One-step method for synthesizing circular RNA

Abstract

Provided is a method of preparing a circular RNA, comprising providing a template DNA in a reaction solution wherein the template DNA comprises a sequence encoding a precursor RNA to allow synthesis of the precursor RNA by in vitro transcription of the template DNA and to allow the precursor RNA to self-splice, thereby producing a circular RNA wherein in vitro transcription of the template DNA and self-splice of the precursor RNA (i.e., a sequence encoding the template DNA) of the template DNA are separated from each other. Cyclization) is carried out in the same reaction solution under the same reaction conditions (e.g., the same reaction temperature). The method can be carried out in a single reaction vessel and does not require a step that allows the precursor RNA to be purified prior to self-splicing.

Description

Technical Field

[0001] The present disclosure relates to circular RNA and methods of making and using circular RNA. Background Art

[0002] Messenger RNA (mRNA) is a class of single-stranded RNA involved in protein synthesis. In vitro transcribed (IVT) mRNA has recently attracted widespread attention as a new type of drug with great therapeutic potential. In particular, the successful use of COVID-19 mRNA vaccines has demonstrated its in vivo safety and efficacy. However, the use of mRNA in non-vaccine therapies such as protein replacement therapy is limited by several factors, including mRNA stability, its persistence of expression in vivo, immunogenicity, and expressing cell types.

[0003] Circular RNA (circRNA) has become a therapeutic agent. CircRNA is a class of single-stranded RNA that forms a 3'-5' covalent closed loop. CircRNA is produced by an atypical splicing process called "backsplicing", in which the spliceosome fuses the splice donor site in the downstream exon with the splice acceptor site in the upstream exon. Although circRNA is generally non-coding, several studies have provided evidence that some circRNAs can be translated into proteins. Unlike linear mRNA, circRNA does not require a 5'-cap or 3'-poly (A) tail to maintain its stability. In addition, circRNA has beneficial features that mRNA does not have, such as reduced immunogenicity and extended translation duration. For these reasons, circRNA has been explored as a therapeutic agent.

[0004] Researchers have developed several methods to connect the ends of linear RNA precursors into closed circular RNA in vitro. The most commonly used methods include enzyme ligation and ribozyme ligation. Enzyme ligation-mediated cyclization usually requires complementary splints (DNA or RNA oligonucleotides) to bring the two ends of the RNA molecule closer, and utilizes enzymes from bacteriophage T4 (such as T4 DNA ligase, T4 RNA ligase 1 and T4 RNA ligase 2) catalysis. However, the cyclization efficiency of all these ligase-mediated cyclizations is not effective enough, especially for large RNA molecules. In addition, the generation of intermolecular end-joining byproducts in the ligation reaction cannot be completely avoided, resulting in complex system optimization and being unfavorable for expanded production.

[0005] Alternatively, circular RNA can also be prepared by ribozyme ligation. Ribozyme-mediated RNA cyclization is usually carried out by a permuted intron and exon (PIE) method based on a modified type I intron self-splicing system. Type I introns are large self-splicing ribozymes. Natural type I introns do not require the help of spliceosomes or other proteins to self-splicing, but rely on magnesium and free guanosine nucleotides to initiate and complete the reaction. This process leads to the connection of exons flanking introns and internal intron cyclization, thereby producing intron circRNA. Previous studies have designed a PIE system using modified type I introns, including placing the 5' half of the type I intron at the tail of the exon and transferring the remaining 3' half to the head of the same exon (Puttaraju et al., 1992, "Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons", Nucleic Acids Res 20, 5357-5364; Wesselhoeft et al., 2018, "Engineering circular RNA for potent and stable translation in eukaryotic cells", Nature Communications 9, 2629). This method achieves RNA cyclization through a conventional type I intron self-splicing reaction, which includes two ester exchange reactions at a defined splicing site. The attack of free GTP on the 5' splice site results in the release of the 3' end sequence (5' half region intron) of the PIE construct (the first ester exchange). In the second transesterification reaction, the free 3'OH group of the newly generated 3' half-region exon attacks the 3' splice site, thereby causing the release of circRNA and 3' half-region intron.

[0006] Compared with enzyme-ligation-mediated cyclization, the PIE method can be used to cyclize larger linear RNA precursors, which does not require the addition of additional protein ligases, and its reaction conditions and purification methods are relatively easier to develop and optimize. The circular RNA encoding exogenous proteins synthesized by the PIE method has been verified in vitro and in vivo, and they retain the characteristics of low immunogenicity and long translation duration, thereby broadening its scope of application (Wesselhoeft et al., 2019, "RNA circularization diminishes immunogenicity and can extend translation duration in vivo." Mol. Cell. 74, 508-520; Qu et al., 2022, "Circular RNA vaccines against SARS-CoV-2 and emerging variants", Cell 185, 1728-1744). Based on these advantages, the PIE system has become the most studied and widely used RNA cyclization method.

[0007] The in vitro generation of circRNAs by the PIE method requires a construct of a sequence of interest flanked by the 3' and 5' introns of the replaced type I catalytic intron. The PIE sequence vector is linearized by a single enzyme digestion and used as a template for in vitro transcription. The precursor RNA is obtained by a standard in vitro transcription (IVT) reaction mediated by T7 polymerase and then purified. The yield of the IVT reaction and the purity of the purified product need to be optimized and guaranteed during the production process, and they are also the technical core of nucleic acid drug production. The purified precursor RNA needs to be pre-denatured at about 70°C and then rapidly cooled for renaturation before subsequent reactions to ensure effective cyclization. Special pre-denaturation equipment may be required in industrial scale-up production to achieve sufficient and uniform heating of larger reaction systems and to ensure a rapid and accurate cooling process. The optimal temperature for cyclization should be set at about 55°C and carried out in a specific buffer system (containing 2mM GTP, 50mM Tris HCl, 10mM MgCl2, 1mM DTT, pH 7.5). In addition, the concentration of the precursor RNA needs to be optimized to achieve cyclization. When the concentration of precursor RNA is low, it can be fully circularized, but the reaction volume increases exponentially in the subsequent production amplification, which poses a huge challenge to the control of reaction temperature and the selection of reaction vessels. Although increasing the concentration of precursor RNA can reduce the reaction volume and thus reduce the pressure of amplification production, the cost is the sacrifice of circularization speed. According to the established process flow, it takes at least two days from IVT of linearized plasmid to precursor purification in amplification production, accompanied by possible RNA loss. The purified precursor RNA requires special denaturation and reaction equipment compatible with 55°C conditions for circularization, followed by rapid termination of the reaction and subsequent column chromatography purification. These steps take at least two days. Therefore, it takes at least four days to obtain purified circRNA by traditional processes. There are still technical challenges and difficulties in amplifying the production of circular RNA in the traditional two-step process.

[0008] There is a need for ribozyme-mediated cyclization processes that are simpler, faster, and more efficient than traditional processes. Summary of the invention

[0009] In one aspect, the present invention provides a method for preparing circular RNA (method 1.0), which includes providing a template DNA (wherein the template DNA comprises a sequence encoding a precursor RNA) in a reaction solution to allow the synthesis of the precursor RNA by in vitro transcription of the template DNA, and allowing the precursor RNA to self-splicing, thereby producing a circular RNA, wherein the in vitro transcription of the template DNA and the self-splicing (i.e., cyclization) of the precursor RNA are carried out in the same reaction solution under the same reaction conditions (e.g., the same reaction temperature). The method can be carried out in a single step in a single reaction vessel, and is not limited to the step of purifying the precursor RNA before allowing the precursor RNA to self-splicing.

[0010] In some embodiments, the DNA template comprises the following elements operably connected to each other and arranged in the following order: RNA polymerase promoter, optionally 5' homology arm, 3' I-type intron fragment containing 3' splice site dinucleotide, optionally 5' spacer sequence, insertion sequence, optionally 3' spacer sequence, 5' I-type intron fragment containing 5' splice site dinucleotide and optionally 3' homology arm. In some embodiments, the insertion sequence comprises a protein coding sequence, optionally wherein the insertion sequence comprises an IRES (internal ribosome entry site) sequence operably connected to the protein coding sequence.

[0011] In some embodiments, the reaction solution comprises Mg at a concentration greater than 26 mM (e.g., greater than 30 mM or greater than 35 mM). 2+ In certain embodiments, the solution contains Mg 2+ The concentration is from 38mM to 66mM.

[0012] In some embodiments, the reaction solution comprises pyrophosphatase at a concentration of from 1 U / ml to 5 U / ml, e.g., from 1 U / ml to 4 U / ml, from 1.5 U / ml to 3 U / ml, from 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml, or about 4 U / ml.

[0013] In some embodiments, the reaction solution contains 38-66 mM Mg 2+ , optionally 1-4 U / ml pyrophosphatase, RNA polymerase, ribonuclease inhibitor, ATP, GTP, CTP, UTP, DTT and monovalent cations (Na + or K + ).

[0014] In some embodiments, the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) are performed at a temperature of from 37° C. to 55° C. In some embodiments, the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) are performed at a temperature above 37° C., such as from 39° C. to 55° C., from 41° C. to 55° C., from 43° C. to 55° C., from 39° C. to 50° C., from 41° C. to 50° C., from 43° C. to 50° C., from 39° C. to 47° C., from 41° C. to 47° C., from 43° C. to 47° C., from 47° C. to 55° C., from 50° C. to 55° C., from 39° C. to 43° C., about 39° C., about 41° C., about 43° C., about 47° C., about 53° C., or about 55° C.

[0015] In some embodiments, the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) can be carried out for at least 1 hour, such as at least 1.5 hours, at least 2.5 hours, at least 3 hours, 1 hour to 3 hours, 1.5 hours to 3 hours, 2 hours to 3 hours or 2.5 hours to 3 hours. In certain embodiments, the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) are carried out for 2.5-3 hours.

[0016] In some embodiments, the method further comprises the step of removing the DNA template after the RNA self-splicing, optionally wherein the DNA template is removed by adding deoxyribonuclease I, for example at 37°C for 30 minutes.

[0017] In some embodiments, the method further comprises a step of purifying the circular RNA after the RNA self-splicing, or if the method comprises a step of removing the DNA template, after the step of removing the DNA template. In some embodiments, the purification step is selected from a precipitation step, a tangential flow filtration step, and a chromatography step, and combinations thereof.

[0018] In another aspect, the present invention provides a reaction solution for one-step synthesis of circular RNA, comprising Mg at a concentration greater than 26 mM (e.g., from 38 mM to 66 mM). 2+ , optionally 1-4 U / ml pyrophosphatase, RNA polymerase, ribonuclease inhibitor, ATP, GTP, CTP, UTP, DTT and monovalent cations (Na + or K + ).

[0019] Further areas of applicability of the present disclosure will become apparent from the detailed description provided hereinafter.It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the present disclosure, are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram showing the circRNA precursor used in each example.

[0021] Figure 2 Schematic diagram showing the conventional process (left) and the one-step method (right).

[0022] Figure 3 Fragment analysis of circularized products with and without pre-denaturation is shown. Peaks corresponding to intronic sequences at both ends of the precursor, circularized RNA (indicated by circles) and remaining precursor (indicated by curves) are shown. The ratio of circular RNA to precursor is marked.

[0023] Figure 4AThe effect of Mg2+ concentration on circularization efficiency (% of total CircRNA) in the IVT system is shown. The dotted line indicates 40% circularization rate.

[0024] Figure 4B Shown in different Mg 2+ Fragment analysis of IVT products at 1:1 concentration. Peaks corresponding to intronic sequences, circularized RNA (indicated by circles), and remaining precursors (indicated by curves) are shown.

[0025] Figure 4C The effect of Mg2+ concentration on the yield (total RNA) in the IVT system is shown. The dotted line indicates a 200 μg yield.

[0026] Figure 5A The cyclization efficiency at different reaction durations is shown. The dotted line indicates 40% cyclization rate.

[0027] Figure 5B The yield (total RNA) in the IVT system is shown for different reaction durations. The dotted line indicates 200 μg yield.

[0028] Fig. 6A Pareto chart showing the normalized effect of different factors or combinations of specific factors on the yield (total RNA) in the IVT system. In the Pareto chart, bars that intersect the reference line are statistically significant.

[0029] Figure 6B Main effects plots for Yield (total RNA) / reaction are shown.

[0030] Figure 7 Shown is an interaction plot showing the average yield (total RNA) of the two T7 RNAP types versus temperature.

[0031] Fig. 8A A Pareto chart showing the normalized effect of different factors or a specific combination of factors on the cyclization efficiency. In the Pareto chart, the bars that intersect the reference line are statistically significant.

[0032] Figure 8B Main effects plot showing cyclization efficiency / reaction.

[0033] Fig. 9 The average cyclization efficiency of the three pyrophosphatase concentrations is shown as a function of Mg 2+ Interaction plot of concentration.

[0034] Fig.10 Shows different temperatures and Mg 2+ Line graph of % dsRNA in the one-step IVT reaction product. DETAILED DESCRIPTION

[0035] The following description of the preferred embodiment(s) is merely exemplary in nature and is in no way intended to limit the present disclosure, its application, or uses.

[0036] As used throughout, ranges are used as shorthand for describing each value within the range. Any value within the range may be selected as the endpoint of the range. In addition, all references cited herein are hereby incorporated by reference in their entirety. If a definition in the present disclosure conflicts with a definition in the cited reference, the present disclosure shall prevail.

[0037] The traditional PIE-based cyclization process includes two independent steps: an in vitro transcription (IVT) step and a cyclization step. According to previous studies (Wesselhoeft et al., (2018), "Engineering ring RNA for potent and stable Translation in eukaryotic cells, Nature Communications 9, 2629), 2mM GTP and 10mM MgCl2 are required for the cyclization of IVT-derived precursor RNA by the self-splicing process of type I introns, while the conventional IVT reaction solution contains 10mM GTP and 26mM MgCl2. Studies have found that precursor RNA cannot be effectively cyclized under traditional IVT conditions (10mM GTP and 26mM MgCl2). Therefore, it is necessary to optimize the traditional IVT conditions for a one-step reaction system for circular RNA synthesis. In the present invention, it was found that adding Mg 2+ Increasing the concentration from 26 mM to 36 mM can not only promote the cyclization of circRNA precursors, but also increase the yield of IVT. In the present invention, the conditions of the one-step reaction system are also optimized. For example, studies have found that adding pyrophosphatase to the reaction solution can increase the IVT yield while also increasing the cyclization efficiency. It is believed that this is because pyrophosphatase helps maintain a stable magnesium ion concentration throughout the IVT / cyclization process. After multi-factor optimization, the one-step process can achieve a cyclization efficiency similar to that of the traditional process and reduce the production of double-stranded RNA (dsRNA).

[0038] The one-step circular RNA synthesis system of the present invention has significant advantages over the traditional circular RNA synthesis process, such as improved cyclization efficiency, reduced byproducts that may cause inflammation (e.g., dsRNA), and shortened production cycle, all of which are conducive to process scale-up and technology transfer. Using the traditional process to synthesize circRNA, IVT is first required to generate precursors, and after purification, the cyclization of the purified precursor requires additional introduction of GTP and Mg-containing 2+buffer to initiate cyclization. In addition, more than 70% cyclization efficiency can only be achieved when the precursor is pre-denatured at 70°C before cyclization. Therefore, the traditional process requires five steps in sequence, including IVT, first purification, pre-denaturation, cyclization, and second purification, to obtain the final circRNA ( Figure 1 ). In contrast, in the method of the present invention, IVT and cyclization are performed in one step. In addition, the one-step process does not require a pre-denaturation step before cyclization. Therefore, the one-step process does not require special denaturation equipment and high temperature reaction conditions of 55°C, is significantly simpler than the traditional process, and shortens the circRNA synthesis cycle from 4 days to 2 days ( Figure 1 ).

[0039] In one aspect, the present invention provides a method for preparing circular RNA (method 1.0), which includes providing a template DNA (wherein the template DNA comprises a sequence encoding a precursor RNA) in a reaction solution to allow synthesis of the precursor RNA by in vitro transcription of the template DNA, and allowing the precursor RNA to self-splicing, thereby producing circular RNA, wherein the in vitro transcription of the template DNA and the self-splicing (i.e., circularization) of the precursor RNA are carried out in the same reaction solution under the same reaction conditions (e.g., the same reaction temperature).

[0040] For example, the present invention includes:

[0041] 1.1. Method 1.0, wherein the method does not include a step of purifying the precursor RNA prior to allowing the precursor RNA to self-splicing.

[0042] 1.2. As in method 1.0 or 1.1, wherein the template DNA is circular, optionally wherein the circular template DNA is a DNA plasmid.

[0043] 1.3. As in method 1.0 or 1.1, wherein the template DNA is linear, optionally wherein the linear template DNA is prepared by linearizing a DNA plasmid, for example, using a restriction endonuclease.

[0044] 1.4. As any of the aforementioned methods, the DNA template comprises the following elements operably linked to each other and arranged in the following order: an RNA polymerase promoter, optionally a 5' homology arm, a 3' type I intron fragment containing a 3' splice site dinucleotide, optionally a 5' spacer sequence, an insertion sequence, optionally a 3' spacer sequence, a 5' type I intron fragment containing a 5' splice site dinucleotide, and optionally a 3' homology arm.

[0045] 1.5. The method of 1.4, wherein the inserted sequence comprises a non-coding sequence having biological activity, optionally wherein the non-coding sequence is a microRNA or Inc (long non-coding) RNA.

[0046] 1.6. The method of 1.4, wherein the inserted sequence comprises a protein coding sequence, optionally wherein the inserted sequence comprises an IRES sequence operably linked to the protein coding sequence.

[0047] 1.7. The method of 1.6, wherein the protein coding sequence encodes an antibody.

[0048] 1.8. The method of 1.6-1.7, wherein the IRES sequence is selected from the group consisting of the IRES sequences of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invictavirus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stallintestine virus, Kashmir bee virus, Human rhinovirus 2, Human rhinovirus B, Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, human enterovirus B, equine rhinitis virus, Ectropis obliqua picoma-likevirus, encephalomyocarditis virus (EMCV), Drosophila C virus, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia gene (Drosophilaantennapedia), human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n-myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, salivary virus, Cosavirus, Parechovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian picornavirus, Turnip crinkle virus, eIF4G aptamer, Coxsackievirus B3 B3, CVB3) or Coxsackievirus A (CVB1 / 2).

[0049] 1.9. The method of 1.8, wherein the IRES sequence is the IRES sequence of Coxsackievirus B3 (CVB3).

[0050] 1.10. Any of the preceding methods, wherein the RNA polymerase promoter is a T7 viral RNA polymerase promoter, a T6 viral RNA polymerase promoter, an SP6 viral RNA polymerase promoter, a T3 viral RNA polymerase promoter, or a T4 viral RNA polymerase promoter.

[0051] 1.11. Any of the preceding methods, wherein the RNA polymerase promoter is a T7 viral RNA polymerase promoter.

[0052] 1.12. Any of the preceding methods, wherein the 3' group I intron fragment is a continuous sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95% or 100%) homology to the 3' proximal fragment of a native group I intron.

[0053] 1.13. Any of the preceding methods, wherein the 5' group I intron fragment is a contiguous sequence that has at least 80% (eg, at least 85%, at least 90%, at least 95% or 100%) homology to the 5' proximal fragment of a native group I intron.

[0054] 1.14. Methods 1.12-1.13, wherein the natural group I intron is derived from the T4 bacteriophage gene td or the cyanobacterium Anabaena precursor-tRNA-Leu gene.

[0055] 1.15. Any of the preceding methods, wherein the 5' homology arm and the 3' homology arm are perfectly complementary to each other.

[0056] 1.16. Any of methods 1.0-1.14, wherein the 5' homology arm and the 3' homology arm are partially (eg, at least 80%, at least 85%, at least 90% or at least 95%) complementary to each other.

[0057] 1.17. Any of the preceding methods, wherein the 5' homology arm is from 5 to 50 nucleotides in length, for example, from 9 to 19 nucleotides in length.

[0058] 1.18. Any of the preceding methods, wherein the 3' homology arm is from 5 to 50 nucleotides in length, for example, from 9 to 19 nucleotides in length.

[0059] 1.19. Any of the preceding methods, wherein the DNA template comprises a 5' spacer sequence between the 3' group I intron fragment and the insertion sequence, optionally wherein the 5' spacer sequence is from 5 to 50 nucleotides in length, for example, from 10 to 20 nucleotides in length.

[0060] 1.20. Any of the preceding methods, wherein the 5' spacer sequence is a poly A sequence or a poly AC sequence.

[0061] 1.21. Any of the preceding methods, wherein the DNA template comprises a 3' spacer sequence between the insert sequence and the 5' group I intron fragment, optionally wherein the 3' spacer sequence is from 5 to 50 nucleotides in length, for example, from 10 to 20 nucleotides in length.

[0062] 1.22. Any of the preceding methods, wherein the 3' spacer sequence is a poly A sequence or a poly AC sequence.

[0063] 1.23. Any of the preceding methods, wherein the reaction solution comprises Mg at a concentration greater than 26 mM, such as greater than 30 mM or greater than 35 mM. 2+ .

[0064] 1.24. Any of the preceding methods, wherein the solution contains Mg 2+The concentration of Mg in the solution is from 30mM to 100mM, for example, from 30mM to 90mM, from 30mM to 80mM, from 30mM to 70mM, from 30mM to 60mM, from 30mM to 50mM, from 30mM to 40mM, from 35mM to 100mM, from 35mM to 90mM, from 35mM to 80mM, from 35mM to 70mM, from 35mM to 60mM, from 35mM to 50mM, from 35mM to 40mM, from 38mM to 66mM, for example, about 38mM, optionally wherein the solution contains Mg 2+ The concentration is from 38mM to 66mM.

[0065] 1.25. Any of the preceding methods, wherein the reaction solution comprises pyrophosphatase at a concentration of 1 U / ml to 5 U / ml, such as 1 U / ml to 4 U / ml, 1.5 U / ml to 3 U / ml, 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml or about 4 U / ml.

[0066] 1.26. As any of the preceding methods, the reaction solution comprises RNA polymerase, ribonuclease inhibitor, ATP, GTP, CTP, UTP, DTT and monovalent cations (Na + or K + ).

[0067] 1.27. As any of the preceding methods, the reaction solution comprises 5 U / μl RNA polymerase, 1 U / μl ribonuclease inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT and 5 mM monovalent cations (Na + or K + ).

[0068] 1.28. Any of the preceding methods, wherein the reaction solution comprises 38-66 mM Mg 2+ , 1-4 U / ml pyrophosphatase, RNA polymerase, ribonuclease inhibitor, ATP, GTP, CTP, UTP, DTT and monovalent cations (Na + or K + ).

[0069] 1.29. Any of the preceding methods, wherein the reaction solution comprises 38 mM Mg 2+ , 2U / ml pyrophosphatase, RNA polymerase, ribonuclease inhibitor, ATP, GTP, CTP, UTP, DTT and monovalent cations (Na + or K + ).

[0070] 1.30. Any of the preceding methods, wherein the reaction solution comprises 38-66 mM Mg 2+ , optionally 1-4 U / ml pyrophosphatase, 5 U / μl RNA polymerase, 1 U / μl ribonuclease inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT and 5 mM monovalent cations (Na + or K + ).

[0071] 1.31. Any of the preceding methods, wherein the reaction solution comprises 38 mM Mg 2+ , 2U / ml pyrophosphatase, 5U / μl RNA polymerase, 1U / μl ribonuclease inhibitor, 10mM ATP, 10mM GTP, 10mM CTP, 10mM UTP, 10mM DTT and 5mM monovalent cations (Na + or K + ).

[0072] 1.32. Any of the preceding methods wherein the reaction solution comprises a buffer.

[0073] 1.33. Any of the preceding methods, wherein the pH of the reaction solution is 6 to 8, such as 7 to 8, or about 7.5.

[0074] 1.34. Any of the preceding methods, wherein the reaction solution comprises an RNA polymerase selected from the group consisting of T7 viral RNA polymerase, T6 viral RNA polymerase, SP6 viral RNA polymerase, T3 viral RNA polymerase, or T4 viral RNA polymerase.

[0075] 1.35. Any of the preceding methods, wherein the RNA polymerase promoter in the DNA template is a T7 viral RNA polymerase promoter and the reaction solution comprises a T7 viral RNA polymerase.

[0076] 1.36. Any of the preceding methods, wherein the in vitro transcription of the template DNA and circularization (i.e., self-splicing) of the precursor RNA is performed at a temperature of from 37°C to 55°C, e.g., from 39°C to 55°C, from 41°C to 55°C, from 43°C to 55°C, from 37°C to 50°C, from 39°C to 50°C, from 41°C to 50°C, from 43°C to 50°C, from 37°C to 47°C, from 39°C to 47°C, from 41°C to 47°C, from 43°C to 47°C, from 47°C to 55°C, from 50°C to 55°C, from 39°C to 43°C, about 37°C, about 39°C, about 41°C, about 43°C, about 47°C 7°C, about 53°C or about 55°C, optionally wherein the in vitro transcription of the template DNA and the circularization (i.e., self-splicing) of the precursor RNA are carried out at a temperature above 37°C, such as from 39°C to 55°C, from 41°C to 55°C, from 43°C to 55°C, from 39°C to 50°C, from 41°C to 50°C, from 43°C to 50°C, from 39°C to 47°C, from 41°C to 47°C, from 43°C to 47°C, from 47°C to 55°C, from 50°C to 55°C, from 39°C to 43°C, from about 39°C, about 41°C, about 43°C, about 47°C, about 53°C or about 55°C.

[0077] 1.37. Any of the preceding methods, wherein in vitro transcription of the template DNA and circularization (i.e., self-splicing) of the precursor RNA are carried out at a temperature of from 47°C to 55°C, such as from 50°C to 55°C, about 47°C, about 53°C or about 55°C, and the RNA polymerase is a thermostable polymerase (e.g., T7 Toyobo).

[0078] 1.38. Any of the preceding methods, wherein the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) are performed for at least 1 hour, such as at least 1.5 hours, at least 2.5 hours, at least 3 hours, 1 hour to 3 hours, 1.5 hours to 3 hours, 2 hours to 3 hours, or 2.5 hours to 3 hours, optionally wherein the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) are performed for 2.5-3 hours.

[0079] 1.39. Any of the preceding methods, wherein the method further comprises a step of removing the DNA template after synthesizing the precursor RNA, optionally wherein the DNA template is removed by adding deoxyribonuclease I, for example, at 37°C for 30 minutes.

[0080] 1.40. Any of the preceding methods, wherein the method further comprises a step of purifying the circular RNA synthesized thereby, for example, if the method comprises a step of removing the DNA template, the purification is performed after the step of removing the DNA template.

[0081] 1.41. A method as described in 1.40, wherein the purification step is selected from a precipitation step, a tangential flow filtration step and a chromatography step, and combinations thereof.

[0082] 1.42. The method of 1.41, wherein the precipitation step is an alcohol precipitation step or a LiCl precipitation, optionally wherein the precipitation step is a LiCl precipitation.

[0083] 1.43. The method of 1.41, wherein the tangential flow filtration step is a diafiltration step using tangential flow filtration and / or a concentration step using tangential flow filtration.

[0084] 1.44. A method as described in 1.41, wherein the chromatography step is selected from HPLC, anion exchange chromatography, affinity chromatography, hydroxyapatite chromatography, magnetic bead chromatography and core bead chromatography, optionally wherein the chromatography step is magnetic bead chromatography.

[0085] 1.45. Any of the preceding methods, wherein the precursor RNA is unmodified, ie, contains only naturally occurring nucleosides, such as adenosine, guanosine, cytidine and uridine.

[0086] 1.46. Any of methods 1.0-1.44, wherein the precursor RNA is partially modified or fully modified, ie, contains nucleosides other than adenosine, guanosine, cytidine and uridine.

[0087] 1.47. Method 1.46, wherein the precursor RNA comprises a nucleoside selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methylcytidine, N6-methyladenosine, and combinations thereof.

[0088] 1.48. As described in method 1.46, part or all of the nucleoside triphosphates in the reaction solution contain nucleoside triphosphates other than adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP).

[0089] 1.49. As in method 1.46, part or all of the nucleoside triphosphates in the reaction solution contain modified nucleoside triphosphates, for example, wherein the modified nucleoside triphosphates are selected from pseudouridine-5'-triphosphate, 1-methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, N6-methyladenosine-5'-triphosphate and combinations thereof.

[0090] 1.50. Method 1.46, wherein the nucleosides in the precursor RNA do not comprise uridine, but comprise nucleosides selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4-thiouridine, and combinations thereof.

[0091] 1.51. Method 1.46, wherein the nucleosides in the precursor RNA do not comprise cytidine but comprise 5-methylcytidine.

[0092] 1.52. Any of the preceding methods wherein the cyclization efficiency is at least 70%.

[0093] 1.53. Any of the preceding methods, wherein the percentage of dsRNA relative to total RNA in the final product is less than 1%, such as less than 0.1%.

[0094] 1.54. Any of the preceding methods, wherein the reaction solution is a reaction solution conforming to Reaction Solution 2, etc. described below.

[0095] In one embodiment, the reaction solution according to the process of the present invention comprises Mg 2+ 。 The reaction solution may further contain pyrophosphatase.

[0096] In further embodiments, the reaction solution may contain nucleoside triphosphates.

[0097] In yet further embodiments, the reaction solution may comprise a reducing agent.

[0098] In yet another embodiment, the reaction solution may contain RNA polymerase.

[0099] In another embodiment, the reaction solution may contain a ribonuclease inhibitor.

[0100] In another embodiment, the reaction may include monovalent cations.

[0101] The selection and / or concentration of each component is as described herein. The concentration is based on the reaction solution / mixture as used in the method.

[0102] For example, Mg in solution 2+ The concentration of the pyrophosphatase in the solution is greater than 26mM, in particular from 38 to 66mM, more specifically 38mM. For example, the concentration of pyrophosphatase in the solution is from 1U / ml to 5U / ml, in particular from 1U / ml to 4U / ml, more particularly 2U / ml. For example, the nucleoside triphosphates include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP), and the concentration of each of them is 10mM. For example, the reducing agent is DTT, and its concentration is 10mM. For example, the RNA polymerase is T7 viral polymerase, and its concentration is 5U / μl. For example, the concentration of the ribonuclease inhibitor is 1U / μl. For example, the monovalent cation is Na + or K+ , and its concentration is 5mM.

[0103] In one embodiment, the reaction solution according to the method of the present disclosure comprises Mg 2+ , RNA polymerase, a ribonuclease inhibitor, nucleoside triphosphates, a reducing agent and a monovalent cation (Na+ or K+), and optionally a pyrophosphatase.

[0104] In one embodiment, the reaction conditions according to the methods of the present disclosure include temperature for in vitro transcription of the template DNA and circularization (ie, self-splicing) of the precursor RNA.

[0105] In further embodiments, the reaction conditions according to the methods of the present disclosure include the duration of in vitro transcription of the template DNA and circularization (ie, self-splicing) of the precursor RNA.

[0106] The selection of each condition is as described herein. For example, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) are performed at a temperature of from 37°C to 55°C, particularly 39°C to 50°C or 47°C to 55°C using a thermostable polymerase (e.g., T7 Toyobo). For example, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) are performed for 2.5-3 hours.

[0107] In one embodiment, the reaction conditions according to the methods of the present disclosure include the temperature and duration of in vitro transcription of the template DNA and circularization (ie, self-splicing) of the precursor RNA.

[0108] In a most particular embodiment, the reaction solution of the method of the present disclosure comprises 38 mM Mg 2+ , 2U / ml pyrophosphatase, 5U / μl RNA polymerase, 1U / μl ribonuclease inhibitor, 10mM ATP, 10mM GTP, 10mM CTP, 10mM UTP, 10mM DTT, 5mM monovalent cation (Na + or K + ); in vitro transcription of template DNA and circularization (ie, self-splicing) of precursor RNA are performed at a temperature of 37° C. to 55° C. and for 2.5-3 hours.

[0109] The present disclosure also provides a circular RNA obtained by any one of method 1 and the like.

[0110] The present disclosure also provides a pharmaceutical composition comprising the circular RNA obtained by any one of method 1 and the like.

[0111] The present disclosure also provides a reaction solution for one-step circular RNA synthesis, which comprises Mg at a concentration greater than 26 mM.2+ , pyrophosphatase, RNA polymerase, ribonuclease inhibitor, nucleoside triphosphate, reducing agent and monovalent cation (for example, selected from Na + , K + and combinations thereof) (reaction solution 2), for example,

[0112] 2.1. Reaction solution 2, wherein the reaction solution contains Mg at a concentration greater than 30 mM or greater than 35 mM. 2+ .

[0113] 2.2. A reaction solution as described above, wherein the solution contains Mg 2+ The concentration of Mg in the solution is from 30mM to 100mM, for example from 30mM to 90mM, from 30mM to 80mM, from 30mM to 70mM, from 30mM to 60mM, from 30mM to 50mM, from 30mM to 40mM, from 35mM to 100mM, from 35mM to 90mM, from 35mM to 80mM, from 35mM to 70mM, from 35mM to 60mM, from 35mM to 50mM, from 35mM to 40mM, from 38mM to 66mM, for example about 38mM, optionally wherein the solution contains Mg 2+ The concentration ranged from 38 mM to 66 mM.

[0114] 2.3. A reaction solution as described in any preceding claim, wherein the reaction solution comprises pyrophosphatase at a concentration from 1 U / ml to 5 U / ml, for example, from 1 U / ml to 4 U / ml, from 1.5 U / ml to 3 U / ml, from 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml or about 4 U / ml.

[0115] 2.4. A reaction solution as described in any of the foregoing, wherein the nucleoside triphosphates are selected from the group consisting of: ATP, GTP, CTP, UTP, pseudouridine-5'-triphosphate, 1-methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, N6-methyladenosine-5'-triphosphate and combinations thereof.

[0116] 2.5. A reaction solution as described in any preceding claim, wherein the nucleoside triphosphates include ATP, GTP, CTP and UTP.

[0117] 2.6. A reaction solution as described in any of the preceding claims, wherein the reaction solution comprises pyrophosphatase at a concentration from 1 U / ml to 5 U / ml, for example, from 1 U / ml to 4 U / ml, from 1.5 U / ml to 3 U / ml, from 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml or about 4 U / ml.

[0118] 2.7. A reaction solution as in any preceding claim, wherein the nucleoside triphosphates include pseudouridine-5′-triphosphate, ATP, GTP, and CTP.

[0119] 2.8. A reaction solution as in any preceding claim, wherein the nucleoside triphosphates include 1-methylpseudouridine-5'-triphosphate, ATP, GTP and CTP.

[0120] 2.9. Any preceding reaction solution, wherein the reducing agent is DTT.

[0121] 2.10. A reaction solution as described in any of the foregoing, wherein the nucleoside triphosphates include pseudouridine-5'-triphosphate, 1-methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, or a combination thereof.

[0122] 2.11. A reaction solution as described in any of the preceding claims, wherein the reaction solution comprises 5 U / μl RNA polymerase, 1 U / μl ribonuclease inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT and 5 mM monovalent cations (Na + or K + ).

[0123] 2.12. A reaction solution as described in any preceding claim, wherein the reaction solution comprises 38-66 mM Mg 2+ , optionally 1-4 U / ml pyrophosphatase, RNA polymerase, ribonuclease inhibitor, ATP, GTP, CTP, UTP, DTT and monovalent cations (Na + or K + ).

[0124] 2.13. A reaction solution as described in any preceding claim, wherein the reaction solution comprises 38 mM Mg 2+ , 2U / ml pyrophosphatase, RNA polymerase, ribonuclease inhibitor, ATP, GTP, CTP, UTP, DTT and monovalent cations (Na + or K + ).

[0125] 2.14. A reaction solution as described in any preceding claim, wherein the reaction solution comprises 38-66 mM Mg 2+ , optionally 1-4 U / ml pyrophosphatase, 5 U / μl RNA polymerase, 1 U / μl ribonuclease inhibitor, 10 mM ATP, 10 mM GTP, 10 mM CTP, 10 mM UTP, 10 mM DTT and 5 mM monovalent cations (Na + or K +).

[0126] 2.15. A reaction solution as described in any preceding claim, wherein the reaction solution comprises 38 mM Mg 2+ , 2U / ml pyrophosphatase, 5U / μl RNA polymerase, 1U / μl ribonuclease inhibitor, 10mM ATP, 10mM GTP, 10mM CTP, 10mM UTP, 10mM DTT and 5mM monovalent cations (Na + or K + ).

[0127] 2.16. Any preceding reaction solution, wherein the reaction solution comprises a buffer.

[0128] 2.17. A reaction solution as in any preceding claim, wherein the pH of the reaction solution is from 6 to 8, such as from 7 to 8, or about 7.5.

[0129] 2.18. A reaction solution as in any preceding claim, wherein the reaction solution comprises an RNA polymerase selected from the group consisting of T7 viral RNA polymerase, T6 viral RNA polymerase, SP6 viral RNA polymerase, T3 viral RNA polymerase, or T4 viral RNA polymerase.

[0130] The present disclosure also provides use of any one of reaction solutions 2, etc. in a one-step circular RNA synthesis method, for example, in a method according to any one of method 1, etc.

[0131] In the method of the present invention, the precursor RNA is synthesized by in vitro transcription of the template DNA. For example, the DNA template contains a promoter upstream of the region encoding the precursor RNA. The promoter is recognized by an RNA polymerase, such as a T7 promoter, which is recognized by a T7 viral RNA polymerase. In some embodiments, the promoter is a T7 promoter and the RNA polymerase is a T7 viral RNA polymerase; or, the promoter is a T6 promoter and the polymerase is a T6 viral RNA polymerase; or, the promoter is an SP6 viral RNA polymerase promoter and the polymerase is an SP6 viral RNA polymerase; or, the promoter is a T3 viral RNA polymerase promoter and the polymerase is a T3 viral RNA polymerase; or, the promoter is a T4 viral RNA polymerase promoter and the polymerase is a T4 viral RNA polymerase. In certain embodiments, the RNA polymerase promoter is a T7 viral RNA polymerase promoter and the polymerase is a T7 viral RNA polymerase.

[0132] The template DNA can be linear or circular. In some embodiments, the template DNA is prepared by, for example, linearizing a DNA plasmid using a restriction endonuclease. In other embodiments, the template is circular (e.g., a DNA plasmid). The template DNA may be included in an RNA polymerase terminator sequence element downstream of the region encoding the precursor RNA, particularly when the template DNA is circular.

[0133] The template DNA comprises a sequence encoding a precursor RNA. As used herein, "circular precursor RNA" or "precursor RNA" refers to a linear RNA molecule that can self-splicing to produce a circular RNA (circRNA). The precursor RNA comprises a circular RNA sequence and a splicing sequence (e.g., an intron fragment and optional 5' and 3' homology arms) necessary for cyclizing the RNA. These splicing sequences are removed from the precursor RNA during the cyclization process. The precursor RNA may be unmodified, partially modified or completely modified. In some embodiments, the precursor RNA is unmodified, i.e., the nucleoside moiety in the precursor RNA is a naturally occurring nucleoside, such as adenosine, guanosine, cytidine and uridine. In other embodiments, the precursor RNA is modified, that is, the nucleoside portion in the precursor RNA comprises nucleosides in addition to or instead of adenosine, guanosine, cytidine and uridine; for example, the nucleosides include pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4-thiouridine, 5-methylcytidine, N6-methyladenosine, inosine or a combination thereof, for example, wherein uridine is replaced by pseudouridine, 1-methylpseudouridine, 2-thiouridine, 4-thiouridine or 5-methoxyuridine, and / or cytidine is replaced by 5-methylcytidine, and / or adenosine is replaced by N6-methyladenosine.

[0134] In some embodiments, the DNA template comprises the following elements operably linked to each other and arranged in the following order: a promoter recognized by RNA polymerase, optionally a 5' homology arm, a 3' type I intron fragment containing a 3' splice site dinucleotide, optionally a 5' spacer sequence, an insertion sequence containing a sequence of interest, optionally a 3' spacer sequence, a 5' type I intron fragment containing a 5' splice site dinucleotide, and optionally a 3' homology arm. As used herein, the phrase "operably linked" means that the elements are located on the DNA template so that the precursor DNA can be synthesized by in vitro transcription of the template DNA, and then the precursor DNA can be circularized into a circular RNA using the methods disclosed herein.

[0135] In some embodiments, the 3' group I intron fragment is a contiguous sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) homology to the 3' proximal fragment of a native group I intron, including a 3' splice site dinucleotide and optionally an adjacent exonic sequence of at least 1 nucleotide in length (e.g., at least 5 nucleotides in length, at least 10 nucleotides in length, at least 15 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, or at least 50 nucleotides in length).

[0136] In some embodiments, the 5' group I intron fragment is a contiguous sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, or 100%) homology to the 5' proximal fragment of a native group I intron, including a 5' splice site dinucleotide and optionally an adjacent exonic sequence of at least 1 nucleotide in length (e.g., at least 5 nucleotides in length, at least 10 nucleotides in length, at least 15 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length, at least 50 nucleotides in length).

[0137] The natural type I intron can be selected from any type I intron known to self-splicing. Examples of type I introns include, but are not limited to, type I introns from the T4 bacteriophage gene td or the cyanobacterium Anabaena precursor-tRNA-Leu gene.

[0138] In some embodiments, the template DNA comprises 5' homology arms and 3' homology arms at the 5' end and 3' end of the precursor RNA region. Adding 5' homology arms and 3' homology arms at the 5' end and 3' end of the precursor RNA region can increase the cyclization efficiency by making the 5' and 3' splice sites close to each other, especially when the insertion sequence between the 3' I type intron fragment and the 5' I type intron fragment is longer. In some embodiments, the 5' homology arm length is from 5 to 50 nucleotides, for example, the length is from 9 to 19 nucleotides. In some embodiments, the 3' homology arm length is from 5 to 50 nucleotides, for example, the length is from 9 to 19 nucleotides. In some embodiments, the 5' homology arm and the 3' homology arm are fully complementary to each other. In other embodiments, the 5' homology arm and the 3' homology arm are partially (e.g., at least 80%, at least 85%, at least 90% or at least 95%) complementary to each other.

[0139] Highly structured sequences between the 3' type I intron fragment and the 5' type I intron fragment, such as IRES (internal ribosome entry site), may interfere with the folding of the splicing ribozyme, either proximal to the 3' splice site or distal to the 5' splice site through long-range contacts (Wesselhoeft et al., 2018). Adding a 5' spacer sequence between the 3' type I intron fragment and the insert sequence and / or adding a 3' spacer sequence between the insert sequence and the 5' type I intron fragment can improve the circularization efficiency, especially when the insert sequence is highly structured. In some embodiments, the DNA template comprises a 5' spacer sequence between the 3' type I intron fragment and the insert sequence. In some embodiments, the length of the 5' spacer sequence is from 5 to 50 nucleotides. In some embodiments, the length of the 5' spacer sequence is from 10 to 20 nucleotides. In certain embodiments, the 5' spacer sequence is a poly A sequence. In other embodiments, the 5' spacer sequence is a poly AC sequence. In some embodiments, the DNA template comprises a 3' spacer sequence between the insert sequence and the 5' type I intron fragment. In some embodiments, the length of the 3' spacer sequence is from 5 to 50 nucleotides. In some embodiments, the length of the 3' spacer sequence is from 10 to 20 nucleotides. In certain embodiments, the 3' spacer sequence is a poly A sequence. In other embodiments, the 3' spacer sequence is a poly AC sequence.

[0140] The insertion sequence comprises a sequence of interest. The sequence of interest can be a protein coding or non-coding sequence. In some embodiments, the insertion sequence comprises a non-coding sequence with biological activity. Examples of non-coding sequences with biological activity include, but are not limited to, microRNA and Inc (long non-coding) RNA.

[0141] In some embodiments, the inserted sequence comprises a protein coding sequence. The protein coding sequence may encode any protein for therapeutic or diagnostic use. In some embodiments, the protein coding sequence encodes an antibody.

[0142] When the insertion sequence comprises a protein coding sequence, the insertion sequence may also comprise a sequence required for translation, such as an internal ribosome entry site (IRES) sequence upstream of the protein coding sequence. In some embodiments, the insertion sequence comprises an IRES sequence operably linked to a protein coding sequence. As used herein, the phrase "operably linked" means that the IRES sequence is located upstream of the protein coding sequence so that the protein coding sequence can be translated into a protein in vivo (in a eukaryotic cell, such as a human cell) and / or in vitro. The IRES sequence may be any IRES sequence known in the art. In some embodiments, the IRES sequence is selected from the group consisting of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stallintestine virus, Kashmir bee virus, Human rhinovirus 2, Human rhinovirus B, Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, hepatitis C virus, hepatitis A virus, GB hepatitis virus, foot-and-mouth disease virus, human enterovirus 71, human enterovirus B, equine rhinitis virus, Ectropis obliqua picoma-likevirus, encephalomyocarditis virus (EMCV), Drosophila C virus, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virusvirus), classical swine fever virus, human FGF2, human SFTPA1, human AML1 / RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n-myc, mouse Gtx, human p27kipl, human PDGF2 / c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila reaper, canine Scamper, Drosophila Ubx, salivirus, Cosavirus, Parechovirus, human UNR, mouse UtrA, human VEGF-A, human XIAP, Drosophila hairless hairless), Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, human c-src, human FGF-1, simian picornavirus, Turnip crinkle virus, eIF4G aptamer, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1 / 2). In certain embodiments, the IRES sequence is the IRES sequence of Coxsackievirus B3 (CVB3).

[0143] In the method of the present invention, the in vitro transcription of the template DNA and the cyclization (i.e., self-splicing) of the precursor RNA are performed in one step. The method does not include the step of purifying the precursor RNA before allowing the precursor RNA to self-splicing. In other words, in vitro transcription and cyclization are performed in the same reaction solution under the same reaction conditions (e.g., temperature). Therefore, the reaction solution and reaction conditions must be optimized to improve the efficiency of in vitro transcription and cyclization.

[0144] In some embodiments, the reaction solution comprises Mg at a concentration greater than 26 mM (e.g., greater than 30 mM or greater than 35 mM). 2+ In some embodiments, the solution contains Mg 2+ The concentration of Mg is from 30mM to 100mM, for example, from 30mM to 90mM, from 30mM to 80mM, from 30mM to 70mM, from 30mM to 60mM, from 30mM to 50mM, from 30mM to 40mM, from 35mM to 100mM, from 35mM to 90mM, from 35mM to 80mM, from 35mM to 70mM, from 35mM to 60mM, from 35mM to 50mM, from 35mM to 40mM, from 38mM to 66mM, for example, about 38mM. In certain embodiments, the Mg in the solution is from 30mM to 100mM, for example, from 30mM to 90mM, from 35mM to 80mM, from 35mM to 70mM, from 35mM to 60mM, from 35mM to 50mM, from 35mM to 40mM, from 38mM to 66mM, for example, about 38mM. 2+ The concentration is from 38mM to 66mM

[0145] In some embodiments, the reaction solution comprises a concentration of from 1U / ml to 5U / ml, for example from 1U / ml to 4U / ml, from 1.5U / ml to 3U / ml, from 1.5U / ml to 2.5U / ml, about 1U / ml, about 2U / ml or about 4U / ml of pyrophosphatase. As used herein, 1U (unit) pyrophosphatase is defined as the amount of the enzyme that produces 1 μmol of phosphate per minute from inorganic pyrophosphate under standard reaction conditions (at 25°C, in 20mM Tris-HCl pH8.0, 2mM MgCl2 and 2mM PPi for 10 minutes).

[0146] The reaction solution also includes components required for in vitro transcription. In some embodiments, the reaction solution includes RNA polymerase, ribonuclease inhibitor, ATP, GTP, CTP, UTP, DTT and monovalent cations (Na + or K + In certain embodiments, the reaction solution comprises about 5 U / μl RNA polymerase, about 1 U / μl ribonuclease inhibitor, about 10 mM ATP, about 10 mM GTP, about 10 mM CTP, about 10 mM UTP, about 10 mM DTT, and 5 mM monovalent cations (Na + or K + ). The reaction solution may contain a buffer. The pH value of the reaction solution may be from 6 to 8, such as from 7 to 8, or about 7.5.

[0147] The precursor RNA may be unmodified, partially modified or fully modified. In some embodiments, the precursor RNA is unmodified, that is, contains only naturally occurring nucleotides. In other embodiments, the precursor RNA is partially modified or fully modified. A portion or all of at least one nucleoside triphosphate in the reaction solution may be substituted with a modified nucleoside triphosphate to synthesize a partially modified or fully modified precursor RNA. The example of modified nucleoside triphosphate includes but is not limited to pseudouridine-5'-triphosphate, 1-methylpseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate and 5-methylcytidine-5'-triphosphate.

[0148] The RNA polymerase for in vitro transcription can be selected based on the RNA polymerase promoter in the DNA template. For example, if the RNA polymerase promoter in the DNA template is a T7 viral RNA polymerase promoter, the reaction solution can include a T7 RNA polymerase. In some embodiments, the reaction solution includes an RNA polymerase selected from: a T7 viral RNA polymerase, a T6 viral RNA polymerase, a SP6 viral RNA polymerase, a T3 viral RNA polymerase, or a T4 viral RNA polymerase. In certain embodiments, the RNA polymerase promoter in the DNA template is a T7 viral RNA polymerase and the reaction solution includes a T7 viral RNA polymerase.

[0149] In some embodiments, in vitro transcription of template DNA and circularization (i.e., self-splicing) of precursor RNA is performed at a temperature of from 37° C. to 55° C., e.g., from 39° C. to 55° C., from 41° C. to 55° C., from 43° C. to 55° C., from 37° C. to 50° C., from 39° C. to 50° C., from 41° C. to 50° C., from 43° C. to 50° C., from 37° C. to 47° C., from 39° C. to 47° C., from 41° C. to 47° C., from 43° C. to 47° C., from 47° C. to 55° C., from 50° C. to 55° C., from 39° C. to 43° C., about 37° C., about 39° C., about 41° C., about 43° C., about 47° C., about 53° C., or about 55° C. It has been found that the production of the major byproduct dsDNA decreases with increasing temperature. dsRNA can be recognized by cytoplasmic sensors such as RIG-I and MDA5, and then activate the innate immune system (Wu et al., 2020, "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription, RNA 26, 345-360; Olejniczak, 2010, "Sequence-non-specific effects of RNA interference triggers and microRNA regulators, Nucleic Acids Res 38, 1-16). Since the production of ds RNA should be reduced as much as possible, the preferred temperature is above 37°C. In some embodiments, in vitro transcription of template DNA and circularization of precursor RNA (i.e., self-splicing) is carried out at a temperature above 37°C, e.g., from 39°C to 55°C, from 41°C to 55°C, from 43°C to 55°C, from 39°C to 50°C, from 41°C to 50°C, from 43°C to 50°C, from 39°C to 47°C, from 41°C to 47°C, from 43°C to 47°C, from 47°C to 55°C, from 50°C to 55°C, from 39°C to 43°C, about 39°C, about 41°C, about 43°C, about 47°C, about 53°C, or about 55°C.

[0150] If the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) are performed at high temperatures, a genetically modified RNA polymerase (e.g., T7 Toyobo) that exhibits increased thermal stability may be preferred. In some embodiments, the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) are performed at a temperature of from 47°C to 55°C, such as from 50°C to 55°C, about 47°C, about 53°C, or about 55°C, and the RNA polymerase is a thermostable polymerase (e.g., T7 Toyobo).

[0151] The in vitro transcription of template DNA and the cyclization of precursor RNA (that is, from splicing) can be carried out for at least 1 hour, for example, at least 1.5 hours, at least 2.5 hours, at least 3 hours, from 1 hour to 3 hours, from 1.5 hours to 3 hours, from 2 hours to 3 hours or from 2.5 hours to 3 hours. The reaction time is preferably not less than 1.5 hours, to ensure sufficient cyclization. On the other hand, extending the reaction time is likely to increase by-products. Therefore, the optimal reaction time of one-step process can be 2.5-3 hours. In a preferred embodiment, the in vitro transcription of template DNA and the cyclization of precursor RNA (that is, from splicing) are carried out for 2.5-3 hours.

[0152] In some embodiments, the method further comprises the step of removing the DNA template after RNA self-splicing. The DNA template can be removed by adding deoxyribonuclease I, for example, at 37°C for 30 minutes.

[0153] In some embodiments, if the method includes the step of removing the DNA template, the method also includes the step of purifying the circular RNA after RNA self-splicing or after the step of removing the DNA template. In some embodiments, the purification step is selected from a precipitation step, a tangential flow filtration step and a chromatography step, and a combination thereof. The precipitation step can be an alcohol precipitation step or a LiCl precipitation. The tangential flow filtration step can be a diafiltration step performed using tangential flow filtration and / or a concentration step performed using tangential flow filtration. The chromatography step can be selected from HPLC, anion exchange chromatography, affinity chromatography, hydroxyapatite chromatography, magnetic bead chromatography and core bead chromatography. In some embodiments, the purification step includes a precipitation step, such as a LiCl precipitation. In other embodiments, the purification step includes a chromatography, such as a magnetic bead chromatography.

[0154] Example

[0155] Plasmid construction

[0156] The plasmid containing the circRNA precursor sequence was used as a template for IVT (in vitro transcription). The circRNA precursor sequence was designed based on the type I intron system described by Wesselhoeft et al. (Wesselhoeft et al., 2018, “Engineering ring RNA for potent and stable Translation in eukaryotic cells, Nature Communications 9, 2629). The insert sequence containing a coxsackievirus B3 (CVB3) IRES (internal ribosome entry site), a GFP sequence and two short regions corresponding to exon fragments (E1 and E2) is flanked by the 3' and 5' introns of the replacement type I catalytic intron from the Anabaena precursor tRNA gene. The circRNA precursor sequence (SEQ ID NO: 1) was chemically synthesized and cloned into an expression vector containing a T7 polymerase promoter (Genscript). The schematic diagram of the circRNA precursor used in the experiment is shown below.

[0157] Traditional method for circular RNA synthesis

[0158] Linearized plasmid DNA was used as a template for in vitro transcription. The plasmid containing the circRNA precursor sequence was linearized by XbaI digestion, and the circRNA precursor was synthesized by in vitro transcription from the linearized plasmid DNA template using T7 RNA polymerase. The reaction mixture (20 μL in total) was prepared as follows: 1U / μL ribonuclease inhibitor (Novoprotein E125), 6.67mM ATP, 20mM GTP, 6.67mM CTP, 6.67mM UTP, 1X transcription buffer (Novoprotein GMP-EB121, containing 6mM MgCl2), 10mM DTT (Sigma 43816), 4U / mL inorganic pyrophosphatase (Novoprotein GMP-M036), 5mM NaCl (Invitrogen AM9760G), 20mM MgCl2 (Invitrogen M1028), 5U / μL T7 RNA polymerase (Novoprotein GMP-E121), 25ng / μL linearized plasmid. In vitro transcription was performed at 37°C for 3 hours, and then the reaction mixture was treated with deoxyribonuclease I (Novoprotein GMP-E127) at 37°C for 30 minutes to remove the DNA template. After deoxyribonuclease I treatment, the synthesized precursor RNA was purified by precipitation with 7.5 M LiCl.

[0159] The cyclization of the precursor RNA was carried out according to the method described by Wesselhoeft et al. under modified conditions. The cyclization reaction (20 μL) was carried out by subjecting 6 μg of the precursor RNA directly to the cyclization reaction. Alternatively, the precursor mRNA was preheated to 70°C for 5 minutes and immediately placed on ice for 5 minutes. GTP was added to the precursor RNA together with a magnesium-containing buffer (50 mM Tris HCl, 10 mM MgCl2, 1 mM DTT, pH 7.5) to a final concentration of 2 mM. The reaction mixture was incubated at 55°C for 12 minutes, and then the RNA was purified with a column. The RNA product was evaluated by a fragment analyzer.

[0160] The precursor RNA is circularized by a self-splicing reaction that requires GTP and magnesium ions. This process depends on the structure of the group I intron. Figure 2 As shown, when the precursor RNA was directly subjected to circularization without preheating, about 60% of the RNA was circularized, and when the precursor RNA was preheated at 70°C before circularization, a circularization efficiency of about 70% was achieved, indicating that pre-denaturation of the precursor RNA is conducive to the formation of the structure required for group I intron self-splicing. These results show that the traditional method requires high-temperature pre-denaturation of the LiCl-purified IVT product before circularization to promote the circularization of the precursor RNA.

[0161] Circular RNA synthesis via a one-step process

[0162] Linearized plasmid DNA was used as a template for in vitro transcription. The plasmid containing the circRNA precursor sequence was linearized by XbaI digestion. The one-step reaction mixture (20 μL in total) was prepared as follows: 1U / μL ribonuclease inhibitor (Novoprotein E125), 10mM ATP, 10mM GTP, 10mM CTP, 10mM UTP, 1X transcription buffer (Novoprotein GMP-EB121; containing 6mM MgCl2), 10mM DTT (Sigma 43816), 4U / mL inorganic pyrophosphatase (Novoprotein GMP-M036), 5mM monovalent cation (Na + or K +), MgCl2 (Invitrogen M1028) ranging from 20mM to 80mM, 5U / μL T7 RNA polymerase (KactusBio GMP-T7P-EE101-12), 25ng / μL linearized plasmid. The reaction was carried out at 37°C for 3 hours, and then the reaction mixture was treated with DNase I (Novoprotein GMP-E127) at 37°C for 30 minutes to remove the DNA template. After DNase I treatment, RNA was purified by precipitation with 7.5M LiCl. RNA products were evaluated by fragment analyzer.

[0163] According to previous studies (Wesselhoeft et al., 2018), 2 mM GTP and 10 mM MgCl2 are required for circularization of IVT-derived precursor RNAs via the self-splicing process of group I introns, whereas the conventional IVT system contains GTP (here 10 mM) and MgCl2 (here 26 mM). Figure 3 A and 3B show that the synthesized precursor RNA cannot be efficiently circularized under traditional IVT conditions (10 mM GTP and 26 mM MgCl2). Only 7.5% of the precursor RNA was circularized under this condition, indicating that traditional IVT conditions need to be optimized for the circularization of precursor RNA.

[0164] In order to study Mg 2+ Effect of concentration on precursor RNA circularization, changing Mg in a one-step IVT / circularization system 2+ concentrations while the other components were fixed (Table 1).

[0165] Table 1. 2+ One-step IVT / cyclization system (20 μL)

[0166]

[0167]

[0168] The final Mg of the test 2+ The concentrations included 26mM, 36mM, 46mM, 56mM, 66mM and 86mM, and each NTP concentration was adjusted to 10mM (A:G:C:U=1:1:1:1). The RNA product was evaluated by a fragment analyzer. The results are shown in Figure 3 A and Figure 3 B. The results showed that Mg 2+ Increasing the concentration from 26mM to 36mM promoted the circularization of circRNA precursors. 2+ Only 7.5% of the precursor RNA was circularized at 36 mM Mg 2+41.8% of the precursor RNA was circularized under Mg 2+ Further increase in concentration to 86 mM did not significantly change the efficiency of cyclization.

[0169] In the one-step IVT / cyclization system, different Mg 2+ Total RNA yield at concentration ( Figure 3 C) Mg 2+ Increasing the concentration from 26 mM to 36 mM not only promoted the circularization of circRNA precursors but also increased the RNA yield. 2+ Increasing the concentration to 86 mM significantly reduced RNA production.

[0170] In the one-step IVT / circularization process, the circularization of the precursor RNA can occur in a co-transcriptional and / or post-transcriptional manner. We investigated the circularization efficiency and yield at different reaction time points to address this question. 2+ The other IVT conditions were the same as those in Table 1 except that the concentration was changed to 38 mM. The reaction was carried out at 37°C for 1 h, 1.5 h, 2 h, 2.5 h, 3 h, or 3.5 h, and then the reaction mixture was treated with DNase I (Novoprotein GMP-E127) for 30 min at 37°C to remove the DNA template. After DNase I treatment, RNA was purified by precipitation with 7.5 M LiCl.

[0171] The fragment analysis results showed that extending the reaction duration to 2.5 h could improve the efficiency of precursor RNA cyclization ( Figure 4A ). However, the process was almost complete at 2.5 hours. Reaction time exceeding 2.5 hours did not improve the cyclization efficiency. The effect of reaction time on total RNA yield was also studied. The results showed that the yield exceeded 200 μg / reaction when the reaction was carried out for 1 hour, and further extension of the reaction duration did not significantly increase the yield ( Figure 4B ). Prolonging the reaction time may increase the by-products. Therefore, the optimal reaction duration of the one-step process is 2.5-3 hours.

[0172] Optimization of one-step cyclization system

[0173] In order to comprehensively evaluate the factors affecting IVT and cyclization in the one-step process, a multi-level crossover experiment was designed using software targeting four factors: temperature, Mg 2+ The concentration, pyrophosphatase concentration and reaction temperature were calculated. The cyclization efficiency was checked by fragment analyzer, and the yield of total RNA was calculated by measuring the product concentration. The results are shown in Table 2. All data were entered into the software for factorial design analysis.

[0174] Table 2. Factorial Design of Experiments (DOE) for One-Step Cyclization System

[0175]

[0176]

[0177]

[0178] According to the standardized effect analysis, magnesium ion concentration and temperature were the main factors affecting IVT yield, and the interaction between reaction temperature and T7 RNAP type also affected the yield ( Figure 5A Elevated temperatures (above 37°C) will reduce overall IVT yield ( Figure 5B ). 2+ Increasing the concentration from 26 mM to 38 mM significantly increased the average IVT yield, but the Mg 2+ Further increases in concentration did not increase the average yield under all conditions tested ( Figure 5B Adding pyrophosphatase to the reaction increased the yield ( Figure 5B ). It is believed that this is because pyrophosphatase helps maintain a stable magnesium ion concentration throughout the IVT process. The type of T7 RNAP used in IVT had little effect on the average yield of all tested groups, but the effect of temperature on IVT yield depended significantly on the type of T7 RNAP used in IVT (Figure 6). When TOYOBO T7 RNAP (a thermostable T7 RNA polymerase) was used in IVT, the average IVT yield was lowest at 37°C, and the average IVT yields at high temperatures (47, 53, or 55°C) were approximately the same. In contrast, when KACTUS T7 RNAP was used in IVT, the average IVT yield was highest at 37°C, and the average IVT yield was greatly reduced at high temperatures (47, 53, or 55°C), indicating that the enzyme activity of KACTUS T7 RNAP is reduced under high temperature conditions.

[0179] For the cyclization efficiency, Mg 2+ concentration, reaction temperature, pyrophosphatase concentration, and Mg 2+ The interaction with pyrophosphatase affects the circularization of precursor RNA ( Figure 7 A). Increasing the temperature from 37°C to 47°C promoted cyclization, but the efficiency of cyclization decreased at higher temperatures (53°C and 55°C) ( Figure 7 B) Mg 2+ Increasing the concentration from 26 mM to 66 mM promoted cyclization, but higher Mg 2+ concentrations (76 mM and 96 mM) did not further improve the cyclization efficiency ( Figure 7B). Adding pyrophosphatase to the IVT reaction can promote cyclization, but the concentration of pyrophosphatase is not positively correlated with the average cyclization efficiency. The cyclization efficiency is highest when 2 U / mL pyrophosphatase is used ( Figure 7 B) It is worth noting that when Mg 2+ When combined with pyrophosphatase, the cyclization can be further promoted (Figure 8). After multi-factor optimization, the one-step process can achieve a cyclization efficiency similar to that of the traditional process. 2+ When the concentration was 38 mM, the pyrophosphatase concentration was 2 U / mL, and the reaction temperature was 47°C, the cyclization efficiency could be as high as 74.9% (Table 2).

[0180] One of the major byproducts identified in the one-step process is dsRNA, which can be recognized by cytoplasmic sensors such as RIG-I and MDA5, thereby activating the innate immune system. The amount of dsRNA produced under IVT conditions with different temperatures and magnesium concentrations was examined. Antibody-dependent fluorescence resonance energy transfer (FRET) assay was used to detect dsRNA in IVT samples. The specific method was performed according to the kit manual (Cisbio 64RNAPEG). The results are shown in Table 3 and Fig. 9 The results showed that temperature was a factor that directly affected the production of dsRNA (Table 3 and Figure 13). The production of dsRNA was inhibited as the temperature increased, while Mg 2+ The concentration had no significant effect on dsRNA production.

[0181] Table 3. Temperature and Mg 2+ Effect of concentration on dsRNA content

[0182]

[0183]

[0184] Although the present disclosure has been described with respect to specific embodiments, including currently preferred modes of carrying out the present disclosure, it will be appreciated by those skilled in the art that there are numerous variations and permutations of the above-described systems and techniques. It will be appreciated that other embodiments may be utilized and that modifications may be made to structure and function without departing from the scope of the present disclosure. Therefore, the scope of the present disclosure should be interpreted broadly as set forth in the appended claims.

[0185] Sequence Listing

[0186] SEQ ID No: 1: Precursor sequence

[0187]

Claims

1. A method for preparing circular RNA, comprising providing a template DNA in a reaction solution, wherein the template DNA comprises a sequence encoding a precursor RNA, so as to allow the precursor RNA to be synthesized by in vitro transcription of the template DNA, and to allow the precursor RNA to self-splicing, thereby producing circular RNA, wherein the in vitro transcription of the template DNA and the self-splicing of the precursor RNA are performed in the same reaction solution under the same reaction conditions.

2. The method of claim 1, wherein the method does not include a step of purifying the precursor RNA prior to allowing the precursor RNA to self-splicing.

3. The method of any preceding claim, wherein the DNA template comprises the following elements operably linked to each other and arranged in the following order: an RNA polymerase promoter, optionally a 5' homology arm, a 3' type I intron fragment containing a 3' splice site dinucleotide, optionally a 5' spacer sequence, an insert sequence, optionally a 3' spacer sequence, a 5' type I intron fragment containing a 5' splice site dinucleotide, and optionally a 3' homology arm.

4. The method of claim 3, wherein the insert sequence comprises a protein coding sequence, and wherein the insert sequence comprises an IRES sequence operably linked to the protein coding sequence.

5. A method as claimed in any preceding claim, wherein the reaction solution comprises Mg at a concentration greater than 26 mM. 2+ .

6. A method as described in any of the preceding methods, wherein the reaction solution contains Mg at a concentration greater than 35 mM. 2+ , optionally wherein the Mg in the solution 2+ The concentration was from 38 mM to 66 mM.

7. A method as claimed in any preceding claim, wherein the reaction solution comprises pyrophosphatase at a concentration of from 1 U / ml to 5 U / ml, from 1 U / ml to 4 U / ml, from 1.5 U / ml to 3 U / ml, from 1.5 U / ml to 2.5 U / ml, about 1 U / ml, about 2 U / ml or about 4 U / ml.

8. A method as claimed in any preceding claim, wherein the reaction solution comprises 38-66 mM Mg 2+ , optionally 1-4 U / ml pyrophosphatase, RNA polymerase, ribonuclease inhibitor, ATP, GTP, CTP, UTP, DTT and monovalent cations (Na + or K + ).

9. The method of any preceding claim, wherein the in vitro transcription of the template DNA and circularization (ie, self-splicing) of the precursor RNA are performed at a temperature of from 37°C to 55°C.

10. A method as claimed in any preceding claim, wherein the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) are performed at a temperature above 37°C, optionally wherein the in vitro transcription of the template DNA and the circularization of the precursor RNA (i.e., self-splicing) are performed at a temperature from 39°C to 50°C.

11. A method as claimed in any preceding claim, wherein the in vitro transcription of the template DNA and the circularization (i.e., self-splicing) of the precursor RNA are carried out for at least 1 hour, optionally wherein the in vitro transcription of the template DNA and the circularization (i.e., self-splicing) of the precursor RNA are carried out for at least 2.5-3 hours.

12. The method of any preceding claim, wherein the method further comprises the step of removing the DNA template after the precursor RNA synthesis, optionally wherein the DNA template is removed by adding deoxyribonuclease I, for example at 37°C for 30 minutes.

13. The method of claim 12, wherein the method further comprises a step of purifying the circular RNA after the step of removing the DNA template.

14. The method of claim 13, wherein the purification step is selected from the group consisting of a precipitation step, a tangential flow filtration step, and a chromatography step, and combinations thereof.

15. A reaction solution for one-step circular RNA synthesis (e.g., for use in a method according to any preceding claim), the reaction solution comprising Mg2+ at a concentration greater than 26 mM. + , optionally pyrophosphatase (e.g., 1-4 U / ml pyrophosphatase), RNA polymerase, ribonuclease inhibitor, nucleoside triphosphates (e.g., ATP, GTP, CTP, and UTP), a reducing agent (e.g., DTT), and a monovalent cation (e.g., selected from Na + , K + and combinations thereof).

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