attached to a solid support. (In some places of this specification, the “TSP-DNA Template” is referred to as the “Spy Tag DNA.”) Note, in addition to the thermostable peptides described above, any other thermostable peptide can be used hi this method. The method comprises conducting a PCR with an “initial DNA template,” and using a "TSP-DNA Oligo” as a forward primer, and using a poly(T)-containing sequence as a reverse primer. The resulting structure of the “TSP-DNA Template” is as follows: “TSP-DNA Oligo”, a 5’ UTR, a “gene of interest,” a 3’ UTR and a poly(A) sequence. The resulting “TSP-DNA Template” is a linearized product, thus omitting the need for linearization of plasmid using restriction enzymes. The “TSP-DNA Template” is immobilized to a solid support.

[0043] In some embodiments, the open reading frame (ORF) of the “gene of interest” is any sequence used in a vaccine, a gene therapy or in a research tool. Examples of genes of interest used in vaccines are genes encoding for glycoproteins of viruses, e.g., SARS-CoV-2 Spike protein and influenza hemagglutinin. Examples of genes of interest used in gene therapy include the following genes: p53, RPE65, SMN2 and BRCA1. Examples of commonly used research tools include the plasmid map of pUC 19 plasmid (FIG. 3) according to SEQ ID NO: 12, which encodes eGFP, and the luciferase gene according to SEQ ID NO: 13.

[0044] In one example, the “initial DNA template” has at least about 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 12 or 13. In one example, the “TSP-DNA Template” has at least about 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14.

[0045] The “TSP-DNA Template” is capable of serving as a template for in vitro transcription (IVT) of the gene of interest to synthesize mRNA.

[0046] Manufacture of mRNA from TSP- DNA Template

[0047] In one aspect of the invention, a method for manufacturing mRNA of a “gene of interest” from a plasmid construct or synthetic DNA (“initial DNA template”) is provided. The method comprises generating a linearized DNA (“TSP-DNA Template”) immobilized on a solid support, as described above; and using the immobilized “TSP-DNA Template” as a template for in vitro transcription (IVT) reaction to synthesize mRNA product comprising the “gene of interest.” The in vitro transcription reaction includes adding a transcription master mix comprising RNA polymerase, nucleoside triphosphates (NTPs), and a transcription buffer to the immobilized composition.

[0048] The method can further comprise separating the immobilized “TSP-DNA Template” from the in vitro transcription mRNA products. That is, the method further comprises separating the synthesized mRNA from the solid support after transcription, and purifying the synthesized mRNA.

[0049] Multiple rounds of in vitro transcription can be conducted using the same immobilized “TSP-DNA Template” without the need for enzymatic digestion to separate it from the generated mRNA. For example, the immobilized “TSP-DN A Template” is reused for at least three rounds of in vitro transcription, thereby reducing the costs associated with the mRNA production process.

[0050] The utilization of a peptide tag allows DNA immobilization and maintains the accessibility of the DNA to the extent that it can serve as the template for an in vitro transcription (IVT) reaction wherein mRNA is produced. The immobilization of the template DNA also ensures spatial confinement and stability during the transcription process, wherein mRNA can be manufactured in a more efficient manner than is currently possible. The PCR-based mRNA manufacturing processes offer several benefits compared to traditional methods, including speed, precision, scalability, flexibility, consistency, reduced contamination risk, cost-effectiveness, and customization, making them an attractive option for producing mRNA for various applications, including therapeutics, vaccines, and research purposes.

[0051] The PCR product is purified by immobilization onto a solid support (e.g., bead) via the product’s binding partner pair (polypeptide). The solid support-DNA complexes can be thoroughly washed, and no further purification of the PCR product is required. Another advantage of the immobilized DNA system is that immobilized DNA in the system can be reused for several IVT cycles which additionally decreases the costs for plasmid DNA manufacturing, and debottlenecking plasmid DNA supply chain. The solid support-DNA complexes can directly serve as templates for IVT where the beads are resuspended in the IVT master mix (T7 RNA polymerase, nucleoside triphosphates (NTPs), transcription buffer). When IVT is complete, the synthesized RNA can be isolated by removing the solid support-DNA complexes. In conventional workflow, template DNA is typically digested by an enzyme that must be added to purify the reaction, thus adding additional process times and cost to the manufacturing process. The instant invention eliminates the need for enzymatic digestion of the DNA to assist in the purification process and reduces the risk of DNA contamination in the final mRNA product. The solid support-DNA complexes can then be reused by adding fresh reagents and repeating the reaction.

[0052] EXAMPLES

[0053] The examples shown in the following are merely illustrative and shall describe the present invention in a further way. These examples shall not be construed to limit the present invention thereto.

[0054] Example 1: Immobilization of SpyCatcher on magnetic beads for production of SpyCatcher- magnetic beads

[0055] SpyCatcher ligands were immobilized onto a solid support, such as magnetic beads, via covalent linkage between the ligands and activated bead surfaces, as described below. The solid support may be composed of natural polymers (e.g., agarose), synthetic polymers, or glass-based materials.

[0056] 1.1 . Solid support surface preparation

[0057] In certain embodiments, the solid support was activated with epoxy functional groups and used directly for subsequent SpyCatcher ligand immobilization. In other embodiments, the epoxy-activated solid support was further functionalized with maleimide or alkene groups to facilitate alternative conjugation chemistries.

[0058] 1.2. SpyCatcher immobilization via thiol-ene photoclick chemistry

[0059] Alkene-functionalized epoxy solid support (0.3 mL) was suspended in a coupling buffer comprising 0.2 M sodium phosphate (pH 6-8), with or without sodium sulfate (0-1 M), and the final slurry volume was adjusted to approximately 0.8-1.0 mL. The slurry was degassed under nitrogen for 10 minutes. SpyCatcher ligand, pre-treated with 20 mM TCEP for 2 hours in 1 * PBS, was further diluted in 0.05 M sodium phosphate dibasic buffer (pH 6-8). The ligand solution was added to the solid support slurry at a ligand-to-solid ratio of 5-10 mg / mL. The mixture was incubated at room temperature for 21 hours. A photoinitiator, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP, 10 mg in 0.3 mL coupling buffer), was added, followed by nitrogen blanketing. The reaction vial was sealed and irradiated with UV light (365 nm) for 2 hours while rotating. Following immobilization, the solid supports were washed three times with: (i) 1 x PBS containing 0.05% polysorbate 20 (PS20); (ii) lx PBS containing 20% ethanol and 0.01% PS20; and stored in the same buffer as a 20% (v / v) slurry for downstream mRNA production analysis. All immobilization supernatants and wash fractions were collected and analyzed by HPLC to quantify unbound SpyCatcher and determine immobilization efficiency.

[0060] 1.3. SpyCatcher immobilization via cysteine-maleimide linkage

[0061] Maleimide-functionalized solid support (-0.25 mL) was suspended in 0.1-0.2 M sodium phosphate buffer (pH 7.0-7.4), with or without sodium sulfate (0-0.75 M), and adjusted to a final volume of 0.85 mL. The slurry was degassed under nitrogen for 10 minutes. TCEP-treated SpyCatcher ligand (~0.25 mL, 20 mM TCEP, 2 hours, in 1 x PBS) was added to the slurry at a ligand-to-solid ratio of 5.5 mg / mL. The vial was blanketed with nitrogen and incubated on a rocker at room temperature for 4.3 hours. Post-reaction, the solid supports were washed and stored as described in Section 3.2.

[0062] 1.4. SpyCatcher immobilization via cysteine-epoxy linkage

[0063] Epoxy-functionalized solid support (0.3 mL) was suspended in 0.1-0.26 M sodium phosphate buffer (pH 8.1-11), with or without sodium sulfate (0-1 M), and adjusted to a final volume of 1.3 mL. The slurry was degassed under nitrogen for 10 minutes. TCEP-treated SpyCatcher ligand (0.3 mL, 20 mM TCEP, 2 hours, in 1 x PBS) was added to the slurry at a ligand-to-solid ratio of 5.5 mg / mL. The mixture was incubated on a rocker at room temperature for 21 hours. Following immobilization, the solid supports were washed and stored as described in Section 3.2. Supernatants and wash fractions were analyzed by HPLC to determine the amount of immobilized SpyCatcher ligand.

[0064] Example 2: Immobilization of SpyTagDNA on SpyCatcher-magnetic beads

[0065] The goal of this proof-of-principle experiment was to obtain stably immobilized and functional SpyTagDNA. SpyTagDNA was generated and used in IVT to synthesize RN A. As an immobilization strategy, immobilization onto magnetic beads was done via a covalent interaction with SpyCatcher coupled to the magnetic beads. A detailed description of the immobilization procedure and a discussion of the results are provided below.

[0066] 2.1. Design of SpyTag-DNA oligo

[0067] The SpyTag-DNA oligo according to SEQ ID No: 11 is designed to be used in the generation of SpyTagDNA which serves as the template for IVT. The SpyTag (SEQ ID No: 1) is conjugated to the DNA oligo (SEQ ID No: 5) via strain-promoted azide-alkyne cycloaddition. The lysine at the 16thposition of the SpyTag is modified with an azide group. The 5’ end of the DNA oligo is modified with a DBCO group.

[0068] 2.2. Interaction of SpvTag-DNA oligo with SpyCatcher

[0069] To assess the binding of SpyTag-DNA oligo to SpyCatcher, the coupling reaction was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent protein gel staining. The SpyTag-DNA oligo and SpyCatcher was used at a ratio of 1:1 at 50 μM each. The coupling reaction was performed in 10 mM Tris, pH 8.0 for 30 minutes at room temperature. The complex was then analyzed using SDS-PAGE and One-Step Blue® Protein Gel Stain. The result as shown in FIG. 2 demonstrates the formation of the complex between SpyTag-DNA oligo (17.3 kDa) and SpyCatcher (15.7 kDa). It is represented by the band in lane 2 showing the expected size of complex at 33 kDa.

[0070] 2,3. Generation of SpyTagDNA

[0071] Plasmid map of pUC19 plasmid (FIG. 3) according to SEQ ID NO: 12, which encodes eGFP, was used as an example. The open reading frame (ORF) of the gene of interest can be of any sequence used in a vaccine, gene therapies or in a research tool. One other example of a commonly used research tool is the luciferase gene according to SEQ ID NO: 13. The SpyTag DNA (SEQ ID NO: 14) was generated by PCR using a forward primer (SEQ ID NO: 11), a reverse primer (SEQ ID NO: 11), a plasmid DNA as a template (SEQ ID NO: 12), and Q5 high-fidelity 2x master mix. The corresponding plasmid map is shown in FIG. 3. The reaction mixture, prepared in a final volume of 50 μl, contained 1 ng of template DNA, 0.5 μM of forward primer, 0.5 μM of reverse primer, lx of Q5 high-fidelity 2x master mix, and nuclease-free water. Cycling conditions consisted of an initial denaturation at 98°C for 30 sec, followed by 35 cycles of denaturation at 98°C for 10 sec, annealing at 56°C tor 30 sec, and extension at 72°C for 60 sec, with a final extension step at 72°C for 5 min. PCR products were visualized on a 1% agarose gel stained with SYBR green I dye as shown in FIG.

[0072] 4. PCR products were purified by immobilizing to magnetic beads coupled with SpyCatcher.

[0073] 2.4, In vitro transcription using SpyTagDNA as template

[0074] 1 μg of SpyTagDNA was used as template in in vitro transcription. The linearized plasmid DNA (FIG.3) in which the SpyTagDNA was generated from was also used as a control. The SpyTagDNA was incubated in a total volume of 20 μl containing 2 μl of T7 RNA Polymerase Mix, 10 mM each NTP buffer mix, 20 U of RNase inhibitor, and nuclease-free water for 2 hours at 37°C. Subsequently, the DNA template was digested by adding 2 μl DNase I to the IVT reaction mixture, mixing and incubating it for 15 minutes at 37°C. After DNase I digestion, LiCl precipitation was performed. The pellet was resuspended in nuclease-free water and RNA concentration was determined by UV absorbance measurements. The in vitro transcribed RNA was analyzed by RNA agarose gel electrophoresis as shown in FIG. 5.

[0075] 2,5. Immobilization procedure

[0076] 2 μg of SpyTagDNA were transferred into immobilization buffer (10 mM Tris, pH 8.0) comprising Sunresin polymer magnetic beads coupled with SpyCatcher (referred to as “SpyCatcher-magnetic beads”) in a total volume of 100 μl and rotated for 30 min at room temperature to allow immobilization. After immobilization, the SpyTagDNA-bead complex was placed on magnet for 15 seconds and supernatant was collected to determine the amount of immobilized SpyTagDNA. The SpyTagDNA-bead complex was then washed by resuspending in 100 μL of 10 mM Tris, pH 8.0 containing 0.05% Polysorbate 20 for a total of three washing steps. The immobilized SpyTagDNA was used in an RNA in vitro transcription reaction.

[0077] Example 3: Use of immobilized SpyTagDNA for production of in vitro transcribed RNA

[0078] The SpyTagDNA-bead complex obtained according to Example 1 was used to test the suitability of the immobilized SpyTagDNA in IVT reaction. A schematic diagram as shown in FIG. 6 illustrates the assembly of the in vitro transcription system comprising of three components:

[0079] A. Component A of the system, wherein the solid support is selected from the group consisting of agarose, sepharose, magnetic beads, and glass beads. B. Component B of the system, wherein the polypeptide (SpyCatcher) is immobilized on the solid support, and function as binding partner pair for SpyTagDNA.

[0080] C. Component C of the system, wherein the linearized DNA is linked to the thermostable peptide (SpyTagDNA).

[0081] 3.1. RNA in vitro transcription using a DNA template immobilized on magnetic bead via SpyTag- SpyCatcher interaction

[0082] Immobilized SpyTagDNA was used as template in a subsequent RNA in vitro transcription. The linearized plasmid DNA (FIG. 3) in which the SpyTagDNA was generated from was also used as a control. Tite SpyTagDNA-bead complex was incubated with 1400 rpm agitation in a total volume of 80 μl containing 8 μl of T7 RNA Polymerase Mix, 10 mM each NTP buffer mix, 80 U of RNase inhibitor, and nuclease-free water for 2 hours at 37°C. The reaction vessel was placed on magnet for 15-30 seconds, and the supernatant containing the in vitro transcript was transferred to a new, RNase- free vessel. Synthesized mRNA was purified by LiCl precipitation. The RNA concentration was determined by UV absorbance measurements and the in vitro transcribed RNA was analyzed by RNA agarose gel electrophoresis as shown in FIG. 7.

[0083] 3.2. Cleaning and re-use of the immobilized SpyTagDNA template

[0084] After the first cycle of IVT, the SpyTagDNA-bead complex may be reused in successive in vitro transcription reactions by adding new transcription reagent mix to the complex. The complex was incubated in transcription master mix as described in 2.1. Alternatively, tire SpyTagDNA-bead complex can be stored in storage buffer at -20°C for later use.

[0085] For reusability, the SpyTagDNA-bead complex was reused for another 5 cycles of IVT reactions, totaling 6 cycles of IVT. After each cycle, the supernatant containing the in vitro transcript was transferred to a new, RNase-free vessel and the SpyTagDNA-bead complex is incubated with new transcription reagent mix as described in 2.1. The RNA concentration was determined by UV absorbance measurements and the in vitro transcribed RNA was analyzed by RNA agarose gel electrophoresis. As shown in FIG. 8, the RNA synthesized at different cycles are of the same quality and length. Although an average reduction of approximately 8.5% in RNA yield was observed across successive cycles, the cumulative mRNA output (1.61 mg) from the same immobilized template was greater compared to that obtained using a non-immobilized template, which must be digested after a single cycle of in vitro transcription (IVT). In addition, the integrity as well as the purity of the obtained in vitro transcribed RNA across 6 IVT cycles were also assessed by performing an IPRP- HPLC analysis. As shown in FIG. 9, the HPLC chromatograms of purified in vitro tianscript demonstrated nearly identical integrity of the synthesized RNA in all the IVT cycles.

[0086] SEQUENCE LISTING

[0087] SEO ID NO: 1

[0088] Peptide 003

[0089] RGVPHIVMVDAYKRYK

[0090] SEO ID NO: 2

[0091] Peptide 002

[0092] - - VPTIVMVDAYKRYK

[0093] SEO ID NO: 3

[0094] Peptide

[0095] - - - AHIVMVDAYKPTK

[0096] SEO ID NO: 4

[0097] - - - AHIVMVDA

[0098] SEO ID NO: 5

[0099] 5’ GCTTATGCTTCCAGGATTCATCCTACTGACTAATACGACTCACTATAG 3’

[0100] SEO ID NO: 6

[0101] Polypeptide 003 S49C

[0102] MSYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDE

[0103] DGRELAGATMELRDCSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTV

[0104] NEDGQVTVDGEATEGDAHTGSSGS

[0105] SEO ID NO: 7

[0106] Polypeptide 003

[0107] MSYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDE

[0108] DGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTV

[0109] NEDGQVTVDGEATEGDAHTGSSGS

[0110] SEO ID NO: 8

[0111] Polypeptide 002

[0112] MSYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDE

[0113] DGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTV NEQGQVTVNGEATKGDAHTGSSGS

[0114] SEO ID NO: 9

[0115] Polypeptide

[0116] MSYYHHHHHHDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDE

[0117] DGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTV

[0118] NEQGQVTVNGKATKGDAHI

[0119] SEO ID NO: 10

[0120] HHHHHH SEO ID NO: 11

[0121] 5' TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTC 3'

[0122] SEO ID NO: 12

[0123] GACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTT TCTTAGACGTCAGGTGGCACTrrTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTAT

[0124] TTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTC AATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCC TTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAA AAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACA GCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTT TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTC

[0125] GGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAA AGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAG TGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAAC CGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAG CTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCA ACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAAT

[0126] TAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTAT

[0127] CATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACG GGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCA CTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT CAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAA

[0128] AAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCC GTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTA ATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGC ACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA

[0129] GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAA GCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG

[0130] GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGAT GCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGT TCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTG TGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGAC CGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCG CCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACT

[0131] GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCAC CCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAA CAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGGCTGCAGG TCGACTAATACGACTCACTATAGGGAGACAAGCTTGAATTCACATTTGCTTCTGACAC AACTGTGTTCACTAGCAACCTCAAACAGGATCCACCATGGTGAGCAAGGGCGAGGA GCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCA

[0132] CAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCC

[0133] TGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCA

[0134] CCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACG

[0135] ACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAA

[0136] GGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGG

[0137] TGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG

[0138] CACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAG

[0139] AAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT

[0140] GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCT

[0141] GCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA

[0142] AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA

[0143] TGGACGAGCTGTACAAGTAAGCGGCCGCTCGAGGCTCGCTTTCTTGCTGTCCAATTTC

[0144] TATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGG

[0145] CCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCGGGCCCGCTCG

[0146] CTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAA

[0147] CTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTAT

[0148] TTTCATTGTCTAGAAGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0149] AAACCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGG

[0150] AAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTG

[0151] GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAA

[0152] TGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACC

[0153] GCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCG

[0154] ACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGC

[0155] TTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTC

[0156] ATCACCGAAACGCGCGA

[0157] SEO ID NO: 13

[0158] GACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTT

[0159] CTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTT

[0160] TTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAAT

[0161] AATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTT

[0162] TTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGAT

[0163] GCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTA

[0164] AGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT

[0165] CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCG

[0166] CATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTA

[0167] CGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACT

[0168] GCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGC

[0169] ACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGC

[0170] CATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGC

[0171] AAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGAT

[0172] GGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT

[0173] ATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGG

[0174] GCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA

[0175] TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAA CTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTA

[0176] AAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAG

[0177] TTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCC

[0178] TTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGG

[0179] TTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

[0180] GCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAA

[0181] CTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG

[0182] TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC

[0183] AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCT

[0184] ACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG

[0185] GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGA

[0186] GGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC

[0187] TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACG

[0188] CCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT

[0189] TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATA

[0190] CCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

[0191] GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTG

[0192] GCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG

[0193] TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTG

[0194] TGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCA

[0195] AGCTTGCATGCCTGCAGGTCGACTAATACGACTCACTATAGGGAGACAAGCTTGAATTC

[0196] ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGGATCCACCATGGAA

[0197] GACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCG

[0198] CTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGC

[0199] TTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTCGAAATGTCCGT

[0200] TCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTAT

[0201] GCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTG

[0202] CAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGGGCATT

[0203] TCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGC

[0204] AAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAG

[0205] GGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATAC

[0206] GATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTC

[0207] TGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGAT

[0208] TCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAA

[0209] GTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGG

[0210] ATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGG

[0211] ATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGC

[0212] ACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCC

[0213] CTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGC

[0214] AAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGAT

[0215] AAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGG

[0216] ATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTAT

[0217] GATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATG

[0218] GATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATCGTT

[0219] GACCGCCTGAAGTCTCTGATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGA

[0220] ATCCATCTTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACG ATGACGCCGGTGAACTTCCCGCCXiCCGTTGTTGTTTTGGAGCACGGAAAGACGATGAC

[0221] GGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCG

[0222] CGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGC

[0223] AAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTA

[0224] AGCGGCCGCTCGAGGCTCGCTTTCTTGGTGTCCAATTTCTATTAAAGGTTCCTTTGTTCC

[0225] CTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGC

[0226] CTAATAAAAAACATTTATTTTCATTGCGGGCCCGCTCGCTTTCTTGCTGTCCAATTTCTAT

[0227] TAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTT

[0228] GAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGTCTAGAAGACAAAAAA

[0229] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCGGGTACCGAGCTCGAATTC

[0230] ACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATC

[0231] GCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGA

[0232] TCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTC

[0233] TCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCT

[0234] CTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTG

[0235] ACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGC

[0236] TGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGA

[0237] SEO ID NO: 14

[0238] GCTTATGCTTCCAGGATTCATCCTACTGACTAATACGACTCACTATAGGGAGACAAGCT

[0239] TGAATTCACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGGATCCA

[0240] CCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAG

[0241] CTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGA

[0242] TGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGT

[0243] GCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTA

[0244] CCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGT

[0245] CCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG

[0246] TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTC

[0247] AAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA

[0248] CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG

[0249] CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC

[0250] CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCG

[0251] CCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTG

[0252] ACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGCGGCCGCTCGA

[0253] GGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACT

[0254] ACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAA

[0255] CATTTATTTTCATTGCGGGCCCGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCC

[0256] TTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCT

[0257] GGATTCTGCCTAATAAAAAACATTTATTTTCATTGTCTAGAAGACAAAAAAAAAAAAA

[0258] AAAAAAAAAAAAAAAAAAAAAAAAAAA

[0259] SEO ID NO: 15

[0260] 5’-TAATACGACTCACTATAG-3’ SEQ ID NO: 16

[0261] 5'-ATTAACCCTCACTAAAGGG-3'

[0262] SEO ID NO: 17

[0263] 5'-ATTTAGGTGACACTATAG-3'

Claims

1. CLAIMS:

1. A composition (“TSP-DN A Oligo”) comprising: i. a thermostable peptide (“TSP”), wherein the peptide comprises: an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, or an amino acid sequence with at least about 92% sequence identity to a sequence as set forth in SEQ ID NO: 1, or SEQ ID NO:2, or SEQ ID NO: 3, or SEQ ID NO: 4; and ii. a “DNA Oligo” linked to the thermostable peptide through a linker, wherein the linker comprises: i. an azide group, ii. an alkyne group, iii. an isocyanate group, or iv. a tetrazole group; wherein the “DNA oligo” comprises a promoter sequence for a T7, T3, SP6 RNA polymerase, or a known variant of said promoter sequence, or a sequence complementary to said promoter sequence or said variant.

2. The composition of claim 1, wherein said “DNA Oligo” further comprises a spacer sequence of about 20 to 150 additional nucleotides at the 5’ end.

3. The composition of claim 2, wherein the “DNA Oligo” consists of a T7 RNA polymerase promoter and a 30 nucleotide spacer, wherein the “DNA oligo” has at least 95% identity to SEQ ID NO: 5.

4. The composition of claim 1, wherein the linker is selected from the group consisting of: i. a dibenzocyclooctyne (DBCO) group, ii. a maleimide group, and iii. combinations thereof.

5. The composition of claim 4, wherein the thermostable peptide (“TSP”) is modified with an azide or thiol group, and the “DNA Oligo” is modified with a dibenzocyclooctyne (DBCO) or maleimide group to facilitate click chemistry reactions.

6. The composition of claim 1, wherein the composition (“TSP-DNA Oligo”) has at least 95% sequence identity to SEQ ID NO: 11.

7. The composition of claim 1, wherein the thermostable peptide (“TSP”) is immobilized onto a solid support via a binding partner pair involving a polypeptide8. The composition of claim 7, wherein the solid support is selected from the group consisting of agarose, sepharose, magnetic beads and glass beads..

9. The composition of claim 7, wherein the solid support is functionalized to facilitate covalent bonding with the thermostable peptide (“TSP") having at least 95% sequence identity to any one of SEQ ID NOs: 6 to 9.

10. The method of claim 7, wherein the immobilization is achieved through a SpyTag / SpyCatcher system, wherein the SpyTag is part of the thermostable peptide and the SpyCatcher is part of the polypeptide immobilized on the solid support. 11.A method for manufacturing a linearized DNA linked to a thermostable peptide (“TSP- DNA Template”) from a plasmid construct or synthetic construct, the method comprising: conducting a PCR using a plasmid construct or synthetic construct as an initial DNA template, and using the composition of Claim 1 (“TSP-DNA Oligo") as a forward primer, and using a poly(T)-containing sequence as a reverse primer, wherein the resulting structure is a “TSP-DNA Template” comprising: the composition of Claim 1 (“TSP-DNA Oligo”), a 5’ UTR, a “gene of interest”, a 3’ UTR and a poly(A) sequence, wherein the “TSP-DNA Template” is immobilized to a solid support.

12. The method of Claim 11 wherein the “gene of interest” is a gene used in a vaccine, gene therapy or in a research tool.

13. The method of Claim 11 wherein the “TSP-DNA Template” has at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14.

14. The method of Claim 11 wherein the plasmid template has at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 12.

15. The method of Claim 11, wherein the “TSP-DNA Template'” is capable of serving as a template for in vitro transcription (IVT) of the gene of interest to synthesize mRNA.

16. A method for manufacturing mRNA of a “gene of interest” from a plasmid DNA, comprising: i. providing a linearized DNA (“TSP-DNA Template”) immobilized on a solid support, comprising the method of Claim 11; and ii. using the immobilized “TSP-DNA Template” as a template for in vitro transcription (IVT) reaction to synthesize mRNA product comprising the “gene of interest” sequence.

17. The method of claim 16, further comprising separating the immobilized “TSP-DNA Template” from said in vitro transcription reaction and mRNA products.

18. The method of claim 16, further comprising: conducting multiple rounds of in vitro transcription using the same immobilized “TSP-DNA Template” without the need for enzymatic digestion of the “TSP-DNA Template.”19. The method of claim 16, wherein the in vitro transcription reaction includes adding a transcription master mix comprising RNA polymerase, nucleoside triphosphates (NTPs), and a transcription buffer to the immobilized composition.

20. The method of claim 16, further comprising: i. separating the synthesized mRNA from the solid support after transcription, ii. purifying the synthesized mRNA.

21. The method of claim 16, wherein the synthesized mRNA is used for applications selected from the group consisting of vaccines, gene therapies, and research tools.

22. The method of claim 16, wherein the immobilized “TSP-DNA Template” is reused for at least three rounds of in vitro transcription, thereby reducing the costs associated with the mRNA production process.

Images