Cyclic RNA precursor, preparation method thereof, and cyclic RNA obtained thereby

Abstract

The invention provides a circular RNA precursor, a preparation method thereof, and circular RNA obtained thereby. Specifically, the invention provides a preparation method of a circular RNA precursor, the method comprises a step of carrying out in vitro transcription on a transcription reaction system containing a buffer solution, an enzyme required by a transcription reaction, NTP, polynucleotide and a DNA template, the polynucleotide has a structure shown as a formula (I), and each group in the formula is defined in the specification. By adopting the circular RNA prepared by the invention, the technical effects of reducing immunogenicity and realizing high translation can be realized; and meanwhile, two basic group modification strategies can be realized in one ring RNA molecule. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to circular RNA precursors, methods for their preparation, and circular RNAs obtained therefrom. Background Art

[0002] Circular RNAs (or circRNAs or circular RNAs) are single-stranded RNAs that, unlike linear RNAs, form a covalently closed loop. In circular RNAs, the 3' and 5' ends that are normally present in RNA molecules have been joined together. These features endow circular RNAs with many properties, many of which have only recently been discovered.

[0003] Since circular RNAs do not have 5' or 3' ends, they are resistant to exonuclease-mediated degradation and are more stable in cells than most linear RNAs.

[0004] Many types of circular RNAs are generated from other protein-coding genes. Some circular RNAs have been shown to encode proteins. Some types of circular RNAs have also recently shown potential as gene regulators. The biological functions of most circular RNAs are still unclear.

[0005] The application limitation for protein-coding circular RNAs is the in vitro production of circular RNAs, especially the circularization step after obtaining linear RNAs by in vitro transcription. The most commonly used circularization methods are:

[0006] 1) Ligation of the ends of linear RNAs by ligases (Prisca Obi, Y. Grace Chen Methods. 2021 Dec;196:85-103).

[0007] The existing enzymatic circularization technology is to obtain linearized RNA containing 5’PPP by in vitro transcription, then remove 5’PPP by phosphatase to obtain RNA with 5’OH, and then use T4 phosphokinase to obtain linear RNA with 5’ monophosphate (5’P); or use linearized RNA with 5’PPP as a substrate and add Escherichia coli pyrophosphohydrolase (RppH) to obtain linear RNA with 5’ monophosphate (5’P); or add guanosine monophosphate (GMP) during in vitro transcription to obtain linear RNA containing (5’P); finally, a ligase is selected to circularize the RNA.

[0008] The process of adding guanosine monophosphate (GMP) during in vitro transcription to obtain linear RNA containing (5’P) is relatively simple and convenient, requiring only one-step reaction. However, since GTP and GMP compete for binding to the enzyme during the initiation of transcription by RNA polymerase, some linear RNA containing 5’-PPP will still be generated. To increase the proportion of linear RNA with 5’ monophosphate (5’P) in the reaction products, the amount of GMP added needs to be 5 times that of GTP, which can ensure that more than 80% of the products are linear RNA containing (5’P). However, the problems faced are: (a) there is still more than 10% or 20% of linear RNA with 5’-PPP that cannot be circularized and has strong immunogenicity; (b) the yield of RNA transcription reaction decreases.

[0009] In addition to the above methods, linear RNA with 5’ monophosphate (5’P) can also be obtained through post-transcriptional phosphatase, T4 kinase, or RppH enzyme. However, its process is relatively complex, requiring one or two additional steps of reaction, and purification or solution replacement also needs to be added in the middle to obtain the optimal dissolution conditions for the subsequent reaction.

[0010] 2) Using chemical methods, such as click chemistry, by adding a phosphate activation reagent such as cyanogen bromide, or introducing a special functional group to cyclize and form a non-natural bond. Chemical cyclization still faces difficulties such as low yield and difficulty in obtaining a linear RNA precursor containing a special functional group first. Especially for obtaining circular RNA with protein translation function, such as those with a length greater than 1000 nt, no successful examples have been reported so far.

[0011] 3) Circular RNAs linked head-to-tail are obtained through ribozyme-catalyzed RNA splicing reactions. Commonly used ribozymes include group I and group II introns or their variant PIE systems. During the preparation of circular RNAs by this method, linear RNA contaminants containing triphosphates are generated, and intron dimers are produced after splicing during the circularization process. Even after treatment with RNase R, the immunostimulatory response cannot be completely eliminated, thereby reducing the intracellular translation of circular RNAs. Only through HPLC purification and collection of the late circRNA fraction can the immunogenicity be significantly reduced and the intracellular translation effect be improved. In the prior art, linear RNAs reduce the generation of immunogenicity by modifying bases (such as replacing all uracils with N1-methylpseudouridine). For circular RNAs obtained by ribozyme catalysis, if all uracils in the circRNA are replaced with N1-methylpseudouridine, the cleavage activity of the ribozyme will be inhibited, blocking the occurrence of the circularization reaction. If the ligation enzyme method is used to construct circRNAs, N1-methylpseudouridine-modified circRNAs can be obtained. After transfection into cells, it is found that although the immunogenicity is significantly decreased, the N1-methylpseudouridine-modified circRNAs lose their protein translation and expression ability. Circular RNAs modified by m6A in eukaryotic cells are recognized as the cell's own circular RNAs and are not immunogenic; while unmodified circular RNAs are recognized as foreign circular RNAs and are immunogenic. Similarly, for full-length RNA precursors containing IRES, even 10% m6A substitution of natural bases will reduce the ribozyme activity, thereby reducing the circularization efficiency; the circular RNAs obtained after purification have a lower translation efficiency compared to unmodified circular RNAs. For circular RNAs without IRES modified with 10% m6A, there is almost no translation in cells (R Alexander Wesselhoeft et al., Mol Cell., 2019 May 2; 74(3): 508-520.e4). Ribozymes need to have specific secondary and tertiary structures to be active. Similarly, IRES sequences from viral and eukaryotic genomes also need to have specific secondary and tertiary structures to initiate translation. The base modification of RNA can affect the secondary and tertiary structures of RNA. While the modified RNA eliminates immunogenicity, it also loses the ability to circularize and translate; the coding region of circular RNAs modified by m6A also reduces the translation efficiency.

[0012] Therefore, there is a need in the art for a preparation process of circular RNAs that can obtain low immunogenicity and high translation activity without purification. Summary of the Invention

[0013] The present invention is an innovative invention for the preparation of circular RNA precursors. The circular RNA prepared by the present invention can achieve the technical effects of reducing immunogenicity and high translation; at the same time, a strategy of two base modifications can be achieved in one circular RNA molecule.

[0014] Specifically, in the first aspect of the present invention, a method for preparing a circular RNA precursor is provided. The method includes the step of performing in vitro transcription on a transcription reaction system containing a buffer, enzymes required for the transcription reaction, NTPs, a polynucleotide, and a DNA template. The polynucleotide has a structure represented by the following formula (I):

[0015]

[0016] In the formula:

[0017] N1 is 0 or 1;

[0018] B1 to B n are each independently a nucleobase;

[0019] H1 to H n-2 is 0 or 1;

[0020] M1 to M n-1 are each independently -O-, -S-, -NH-, -CH2-, -C(halogen group)2-, or -CH(halogen group)-;

[0021] M n is a hydroxyl group;

[0022] R1 to R n are each independently hydrogen, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxy-substituted alkyl group, -S-alkyl, -NH-alkyl, -O-aryl, -S-aryl, -NH-aryl, a halogen group, and a locked nucleic acid modification;

[0023] X1 to X n are each independently -O-, -S-, -NH-, -CH2-, -C(halogen group)2-, or -CH(halogen group)-;

[0024] Y1 to Y n are each independently oxygen, sulfur, selenium, or boranyl;

[0025] Z1 to Z n are each independently a hydroxyl group, a mercapto group, a boranyl group, an aryl group, an alkyl group, an alkoxy group, or -O-aryl;

[0026] n is 5;

[0027] The polynucleotide satisfies at least one of the following characteristics a) to e): a) The last two bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream +1 and +2 positions of the promoter of the transcription template strand respectively; b) The last two bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream -1 and +1 positions of the promoter of the transcription template strand respectively; c) The last three bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream +1, +2 and +3 positions of the promoter of the transcription template strand respectively; d) The last three bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream -1, +1 and +2 positions of the promoter of the transcription template strand respectively; e) The last four bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream -1, +1, +2 and +3 positions of the promoter of the transcription template strand respectively; and

[0028] At least one of the first, second and third nucleosides at the 5'-end of the polynucleotide has a modification selected from glycosyl modification and base modification.

[0029] The second aspect of the invention provides a circular RNA precursor, which is a linear RNA and contains the polynucleotide described herein.

[0030] The third aspect of the invention provides a method for preparing circular RNA, which includes the step of performing a cyclization reaction on the circular RNA precursor described herein.

[0031] The fourth aspect of the invention provides a circular RNA molecule containing the polynucleotide described herein.

[0032] The fifth aspect of the invention provides the applications of the polynucleotide, circular RNA precursor and circular RNA molecule having the structure shown in formula (I) herein. Description of the Drawings

[0033] Figure 1 : Polynucleotide pA2' Ome Chromatogram of pG.

[0034] Figure 2 : Capillary electrophoresis analysis chart of the linearized RNA precursor in Example 3.

[0035] Figure 3 : Capillary electrophoresis analysis chart of the circular RNA in Example 3.

[0036] Figure 4 : Circularization rate chart of No. 1 in Example 3.

[0037] Figure 5 : Circularization rate chart of No. 13 in Example 3.

[0038] Figure 6: Gel electrophoresis pattern of reaction intermediates for generating circular RNAs using the Anabeana PIE system in Example 4. Lane 1: RNA precursor; Lane 2: Product obtained after cyclization of the RNA precursor; Lane 3: Product obtained after adding RNase R treatment to the RNA precursor after cyclization; Lane 4: RNA precursor generated by adding pA 2MOE pG (in Lane 4, the RNA has already started to cyclize during the IVT reaction, so in addition to the RNA precursor band, there is also a circular RNA band); Lane 5: RNA precursor generated by adding pA 2MOE pG and obtained product after cyclization; Lane 6: Product obtained after adding RNase R treatment to the RNA precursor generated by adding pA 2MOE pG after cyclization. M: RNA ladder (Thermo, SM1823).

[0039] Figure 7 : HPLC analysis pattern of each reaction intermediate during the process of generating circular RNAs using the Anabeana PIE system in Example 4. The product labels are the same as Figure 6 those above.

[0040] Figure 8 : Capillary electrophoresis analysis pattern of the RNA precursor for generating circular RNAs using the Anabeana PIE system in Example 4.

[0041] Figure 9 : Capillary electrophoresis analysis pattern of the product obtained after cyclization of the RNA precursor in Example 4.

[0042] Figure 10 : Chromatogram of the product obtained after purification by ion exchange liquid chromatography.

[0043] Figure 11 : Expression of related immune factors (RIG-1, IFNβ, TNF, and IL-6) caused by circular RNAs #1 - #5, #9, #12 - #23 before purification and after HPLC purification in A549 cells.

[0044] Figure 12 : GFP expression intensity of circular RNAs #1 - #5, #9, #12 - #23 before purification and after HPLC purification in HEK293T cells detected by flow cytometry analysis. Flow cytometry analysis was used to detect the GFP expression intensity of circular RNAs prepared by different methods in HEK293T cells.

[0045] Figure 13 : Fluorescence imaging results at 6 hr, D3, and D10 after intramuscular injection in mice in Example 11. Detailed implementation methods

[0046] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the embodiments) can be combined with each other to form preferred technical solutions.

[0047] Polynucleotide

[0048] As used herein, the term "polynucleotide" refers to a polymer formed by the polycondensation of more than 2 nucleotide monomers through phosphodiester bonds. The phosphodiester bond is usually formed by the esterification of the hydroxyl group on the 3'-carbon of a nucleoside sugar ring with the 5'-phosphate of another nucleoside monomer. Exemplary polynucleotides include dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, etc. A dinucleotide is a dimer formed by the binding / condensation of two monomers through a phosphodiester bond, a trinucleotide is a trimer formed by the binding / condensation of three monomers through a phosphodiester bond, and so on.

[0049] In some embodiments, the polynucleotide used herein has the structure shown in formula (I):

[0050]

[0051] Wherein:

[0052] N1 is 0 or 1;

[0053] B1 to B n are each independently a nucleobase;

[0054] H1 to H n-2 are 0 or 1;

[0055] M1 to M n-1 are each independently -O-, -S-, -NH-, -CH2-, -C(halogen group)2- or -CH(halogen group)-;

[0056] M n is a hydroxyl group;

[0057] R1 to R n are each independently hydrogen, hydroxyl, alkyl, alkoxy, alkoxy-substituted alkyl, -S-alkyl, -NH-alkyl, -O-aryl, -S-aryl, -NH-aryl, halogen group and locked nucleic acid modification;

[0058] X1 to X n are each independently O, S, -NH-, -CH2-, -C(halogen group)2- or -CH(halogen group)-;

[0059] Y1 to Y n are each independently oxygen, sulfur, selenium or boranyl;

[0060] Z1 to Z nEach is independently a hydroxyl group, a mercapto group, a boranyl group, an aryl group, an alkyl group, an alkoxy group or -O-aryl;

[0061] n is 5.

[0062] In this text, halogen includes F, Cl, Br and I; a halogenated group refers to a monovalent group formed by a halogen, namely -F, -Br, -Cl and -I.

[0063] In this text, as a group or as part of other groups, the term "alkyl" refers to a fully saturated straight-chain or branched-chain hydrocarbon chain group, consisting only of carbon atoms and hydrogen atoms, having, for example, 1 to 12 (such as 1 to 8, 1 to 6 or 1 to 4) carbon atoms, and being connected to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, etc.

[0064] In this text, an alkoxy group refers to an alkyl group connected to an oxygen group, that is, an R-O-group, where R represents an alkyl group, such as a C1-C12 alkyl group, a C1-C8 alkyl group, a C1-C6 alkyl group or a C1-C4 alkyl group. Exemplary alkoxy groups include methoxy and ethoxy, etc.

[0065] In this text, as a group or as part of other groups, the term "aryl" means a conjugated hydrocarbon ring system group having 6 to 18 carbon atoms (preferably having 6 to 14 carbon atoms, more preferably having 6 to 10 carbon atoms). An aryl group can be a monocyclic, bicyclic, tricyclic or more-ring ring system. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl, etc.

[0066] In this text, nucleobases include natural nucleobases and modified nucleobases. Natural bases include adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). In this text, the modification of nucleobases can be various modifications well-known in the art, including but not limited to alkylation substitutions at the N1 and N6 positions, such as substitution of N1 by a methyl group and / or substitution of N6 by a methyl group; modification of the 5C position of C and U bases, such as alkylation or alkoxylation substitution; and substitution of O by S, that is, thio modification; and so on. Exemplary modified nucleobases include, but are not limited to, pseudouridine, dihydrouridine, inosine, 7-methyluridine, N6-methyladenosine, N1-methyladenosine, 5me-C, 5me-U, 5OMe-U, 2S-U and methylcytosine, pseudoisocytidine (pseudo-isoC), N1-methylpseudoisocytidine (N1me pseudo-isoC), etc.

[0067] In the polynucleotide represented by Formula I of the present invention, at least one of the first, second, and third nucleosides at the 5'-end (i.e., the end where base B1 is located in Formula I) has a modification selected from glycosyl modification and base modification. In some embodiments, among the first, second, and third nucleosides at the 5'-end, at least one of R1, R2, and R3 is neither H nor a hydroxyl group.

[0068] In some embodiments, B1 to B n are each independently selected from: cytosine, N1-methyluracil, N6-methyladenine, and uracil. In some embodiments, at least one of B1 to B n is N6-methyladenine.

[0069] In some embodiments, M1 to M n-1 are each preferably -O-.

[0070] In some embodiments, R1 to R n-1 are each independently selected from: hydroxyl group, methyl group, ethyl group, methoxy group, ethoxy group, and methoxyethyl group. In some embodiments, R n is a hydroxyl group. In some embodiments, when n≥3, at least one of R1, R2, and R3 is not a hydroxyl group.

[0071] In some embodiments, X1 to X n are each independently -O-.

[0072] In some embodiments, Y1 to Y n are each independently selected from O and S.

[0073] In some embodiments, Z1 to Z n are each independently selected from hydroxyl groups.

[0074] In some embodiments, the polynucleotide is a dinucleotide, and its structure is shown in Formula I-1 as follows:

[0075]

[0076] Wherein:

[0077] N1 is 0 or 1;

[0078] R1 and R2 are each independently hydrogen, hydroxyl group, alkyl group, alkoxy group, S-alkyl group, NH-alkyl group, O-aromatic group, S-aromatic group, NH-aromatic group, halogenated group, and locked nucleoside modification;

[0079] X1 and X2 are each independently -O-, -S-, -NH-, -CH2-, -C(halogenated group)2-, or -CH(halogenated group)-;

[0080] Y1 and Y2 are each independently oxygen, sulfur, selenium or boranyl;

[0081] Z1 and Z2 are each independently hydroxyl, mercapto, boranyl, aryl, alkyl, alkoxy or O-aryl;

[0082] M1 is -O-, -S-, -NH-, -CH2-, -C(halo)2- or -CH(halo)-;

[0083] M2 is hydroxyl;

[0084] B1 and B2 are each independently a natural or modified nucleobase.

[0085] In some embodiments of Formula I-1, B1 to B2 are each independently selected from: cytosine, N1-methyluracil, uracil.

[0086] In some embodiments of Formula I-1, M1 is -O-.

[0087] In some embodiments of Formula I-1, R1 is selected from: hydroxyl, methyl, ethyl, methoxy, ethoxy and methoxyethyl. In some embodiments of Formula I-1, R2 is hydroxyl. In some embodiments of Formula I-1, R1 is selected from methyl, ethyl and methoxyethyl; R2 is hydroxyl.

[0088] In some embodiments of Formula I-1, X1 to X2 are each independently -O-.

[0089] In some embodiments of Formula I-1, Y1 to Y2 are each independently selected from O and S.

[0090] In some embodiments of Formula I-1, Z1 to Z2 are each independently selected from hydroxyl.

[0091] In some embodiments, the polynucleotide can be a trinucleotide, and its structure is shown in the following formula (I-2):

[0092]

[0093]

[0094] Wherein:

[0095] N1 is 0 or 1;

[0096] R1, R2, and R3 are each independently hydrogen, hydroxyl, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl, halo and locked nucleic acid modification;

[0097] X1, X2, and X3 are each independently -O-, -S-, -NH-, -CH2-, -C(halo)2-, or -CH(halo)-;

[0098] Y1, Y2, and Y3 are each independently oxygen, sulfur, selenium, or boranyl;

[0099] Z1, Z2, and Z3 are each independently a hydroxyl group, a mercapto group, a boranyl group, an aryl group, an alkyl group, an alkoxy group, or an O-aryl group;

[0100] M1 and M2 are -O-, -S-, -NH-, -CH2-, -C(halo)2-, or -CH(halo)-;

[0101] M3 is a hydroxyl group;

[0102] B1, B2, and B3 are each independently a natural or modified nucleobase.

[0103] In some embodiments of Formula I-2, B1 to B3 are each independently selected from: cytosine, N1-methyluracil, and uracil.

[0104] In some embodiments of Formula I-2, M1 to M2 are each preferably -O-.

[0105] In some embodiments of Formula I-2, M3 is OH.

[0106] In some embodiments of Formula I-2, R1 to R2 are each independently selected from: hydroxyl group, methyl group, ethyl group, methoxy group, ethoxy group, and methoxyethyl group. In some embodiments of Formula I-2, R3 is a hydroxyl group. In some embodiments of Formula I-2, one of R1 and R2 is not a hydroxyl group.

[0107] In some embodiments of Formula I-2, X1 to X3 are each independently -O-.

[0108] In some embodiments of Formula I-2, Y1 to Y3 are each independently selected from O and S.

[0109] In some embodiments of Formula I-2, Z1 to Z3 are each independently selected from hydroxyl groups.

[0110] In some embodiments, the polynucleotide can be a tetranucleotide, and its structure is shown in Formula I-3 below:

[0111]

[0112] Wherein:

[0113] N1 is 0 or 1;

[0114] R1, R2, R3, and R4 are each independently hydrogen, hydroxy, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl, halo, and locked nucleic acid modification;

[0115] X1, X2, X3, and X4 are each independently -O-, -S-, -NH-, -CH2-, -C(halo)2-, or -CH(halo)-;

[0116] Y1, Y2, Y3, and Y4 are each independently oxygen, sulfur, selenium, or boranyl;

[0117] Z1, Z2, Z3, and Z4 are each independently hydroxy, mercapto, boranyl, aryl, alkyl, alkoxy, or O-aryl;

[0118] M1, M2, and M3 are -O-, -S-, -NH-, -CH2-, -C(halo)2-, or -CH(halo)-;

[0119] M4 is hydroxy;

[0120] B1, B2, B3, and B4 are each independently a natural or modified nucleobase.

[0121] In some embodiments of Formula I-3, B1 to B4 are each independently selected from: cytosine, N1-methyluracil, uracil.

[0122] In some embodiments of Formula I-3, M1 to M3 are each preferably -O-.

[0123] In some embodiments of Formula I-3, M4 is OH.

[0124] In some embodiments of Formula I-3, R1 to R3 are each independently selected from: hydroxy, methyl, ethyl, methoxy, ethoxy, and methoxyethyl. In some embodiments of Formula I-3, at least one of R1 to R3 is not hydroxy.

[0125] In some embodiments of Formula I-3, R4 is hydroxy.

[0126] In some embodiments of Formula I-3, X1 to X4 are each independently -O-.

[0127] In some embodiments of Formula I-3, Y1 to Y4 are each independently selected from O and S.

[0128] In some embodiments of Formula I-3, Z1 to Z4 are each independently selected from hydroxy.

[0129] As used herein, exemplary dinucleotides may be selected from pApG, pN6mApG, pA 2’Ome pG, pN6mA2’Ome pG, pA 2’ OET pG, pN6mA 2’OET pG, pApU, pN6meApU, pA 2’Ome pU, pN6mA 2’Ome pU, pA 2’OET pU, pN6mA 2’OET pU, pApC, pN6meApC, pA 2’Ome pC, pN6mA 2’Ome pC, pA 2’OET pC, pN6mA 2’OET pC, pApA, pN6meApA, pN6meApN6meA, pA 2’Ome pA, pA 2’OET pA, pN6meA 2’Ome pA, pN6meA 2’OET pA, pA 2’Ome pN6meA, pA 2’OET pN6meA, pN6meA 2’ Ome pN6meA, pN6meA 2’OET pN6meA, ApG, N6mApG, A 2’Ome pG, N6mA 2’Ome pG, A 2’OET pG, N6mA 2’OET pG, ApU, N6meApU, A 2’Ome pU, N6mA 2’Ome pU, A 2’OET pU, N6mA 2’OET pU, ApC, N6meApC, pA 2’Ome pC, N6mA 2’Ome pC, A 2’OET pC, N6mA 2’OET pC, ApA, N6meApA, N6meApN6meA, A 2’Ome pA, A 2’OET pA, N6meA 2’Ome pA, N6meA 2’ OET pA, A 2’Ome pN6meA, A 2’OET pN6meA, N6meA 2’Ome pN6meA, N6meA 2’OET pN6meA, pGpG, pG2‘Ome pG, pG 2’OET pG, pGpU, pG 2’Ome pU, pG 2’OET pU, pGpC, pG 2’Ome pC, pG 2’OET pC, pGpA, pG 2Ome pA, pGpN6mA, pG 2’Ome pN6mA, pUpU, pU 2’Ome pU, pU, pUpA, pU 2’Om pA, pUpN6meA, pU 2’Om pN6meA, GpG, G 2’Ome pG, G 2’OET pG, GpU, G 2’Ome pU, G 2’OET pU, GpC, G 2’Ome pC, G 2’OET pC, GpA, G 2’Ome pA, GpN6mA, G 2’Ome pN6mA, UpU, U 2’Ome pU, UpA, U 2’Ome pA, UpN6meA, U 2’Ome pN6meA and one or more of the following.

[0130] In this article, exemplary trinucleotides can be selected from pApApG, pA 2’Ome pApG, pApA 2’Ome pG, pA 2’Ome pA 2’ Ome pG, pN6mApApG, pN6mA 2’Ome pApG,pN6mApA 2’Ome pG,pN6mA 2’Ome pA 2’Ome pG, pN6mApN6mApG, pN6mA 2’Ome pN6mApG, pN6mA 2’Ome pN6mA 2’OmepG, pN6mApN6mA 2’Ome pG, pApN6mApG, pA 2’Ome N6mApG, pA 2’Ome pN6mA 2’Ome pG, pAN6mA 2’Ome pG, pApUpG, pA 2’ Ome pUpG, pApU 2’Ome pG, pA 2’Ome pU 2’Ome pG, ApApG, A 2’Ome pApG, ApA 2’Ome pG, A 2’Ome pA 2’Ome pG, N6mApApG, N6mA 2’Ome pApG, N6mApA 2’Ome pG, N6mA 2’Ome pA 2’Ome pG, N6mApN6mApG, N6mA 2’Ome pN6mApG, N6mA 2’ Ome pN6mA 2’Ome pG, N6mApN6mA 2’Ome pG, ApN6mApG, A 2’Ome N6mApG, A 2’Ome pN6mA 2’Ome pG, AN6mA 2’Ome pG, pApUpG, A 2’Ome pUpG, ApU 2’Ome pG, A 2’Ome pU 2’Ome pG, and one or more of the following.

[0131] In this article, exemplary tetranucleotides can be selected from pApApApG, and one or more of the following.

[0132] In this article, each identifier in the polynucleotide has a meaning well-known in the art. For example, A, U, G, and C represent adenine, uracil, guanine, and cytosine, respectively; "2’Ome" means that the 2’ position is substituted with a methoxy group; "N6m" and "N6me" mean that the N6 of adenosine is methylated; refers to pseudouridine; "N1me" means that the N1 of uridine is methylated; "2’OET" means that the 2’ position is substituted with an ethoxy group.

[0133] In some embodiments, the polynucleotide of the present invention may also contain modifications to the phosphate group and modifications to the sugar group. Modifications to the phosphate group include, but are not limited to, phosphorothioate (PS) modifications. Modifications to the sugar group include, but are not limited to, locked nucleic acid (LNA) modifications, 2’-methoxy (2’OMe) modifications, 2’-ethoxy (2’OET), and 2’-fluoro (2’F) modifications.

[0134] In this article, the polynucleotides described herein can be synthesized using commercially available raw materials and conventional methods. Examples of commercially available raw materials include 5’-O-DMT-2’-O-TBDMS phosphoramidites (rAAc rCAc, rGdmf, U), 2’-O-MOE-3’-O-phosphoramidites (A2’O-MOEAc, C2’O-MOEAc, G2’O-MOEdmf U2’O-MOE), and 2’-O-ethyl-3’-O-phosphoramidites (A2’O-ethyl Ac, C2’O-ethyl Ac, G2’O-ethyl dmf, U2’O-ethyl), which are from Hongene; di-cyanoethyl-N,N-diisopropyl CED phosphoramidite, which is from ChemGenes. The various dinucleotides, trinucleotides, and polynucleotides described herein can be synthesized using the well-known oligonucleotide solid-phase synthesis process in the art. For example, they can be synthesized using a Bioautomation Mermade 12 solid-phase synthesizer. Additionally, purification can be performed using methods well-known in the art. Exemplarily, purification can be performed using DEAE-650s (Toyopearl) and a semi-preparative RT-HPLC column.

[0135] Circular RNA precursor

[0136] This article also provides a circular RNA precursor, which is a linear RNA, and the nucleotide sequence at its 5’ end is the sequence shown in Formula I of the present application. The circular RNA precursor of the present invention has lower immunogenicity compared to a precursor without modifications.

[0137] In some embodiments, in the Formula I sequence contained in the circular RNA precursor, B1 to B nAt least one of them is N6-methyladenine. Compared with the precursor without N6-methyladenine, cyclizing this RNA precursor by the ligase method to generate a circular RNA containing N6-methyladenine has stronger protein translation ability than the circular RNA without N6-methyladenine, such as the protein translation ability is increased by more than 20%, more than 50%, more than 80%, more than 100%, more than 200%, more than 300%, more than 500% or more than 1000%.

[0138] In this article, the protein translation capacity of an mRNA molecule (translation capacity per mRNA molecule) = half-life × translation rate constant × protein length, and its unit is the total number of amino acids / mRNA, which is used to characterize the total number of amino acids translated by an mRNA molecule within its half-life. The translation rate constant refers to the number of protein copies translated by an mRNA molecule within 1 hour (protein copy number / mRNA / h).

[0139] In some embodiments, the circular RNA precursor is a circular RNA precursor prepared by using the preparation method of the circular RNA precursor described in any embodiment of the present application.

[0140] RNA enzyme

[0141] In this article, RNA polymerase refers to an enzyme that synthesizes RNA by polymerization through phosphodiester bonds using a DNA strand or RNA as a template and ribonucleoside triphosphates as substrates. Because it is related to the transcription of genetic information of gene DNA into RNA in cells, it is also called transcriptase. There are no special restrictions on RNA enzymes in this article, and any RNA enzyme well-known in the art can be used to implement the various technical solutions described in this article. Exemplary RNA enzymes include phage-derived RNA polymerases and mitochondrial-derived RNA polymerases, including but not limited to common T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase, etc. Commercially available RNA enzymes can be used to implement the present invention, or RNA enzymes can be prepared by oneself.

[0142] DNA template

[0143] In this article, DNA is used as a template, and an in vitro transcription system is used to synthesize circular RNA precursors.

[0144] A DNA template usually has two strands. One strand is called the coding strand, also known as the non-template strand or sense strand, which is the strand in the DNA template that carries codons for translation in the 5'-to-3' direction. When double-stranded DNA transcribes RNA, only one DNA strand will be transcribed into RNA, and the sequence of this DNA strand has the same deoxyribonucleotide sequence as the RNA (U is replaced by T). During RNA transcription, this sense DNA strand only provides coding and is not the template for the transcription process. The other DNA strand is called the antisense strand, also known as the template strand, which is the strand in double-stranded DNA that does not have codons for translation in the 5'-to-3' direction and serves as the template for ribonucleotide base pairing during RNA synthesis. Eventually, the mRNA will be translated into a protein.

[0145] In this article, base complementary pairing refers to the phenomenon in which the bases of each nucleotide residue in a nucleic acid molecule are connected to each other by hydrogen bonds according to the corresponding relationship of A with T or U, and G with C. There is a principle of base complementary pairing in DNA replication and in the process of transcription, in the relationship between DNA and RNA. Degenerate bases, according to the degeneracy of codons, are commonly represented by a single symbol for two or more bases.

[0146] There are mainly two ways to synthesize circular RNA in vitro: direct intramolecular ligation into a ring catalyzed by a ligase, and self-splicing into a ring based on type I or type II intron ribozymes. The self-splicing of the type I intron ribozyme into a ring includes the self-splicing of the T4 phage into a ring and the self-splicing of Anabeana. Therefore, the DNA template applicable to this application may contain different elements according to the different ring-forming methods used.

[0147] Generally speaking, regardless of the subsequent method of forming a ring, the DNA template needs to contain a promoter. A promoter is a DNA sequence located upstream of the gene DNA that enables a specific gene to be transcribed. The promoter can be recognized by RNA polymerase and initiate the transcription to synthesize RNA. The promoter sequence is usually located upstream or at the 5' end of the transcription start site. A promoter that can be recognized by the used RNA polymerase and initiate transcription can be selected according to the used RNA polymerase. Exemplary promoters include but are not limited to the T7 promoter, SP6, and T3 promoters. Exemplary T7 promoters include but are not limited to those with sequences shown in any one of SEQ ID NO: 5, 8, and 9-12.

[0148] Therefore, when based on type I or type II intron ribozymes, the DNA template may sequentially contain, from the 5’ to the 3’ end: a promoter, the 5’ sequence of the Anabeana intron splicing system, an IRES sequence, a coding sequence containing a start codon and a stop codon, and the 3’ sequence of the Anabeana intron splicing system.

[0149] Circular RNAs do not have a 5'-end cap structure, so they cannot adopt the classical translation pathway that depends on cap structure initiation. However, circular RNAs can utilize the IRES sequence added before the translation promoter to achieve intracellular and in vivo translation (Molecular Cell 74, 508–520, May 2, 2019). Therefore, in some embodiments, when ligated into a circle using a ligase (such as T4 RNA ligase), the DNA template of the present application may also contain an IRES sequence, which is usually located upstream of the promoter. The IRES sequence is also known as the Internal Ribosome Entry Site sequence, with a length usually of 150-1000 nt. It can fold into a certain secondary and tertiary structure, thereby mediating the binding of ribosomes to RNA and initiating protein translation. IRES is a non-translated RNA. The IRES sequence can be from viruses (including RNA viruses and DNA viruses), or it can be the 5' UTR of some eukaryotic linear RNAs, such as IRES sequences from mammals, plants, and yeast. The present application can use various IRES sequences well-known in the art to implement the solutions of the present application. An exemplary IRES sequence can be the IRES sequence from Encephalomyocarditis virus (EMCV). In some embodiments, the nucleotide sequence of the IRES is as shown in SEQ ID NO:1.

[0150] In addition, m6A upstream of the circular RNA translation promoter can also initiate protein translation. As reported in the Cell Research (2017) 27: 626-641 literature, the RRm6ACH (R = G or A; H = A, C or U) motif sequence can initiate the translation of circular RNAs. The NATURE COMMUNICATIONS|(2022) 13: 3751 literature reports that in addition to the RRm6ACH (R = G or A, H = A, C or U) motif sequence, there are also many other short sequences that can initiate the translation of circular RNAs. These sequences include short sequences with a high AU content, especially short sequences composed of A and U, including but not limited to AAAAAA, AUAUAU, UUAUAU, AAUACU, etc. The circular RNAs in these two articles are all produced intracellularly and are a DNA template that contains RNA that can be transcribed into self-splicing circular RNAs; this DNA template is transformed into cells and circularized in the cell body.

[0151] This application may contain m6A modification on polynucleotides. Using this as a substrate, it is incorporated into the RNA strand through in vitro transcription to form a circular RNA precursor, and then ligase is used to form a loop. The incorporated sequence of the polynucleotide may contain the entire sequence of RRm6ACH or a partial sequence with m6A, and then combined with the front and back sequences after circularization to form RRm6ACH, thereby having the function of initiating the translation of circular RNA. The m6A brought in by the polynucleotide, compared with adding m6ATP to partially or completely replace ATP during the RNA transfer process, can achieve precise positioning of m6A modification without affecting other RNA regions. At the same time, among the circular RNAs obtained thereby, the proportion of circular RNAs with the m6A-modified RRm6ACH sequence exceeds 50%, 60%, 70%, 80%, 90%, 99% or 99.9% of all circular RNAs.

[0152] Therefore, the polynucleotide used in this application satisfies at least one of the following characteristics a) to e): a) The last two bases at the 3' end of the polynucleotide are respectively complementary to the bases at positions +1 and +2 downstream of the promoter of the transcription template strand; b) The last two bases at the 3' end of the polynucleotide are respectively complementary to the bases at positions -1 and +1 downstream of the promoter of the transcription template strand; c) The last three bases at the 3' end of the polynucleotide are respectively complementary to the bases at positions +1, +2, and +3 downstream of the promoter of the transcription template strand; d) The last three bases at the 3' end of the polynucleotide are respectively complementary to the bases at positions -1, +1, and +2 downstream of the promoter of the transcription template strand; e) The last four bases at the 3' end of the polynucleotide are respectively complementary to the bases at positions -1, +1, +2, and +3 downstream of the promoter of the transcription template strand.

[0153] In addition, at least one of the first, second, and third nucleotides at the 5' end of the polynucleotide of the present invention has a modification selected from glycosyl modification and base modification.

[0154] Transcription reaction system

[0155] The transcription reaction system applicable herein can be a reaction system well-known in the art for transcription, and generally contains: buffer, enzymes required for transcription reaction, NTP, polynucleotide according to any embodiment herein, DNA template, and water, etc.

[0156] Buffers can be various buffers routinely used in IVT. Exemplary buffers are buffers containing magnesium ions, which usually may contain Tris-HCl, magnesium chloride, spermidine, and DTT. In this buffer, the concentration of Tris-HCl can be 20 - 500 mM, such as 100 - 500 mM or 300 - 500 mM, the concentration of magnesium chloride can be 30 - 500 mM, such as 100 - 500 mM or 300 - 500 mM, the concentration of spermidine can be 0 - 50 mM, such as 5 - 50 mM or 10 - 30 mM, and the concentration of DTT can be 5 - 500 mM, such as 10 - 300 mM or 50 - 150 mM. The pH of Tris-HCl can be between 7.0 - 8.5, such as between 7.0 - 8.0 or between 7.5 - 8.0.

[0157] In the transcription reaction system, the content (volume ratio) of the buffer is usually 5 - 20%.

[0158] Enzymes required for the transcription reaction usually include RNA polymerase and inorganic pyrophosphatase. Enzyme preparations can be formulated for carrying out transcription and translation. The enzyme preparation can also contain nuclease inhibitors well known in the art. Usually, in this enzyme preparation, the concentration of RNA polymerase such as T7 RNA polymerase can be 100 - 500 U / μL, such as 300 - 500 U / μL, the concentration of inorganic pyrophosphatase can be 0.05 - 0.25 U / μL, such as 0.05 - 0.15 U / μL, and the concentration of nuclease inhibitor can be 10 - 30 U / μL.

[0159] In the transcription reaction system, the content (volume ratio) of the enzyme preparation is usually 5 - 20%.

[0160] NTP is a general term for ATP, CTP, UTP, CTP and their corresponding analogs, referring to natural or unnatural nucleotides and their analogs, including but not limited to nucleotides with base or sugar modifications, such as various modified nucleotides described in the "polynucleotide" section above, including but not limited to 5me-UTP, 5me-CTP, pseudo-UTP, N1-me pseudoUTP, pseudo-iso-CTP, N1-me pseudo-iso-CTP, 5moUTP, 6Nme-ATP, 2'-OMe-GTP, 2’-F-UTP, and 5-OMe-UTP, etc. Various NTPs are used in the transcription reaction system in their conventional dosages. For example, in the reaction system, the concentration of each NTP can be 1 - 15 mM, such as 1 - 10 mM.

[0161] In the transcription reaction system, the concentration of the polynucleotide described in any embodiment herein can be 1 - 15 mM, such as 1 - 10 mM or 3 - 8 mM.

[0162] In the transcription reaction system, the concentration of the DNA template can be 10-100 μg / mL.

[0163] Transcription can be carried out under conventional transcription conditions. For example, the transcription temperature can be 35-38 °C. The transcription time can be determined according to specific reaction conditions and is usually in the range of 1-5 h. After the transcription reaction is completed, an appropriate amount of DNase I enzyme can be added to digest the original DNA template. The digestion can be carried out at 35-38 °C. After the digestion is completed, the transcription product can be separated. For example, an appropriate amount of LiCl can be added to the reaction vessel, mixed well, placed at a temperature below -15 °C for a period of time, then centrifuged, and the precipitate is separated and washed with ethanol to obtain the transcription product.

[0164] The transcription reaction product can be purified and the purified product can be used to carry out the cyclization reaction. Alternatively, the transcription reaction product can be directly used to carry out the cyclization reaction without purification.

[0165] Cyclization reaction system

[0166] According to different cyclization methods, different cyclization reaction systems can be used. The cyclization system usually contains the circular RNA precursor and buffer described herein, etc.

[0167] The buffer of the cyclization reaction system can be a buffer well-known in the art for use in cyclization reactions, including but not limited to any one of citrate-sodium citrate buffer, disodium hydrogen phosphate-sodium hydroxide buffer, or tris(hydroxymethyl)aminomethane salt buffer (Tris-HCl buffer). Preferably, the buffer is Tris-HCl buffer. The buffer provides a buffer environment with a pH of 7.0-8.5, preferably 7.3-7.7, for the cyclization reaction system.

[0168] The cyclization reaction system can also contain metal salts conventionally used in the art for cyclization reactions. Exemplary metal salts include but are not limited to manganese chloride, magnesium chloride, cobalt chloride, zinc chloride, manganese sulfate, and manganese nitrate. In some embodiments, the metal salt is magnesium chloride. The concentration of the metal salt in the reaction system can be appropriately selected according to actual reaction conditions, for example, according to the enzyme used. In some embodiments, when using a ligase for the cyclization reaction, the concentration of a metal salt such as magnesium chloride in the reaction system can be 50-150 mM; when the cyclization reaction is a self-splicing cyclization reaction, the concentration of a metal salt such as magnesium chloride in the reaction system can be 0.1-20 mM, such as 5-20 mM, 0.1-2 mM, 0.5-2 mM, etc.

[0169] In a ligase-based cyclization reaction system, the ligase can be a ligase well-known in the art, including RNA ligases and DNA ligases. RNA ligases include T4 RNA ligase, MthRnl (Methanothermobacter thermoautotrophicus RNA ligase). T4 RNA ligase includes T4 RNA ligase 1, T4 RNA ligase 2, truncated T4 RNA ligase 2, etc. DNA ligases include T4 DNA ligase.

[0170] In some embodiments, the cyclization reaction is a direct intramolecular ligation and cyclization reaction catalyzed by T4 RNA ligase. The cyclization reaction system may contain a buffer, ATP, linear RNA obtained by the IVT method described herein (i.e., the circular RNA precursor described herein), metal salts, and T4 RNA ligase. In this cyclization reaction system, the buffer can be a buffer well-known in the art for use in cyclization reactions, such as Tris-HCl. The concentration of the buffer such as Tris-HCl can be 400-600 mM. The pH of the buffer such as Tris-HCl can be between 7.0 and 8.5 (such as 7.3-7.7). The metal salt can be magnesium chloride; the concentration of the metal salt such as magnesium chloride can be 50-150 mM. In this cyclization reaction system, the content of ATP can be 0.01-10 mM, such as 0.5-2 mM. In this cyclization reaction system, the concentration of the linear RNA (i.e., the single-stranded RNA to be cyclized) obtained by the IVT method described herein can be 0.1-1.0 mg / mL, such as 0.2-0.6 mg / mL. In this cyclization reaction system, the dosage of T4 RNA ligase can be 100-500 U / mL. Various T4 RNA ligases well-known in the art can be used to implement the cyclization scope of the present application. The cyclization reaction system may also contain a reagent for stabilizing enzymes containing free sulfhydryl groups, such as DTT (dithiothreitol). The concentration of DTT can be 5-15 mM.

[0171] It is also possible to obtain head-to-tail circular RNA (i.e., the PIE method) by ribozyme-catalyzed RNA splicing reaction. Commonly used ribozymes include type I and type II introns.

[0172] For the cyclization reaction based on group I intron ribozyme for self-splicing and cyclization, the cyclization reaction system may contain linear RNA obtained by the IVT method described herein (i.e., the circular RNA precursor described herein), buffer, metal salt, and GTP. In this reaction system, the concentration of linear RNA can be 0.5 - 1.5 mg / mL. The buffer can be a buffer well-known in the art for use in cyclization reactions, such as Tris-HCl. The concentration of the buffer such as Tris-HCl can be 20 - 200 mM (such as 30 - 100 mM), and the pH can be 7.0 - 8.5 (such as 7.3 - 7.7). The metal salt can be a magnesium salt, such as magnesium chloride; the concentration of the metal salt such as magnesium chloride can be 1 - 20 mM, such as 5 - 20 mM. The concentration of GTP can be 0.1 - 10 mM, such as 1 - 10 mM.

[0173] For the cyclization reaction based on group II intron ribozyme for self-splicing and cyclization, the cyclization reaction system may contain linear RNA obtained by the IVT method described herein, buffer, metal salt, and ammonium salt. In the reaction system, the concentration of linear RNA can be 0.5 - 1.5 mg / mL. The buffer can be a buffer well-known in the art for use in cyclization reactions, such as Tris-HCl. The concentration of the buffer such as Tris-HCl can be 20 - 80 mM, and the pH can be 7.0 - 8.5 (such as 7.3 - 7.7). The metal salt can be a magnesium salt, such as magnesium chloride; the concentration of the metal salt such as magnesium chloride can be 0.1 - 2 mM, such as 0.5 - 2 mM. The ammonium salt can be ammonium chloride, and the concentration of the ammonium salt can be 300 - 600 mM.

[0174] Method for preparing circular RNA

[0175] The present invention provides a method for preparing circular RNA using the polynucleotide described herein. The method for preparing circular RNA herein includes a transcription step and a cyclization step.

[0176] In the transcription step, the transcription reaction system described above can be used. Generally, transcription can be carried out at 35 - 38 °C, and the transcription time can be determined according to the actual reaction conditions, usually 0.5 - 5 h. After transcription, an appropriate amount of DNase I enzyme can be added to digest the original DNA template, and then the transcribed RNA molecules can be separated by conventional methods. For example, a LiCl solution can be added for precipitation, centrifuged, and the precipitate can be washed to obtain the linear RNA required herein.

[0177] In the cyclization step, when using T4 RNA ligase, the cyclization reaction system can be placed at 20 - 28 °C (such as 20 - 28 °C) for a period of time, such as 0.5 - 8 h. After the reaction, the circular RNA of the present invention is separated. When using group I or group II introns, the cyclization reaction system can be placed at 50 - 57 °C for 1 - 10 minutes.

[0178] In some embodiments, after the cyclization reaction, a purification step may also be included. Methods well-known in the art for purifying RNA molecules can be used for purification. For example, purification can be carried out using RNase R enzyme, or ion-exchange liquid chromatography (IEX-HPLC) can be used for purification, or PAGE gel can be used for purification. Specifically, RNase R is a Mg 2+ -dependent 3'→5' ribonuclease exonuclease that can digest single-stranded RNA but not circular RNA. Treating the cyclized product with RNase R can purify circular RNA and remove linear RNA. Exemplarily, an appropriate amount of RNase R is added to the reactant after the cyclization reaction, and the reaction is carried out at about 37 °C for 1 to 5 hours. After the RNase R reaction is completed, an appropriate amount of LiCl solution is added to the tube for precipitation, centrifugation, ethanol washing, etc.

[0179] When using ion-exchange liquid chromatography (IEX-HPLC) for purification, exemplary purification conditions include: dissolving circular RNA in a buffer of Tris, EDTA, and PB; mobile phase A: 50 mM HEPES + 0.5 M Gdn.HCl + 0.1 M NaCl pH 7.5; mobile phase B: 50 mM HEPES + 0.5 M Gdn.HCl + 1.0 M NaCl pH 7.5, and the flow rate is controlled at 5 ml / min.

[0180] When using PAGE gel for purification, cut the band and place the gel slice into a sterilized centrifuge tube. The centrifuge tube is placed in a liquid nitrogen tank for freezing, and the gel slice is pressed into a slurry with a plastic pipette. Transfer the gel slurry to a centrifuge tube containing an appropriate amount of sodium acetate, shake at room temperature and then centrifuge, add an equal amount of phenol, mix well and centrifuge at room temperature. Obtain the supernatant, transfer it to a centrifuge tube, add an equal amount of chloroform, and centrifuge. Transfer the supernatant to a new centrifuge tube, add an appropriate amount of LiCl and pre-frozen 100% ethanol, and mix evenly. Place the centrifuge tube at -80 °C for 2 hours or longer, then centrifuge to take the supernatant, wash the pellet with an appropriate amount of 70% ethanol, remove the ethanol, and dry to obtain purified circular RNA.

[0181] Using the method of the present invention, circular RNA with low immunogenicity and high translation activity (i.e., high translation ability) can be obtained without purification.

[0182] Circular RNA molecule

[0183] This article also provides a circular RNA molecule that contains the polynucleotide shown in Formula I herein. Compared with circular RNA molecules that do not contain the polynucleotide shown in Formula I herein, the circular RNA molecules provided in this application have reduced immunogenicity and / or enhanced protein translation ability.

[0184] In some embodiments, the circular RNA molecule contains the circular RNA precursor described herein, or is prepared from the circular RNA precursor. In some embodiments, the circular RNA molecule is prepared from the circular RNA precursor via the method described herein. In some embodiments, the circular RNA prepared by the ligase method contains the RRm6ACH sequence pattern, where R is G or A, H is A, C or U, and m6A is from the polynucleotide shown in Formula I.

[0185] The circular RNA molecule described herein can be a nucleic acid-based therapeutic agent or active agent, and can be selected from: messenger RNA (mRNA), antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), ribozyme and aptamer. In some embodiments, the circular RNA molecule can encode a protein of interest, and such proteins can be, for example, proteins with therapeutic, prophylactic or improving physiological functions of an organism, including but not limited to antigens, antibodies and proteins with biological functions known in the art.

[0186] Lipid nanoparticles

[0187] The circular RNA molecule described herein, when used as an active agent or therapeutic agent, can be encapsulated in lipid nanoparticles. Therefore, in some embodiments, the present application provides lipid nanoparticles containing the circular RNA molecule described herein.

[0188] In addition to containing the circular RNA molecule described herein, the lipid nanoparticles generally also contain cationic lipids, one or more auxiliary lipid molecules, one or more cholesterol or cholesterol derivatives and / or one or more polymer-conjugated lipid molecules.

[0189] In this article, cationic lipids are also referred to as ionizable lipids. Cationic lipids commonly used in the art for preparing liposomes can all be used in the present invention. Exemplary cationic lipids include but are not limited to monovalent cationic lipids DOTAP, DOTMA, DIMRIE and DOTIM, multivalent cationic lipids DOGS and DOSPA, and cationic cholesterol derivatives DC-Chol and BGTC, etc.

[0190] In this text, the auxiliary lipid molecules generally refer to neutral lipid molecules. Neutral lipids refer to lipid substances that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to: phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), and ceramides. Neutral lipids can be synthetic or of natural origin. In some embodiments, the auxiliary lipid molecule is DSPC.

[0191] In this text, steroids are compounds that contain the following carbon skeleton:

[0192]

[0193] Non-limiting examples of steroids include cholesterol and the like.

[0194] In this text, cholesterol derivatives can be cholesterol derivatives well-known in the art for preparing liposomes. Exemplary cholesterol derivatives include the commonly used cholesterol, CAS: 57-88-5.

[0195] In this text, polymer-conjugated lipids refer to molecules that contain a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a polyethylene glycol (PEG)-ylated lipid. The term "PEG-ylated lipid" refers to a molecule that contains a lipid moiety and a polyethylene glycol moiety. PEG-ylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), PEG-DAG (diacylglycerol), PEG-PE (phosphatidylethanolamine), PEG-diacylglycylamide (PEG-S-DAG), PEG-DSPE (-distearoyl phosphatidylethanolamine), PEG-cer (ceramide), PEG-dialkoxypropyl carbamate, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, PEG-distearoyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-1,2-dimyristoylpropyl-3-amine (PEG-c-DMA), etc. In some embodiments, the lipid moiety of the polyethylene glycol (PEG)-ylated lipid includes a moiety having a length of C14 to C22, such as a length of C14 to about C16. In some embodiments, the size of the PEG moiety is about 1000, 2000, 5000, 10,000, 15,000, or 20,000 daltons.

[0196] In the lipid nanoparticles described herein, the molar ratio of the cationic lipid, the co-lipid molecule, cholesterol or cholesterol derivative, and the polymer-conjugated lipid molecule can be 45 to 55:5 to 15:35 to 45:0.5 to 3, and preferably can be 48 to 52:8 to 12:36 to 40:1 to 3. In some embodiments, the lipid nanoparticles described herein contain a cationic lipid, DSPC, cholesterol, and PEG-lipid, and their molar ratio is 48 to 52:8 to 12:36 to 40:1 to 3.

[0197] As used herein, the "lipid nanoparticles" described herein refer to particles having at least one dimension in the nanometer range (e.g., 1 - 1000 nm). As is well known in the art, the average diameter of lipid nanoparticles can be from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 nm to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm or about 150 nm, and the lipid nanoparticles are substantially non-toxic.

[0198] The lipid nanoparticles can be included in a formulation for delivering the circular RNAs described herein, which are used as active agents or therapeutic agents, to a target site (such as cells, tissues (such as diseased tissues like tumor tissues), organs).

[0199] Lipid nanoparticles containing nucleic acids and methods for their preparation are known in the prior art. For example, see CN102712935A or related patents, etc., the entire disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0200] The morphology of the lipid nanoparticles of the present invention is not particularly limited, but examples of the morphology in which the ionizable lipids of the present invention are dispersed in an aqueous solvent include unilamellar liposomes, multilamellar liposomes, or unspecified lamellar structures, etc.

[0201] In some embodiments, the lipid nanoparticles described herein are used to deliver the circular RNA molecules described herein for expressing a protein of interest to produce a therapeutic or prophylactic effect in a subject. In some embodiments, the lipid nanoparticles described herein are used to deliver the circular RNA molecules described herein to produce corresponding antisense oligonucleotides (ASO), small interfering RNAs (siRNA) or microRNAs (miRNA) in a subject to regulate the expression of a target gene, such as upregulating the expression of an endogenous protein or downregulating (such as silencing) the protein level and / or mRNA level of the target gene to achieve a therapeutic or prophylactic purpose.

[0202] Accordingly, in some embodiments, the present disclosure also provides a pharmaceutical composition comprising the circular RNA molecules described herein. In some embodiments, the pharmaceutical composition described herein comprises the lipid nanoparticles described herein. The pharmaceutical composition may further comprise various pharmaceutically acceptable carriers or excipients well known in the art. Suitable pharmaceutically acceptable carriers and excipients can be selected according to the specific dosage form and route of administration. For example, as a solid composition for oral administration, the pharmaceutical composition can be formulated into the form of powder, granule, compressed tablet, pill, capsule, chewing gum, wafer, etc. Such solid compositions generally contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth or gelatin; excipients such as starch, lactose or dextrin, disintegrants such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silica; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate or orange essence; and coloring agents. In some embodiments, the pharmaceutical composition is a vaccine. The vaccine may contain various excipients commonly contained in vaccines well known in the art, such as adjuvants.

[0203] Therapeutic methods and uses

[0204] The lipid nanoparticles and pharmaceutical compositions described herein can be used for in vivo and in vitro delivery of the circular RNA molecules described herein as therapeutic or active agents for treating or preventing diseases or disorders in a subject.

[0205] Accordingly, the present disclosure provides the use of the circular RNA molecules described herein in the preparation of lipid nanoparticles or pharmaceutical compositions for treating or preventing diseases or disorders in a subject, and the circular RNA molecules, lipid nanoparticles or pharmaceutical compositions described herein for treating or preventing diseases or disorders in a subject. The present disclosure also provides methods for treating or preventing diseases, including administering to a subject in need thereof a therapeutically or prophylactically effective amount of the circular RNA molecules described herein, lipid nanoparticles or pharmaceutical compositions comprising the circular RNA molecules, or pharmaceutical compositions comprising the lipid nanoparticles.

[0206] As used herein, "therapeutically effective amount" and "prophylactically effective amount" refer to an amount sufficient to achieve treatment or prophylaxis in a mammal, preferably a human, when administered to the mammal. The "therapeutically effective amount" and "prophylactically effective amount" of the circular RNA molecules described herein will vary depending on factors such as the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those of ordinary skill in the art based on their knowledge and the present disclosure.

[0207] As used herein, the "individual" or "subject" generally refers to mammals, including humans and domestic animals such as laboratory animals and household pets (such as cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals (such as wild animals, etc.).

[0208] The diseases and disorders described herein can be various diseases and disorders known in the art that are suitable for nucleic acid therapy and prevention, and depend on the specific biological functions of the circular RNA molecules described herein. In some embodiments, the pharmaceutical composition is a vaccine, and the method includes immunizing an individual to render the individual immune to the corresponding disease or disorder, which includes but is not limited to influenza, hepatitis B, hepatitis C, infections caused by the novel coronavirus, etc. The disease or disorder also includes, for example, tumors, including solid tumors and hematological tumors, various inflammations, etc.

[0209] The routes and methods of administration are well known in the art and as described above, such as including but not limited to oral, topical, transdermal, inhalation, intraperitoneal, sublingual, buccal, rectal, vaginal, and intranasal. In some embodiments, the intraperitoneal administration route includes: subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques.

[0210] In some embodiments, the present application also provides the following applications: the application of the polynucleotide of formula (I) described in any embodiment herein in the preparation of a circular RNA precursor or circular RNA; the application of the circular RNA precursor described in any embodiment herein in the preparation of circular RNA; the application of the polynucleotide of formula (I) described in any embodiment herein in reducing the immunogenicity of circular RNA molecules and / or enhancing the protein translation ability of circular RNA molecules; the application of the circular RNA precursor described in any embodiment herein in reducing the immunogenicity of circular RNA molecules and / or enhancing the protein translation ability of circular RNA molecules.

[0211] The advantages of the present invention include:

[0212] The main rate-limiting bottleneck in the transcriptional synthesis of RNA is the initiation of transcription. The polynucleotides disclosed herein have a stronger initiation potency compared to GMP and NTP, which can be considered an effect of enhanced initiation, thereby enabling a higher RNA transcription yield. In some embodiments of the present invention, relative to rNTP, the use of the polynucleotides of the present invention increases RNA synthesis by more than 10%; in some embodiments, RNA synthesis increases by more than 20%, more than 40%, more than 60%, more than 80%, more than 100%, more than 200%, more than 200%, more than 400%, or more than 800%.

[0213] Introducing a monophosphate at the 5' end of a polynucleotide can avoid obtaining 5'-monophosphorylated (5'P) linear RNA by using phosphatases, T4 kinases, or the RppH enzyme after the transcription reaction. Thus, the present invention can increase the proportion of 5'P monophosphate in the products obtained after the transcription reaction to more than 80% while increasing the yield; in some embodiments, it is increased to more than 90%, more than 95%, more than 99%, or up to 99.9%.

[0214] The research on whether exogenous circular RNAs can trigger immunity started in 2017. Howard Y. Chang (Molecular Cell 67, 228–238, July 20, 2017) mentioned that eukaryotic cell intron sequences are used to distinguish endogenous circular RNAs from exogenous circular RNAs. Mature endogenous circular RNAs will bind to different RNA-binding proteins, while exogenous circRNAs cannot, so they can strongly trigger the immune signals of cells. Among them, RIG-1 is the signaling pathway that causes immune responses. In 2019, Daniel G. Anderson (Molecular Cell 74, 508–520, May 2, 2019) mentioned that HPLC-purified late CircRNAs do not trigger TLR and RIG-I-mediated immune responses, while early CircRNAs, because they contain linear triphosphate RNA impurities, will trigger immune responses. In 2019, when Howard Y. Chang (Molecular Cell 76, 96–109, October 3, 2019) repeated Daniel G. Anderson's experiment of HPLC-purifying circular RNAs, it was found that late CircRNAs still had immunogenicity and could not reproduce Daniel G.'s experiment. In 2021, Ling-Ling Chen (Molecular Cell 82, 420-434, January 20, 2022) used three different circularization methods to draw the conclusion that circRNAs formed using type I introns would introduce additional sequences and trigger immune responses, while circRNAs formed using T4 RNA ligase had the weakest immunogenicity because they did not contain additional sequences. However, Ling-Ling Chen did not use the method of T4 RNA ligase to prepare circRNAs containing the same additional sequences. Therefore, as for the fact that HPLC-purified or PAGE-gel-purified circRNAs still have immunogenicity, whether it is because circRNAs contain extremely trace linear RNAs or extremely trace intron short fragments that are difficult to detect and thus produce immune responses, or whether it is the secondary or tertiary structures formed by certain special sequences in circRNAs that produce immune responses, there is still no conclusion. But one thing is certain, in the prior art, the preparation of circular RNAs requires RNase R to degrade linear RNAs, phosphatase to degrade the 5'-terminal triphosphate of linear RNAs, and then HPLC purification or PAGE-gel purification, which can remove part of the immunogenicity. However, neither the HPLC nor the PAGE-gel purification method is conducive to large-scale industrial preparation. Different from the prior art, the present invention can obtain better translation effects without using the above purification methods.

[0215] The circular RNA precursor prepared by the method of the present invention has glycosyl modification and / or base modification at the first, second, or third nucleoside at the 5' end of the RNA. After the precursor is further prepared into circular RNA, the immunogenicity of the uncircularized linear RNA impurities is lower; if the circularization method using type I intron or type II intron is adopted, the immunogenicity of the generated intron and intron dimer impurities is also significantly reduced. In some embodiments, for the circular RNA generated from the circular RNA precursor prepared by the present invention, compared with the circular RNA prepared after the transcription reaction of the prior art, the expression of RIG-1 decreases by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, or decreases by 99.9%; the expression of IFNβ decreases by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, or decreases by 99.9%; the expression of TNF decreases by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, or decreases by 99.9%; the expression of IL-6 decreases by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, or decreases by 99.9%. The decrease in immunogenicity leads to an increase in the amount of protein translation in vitro and in vivo by more than 20%, more than 50%, more than 80%, more than 100%, more than 200%, more than 300%, more than 500%, or more than 1000%.

[0216] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0217] Preparation Example

[0218] Synthesis of polynucleotides: The raw materials 5'-O-DMT-2'-O-TBDMS phosphoramidites (rAAc, rCAc, rGdmf, U) and 2'-O-MOE-3'-O-phosphoramidites (A2'O-MOEAc, C2'O-MOEAc, G2'O-MOEdmf, U2'O-MOE) and 2'-O-ethyl-3'-O-phosphoramidites (A2'O-ethyl Ac, C2'O-ethyl Ac, G2'O-ethyl dmf, U2'O-ethyl) are from Hongene, and Bis-cyanoethyl-N,N-diisopropyl CED phosphoramidite is from ChemGenes. The dinucleotides, trinucleotides and polynucleotides are synthesized in sequence using the common oligonucleotide solid-phase synthesis process in the industry (Bioautomation Mermade 12 solid-phase synthesizer), and the finished products are obtained after purification by DEAE-650s (Toyopearl) and semi-preparative RT-HPLC column.

[0219] pApG, pA2’OmepG, pA2oETpG are synthesized respectively by the above method. P6mApG, p6mApApG, p6mA2omepApG, pGpGpG and Figure 1 is the chromatogram of polynucleotide pA2’OmepG.

[0220] Example 1

[0221] This example provides a T7 transcription method containing GMP to obtain linear RNA containing (5’P).

[0222] Preparation of buffer solution containing magnesium ions (10X): Mix Tris-HCl (pH = 7.9), MgCl2, spermidine and DTT to prepare a buffer solution containing magnesium ions (10X); among them, the concentration of Tris-HCl is 400 mM, the concentration of MgCl2 is 400 mM, the concentration of spermidine is 20 mM, and the concentration of DTT is 100 mM.

[0223] Preparation of mixed enzymes: Mix T7 RNA polymerase, inorganic pyrophosphatase and nuclease inhibitor to prepare a mixed enzyme system; among them, the concentration of T7 RNA polymerase is 400 U / μL, the concentration of inorganic pyrophosphatase is 0.1 U / μL, and the concentration of nuclease inhibitor is 20 U / μL.

[0224] Preparation of the transcription reaction solution: Mix 200 μL of a buffer containing magnesium ions (10X), 100 μL of 100 mM ATP, 100 μL of 100 mM GMP, 20 μL of 100 mM GTP, 100 μL of 100 mM CTP, 100 μL of 100 mM UTP, 100 μL of a mixed enzyme, and 100 μL of a 1 μg / μL linearized DNA template (this template contains the T7 promoter shown in SEQ ID NO:5, the 5’ UTR sequence shown in SEQ ID NO:3, the IRES sequence from EMCV shown in SEQ ID NO:1, the eGFP sequence shown in SEQ ID NO:2, and the 3’ UTR sequence shown in SEQ ID NO:4). Then add nuclease-free water to make up to 2 mL to obtain the transcription reaction solution.

[0225] After the transcription reaction of the transcription reaction solution at 37 °C for 2 h, add 100 μL of 1 U / μL DNase I enzyme and digest the original DNA template at 37 °C for 30 min.

[0226] After the DNase I reaction, add 2 mL of LiCl to the tube for precipitation. Mix well and place it in a -20 °C refrigerator for 30 min. Then centrifuge at 12000 rpm and 4 °C for 25 min. Wash the precipitate twice with 75% ethanol. After air-drying, add 2 mL of water to dissolve it completely. After quantitative determination, the RNA yield is 2.04 mg.

[0227] RNase H (ribonuclease H) is an endoribonuclease. When RNA hybridizes with DNA, RNase H specifically hydrolyzes the phosphodiester bond of RNA at the hybridization site. Utilizing this feature, a specific biotin probe is annealed to the IVT product RNA through base complementary pairing, and then the specific site of the RNA is digested with RNase H to obtain small RNA fragments with a uniform 5’ end. Subsequently, purification is carried out using avidin magnetic beads conjugated with biotin to obtain small RNA fragments. Finally, the molecular weight of the 5’-end small RNA fragments is analyzed by liquid chromatography and LC-MS to determine the proportion of monophosphates. After conducting experiments by this method, it is determined that the proportion of linear RNA containing monophosphates is 78.6%.

[0228] Example 2

[0229] Preparation of the buffer containing magnesium ions (10X): Mix Tris-HCl (pH = 7.9), MgCl2, spermidine, and DTT to obtain the buffer containing magnesium ions (10X); among them, the concentration of Tris-HCl is 400 mM, the concentration of MgCl2 is 400 mM, the concentration of spermidine is 20 mM, and the concentration of DTT is 100 mM.

[0230] Preparation of the mixed enzyme: The T7 RNA polymerase, inorganic pyrophosphatase, and nuclease inhibitor were mixed to obtain a mixed enzyme system; among them, the concentration of T7 RNA polymerase was 400 U / μL, the concentration of inorganic pyrophosphatase was 0.1 U / μL, and the concentration of nuclease inhibitor was 20 U / μL.

[0231] Preparation of the transcription reaction solution: 200 μL of a buffer containing magnesium ions (10X), 100 μL of 100 mM ATP, 100 μL of 100 mM GTP, 100 μL of 100 mM CTP, 100 μL of 100 mM UTP, 100 μL of 100 mM polynucleotide (see Table 1 below), 100 μL of the mixed enzyme, and 100 μL of 1 μg / μL DNA template (the sequence of the T7 promoter is shown in Table 1 below, and other sequences are the same as in Example 1) were mixed and then nuclease-free water was added to 2 mL to obtain the transcription reaction solution.

[0232] After the transcription reaction of the transcription reaction solution at 37 °C for 2 h, 100 μL of 1 U / μL DNase I enzyme was added and the original DNA template was digested at 37 °C for 30 min.

[0233] After the DNase I reaction ended, 2 mL of LiCl solution was added to the tube for precipitation. After mixing, it was placed in a -20 °C refrigerator for 30 min, then centrifuged at 12000 rpm and 4 °C for 25 min. The precipitate was washed twice with 75% ethanol, air-dried, and then 5 mL of water was added to each to dissolve the precipitate. After complete dissolution, the RNA yield was quantified using a spectrophotometer at OD260. The yield is shown in Table 1 below.

[0234] The method for measuring the proportion of linear RNA containing monophosphate was the same as in Example 1, and the results are shown in Table 1 below.

[0235] Table 1

[0236]

[0237]

[0238] Example 3

[0239] In this example, T4 RNA Ligase 1 was used to circularize the RNA obtained by IVT in Examples 1 and 2.

[0240] Preparation of 10xT4 buffer: Tris-HCl (pH 7.5), MgCl2, and DTT were mixed to obtain a buffer containing magnesium ions (10X). Among them, the Tris concentration was 500 mM, the MgCl2 concentration was 100 mM, and the DTT concentration was 10 mM.

[0241] Preparation of T4 enzyme ligation reaction solution: Mix 500 μL of magnesium ion-containing buffer, 50 μL of 100 mM ATP, and 2 mg of ssRNA (the ssRNAs prepared in Examples 1 and 2 respectively), add 1000 U of T4 RNA Ligase 1, make up to 5 mL with nuclease-free water, mix well, and react at 25 °C for 4 hours. After the reaction, capillary electrophoresis was used to analyze the cyclization results. Exemplary results are as shown in Figure 2 and 3 shown.

[0242] The linear mRNA (linear) and circularized circular mRNA (circ) of IVT were separated chromatographically using IP-RP HPLC. The circ mRNA chromatographic peak and the linear mRNA chromatographic peak on the liquid chromatogram characterized by gel electrophoresis were used as the active ingredients of the sample, and other peaks were impurity peaks; the sum of the peak areas of the two peaks of circ mRNA and linear mRNA was used as the denominator, and their respective peak areas were used as the numerators to calculate their respective percentages. The percentage of the peak area of the circRNA chromatographic peak was defined as the circRNA ratio, that is, the cyclization rate.

[0243] The cyclization rates are shown in Table 2. The cyclization rate of #1 is as shown in Figure 4 shown. The cyclization rate of #13 is as shown in Figure 5 shown.

[0244] Table 2

[0245]

[0246]

[0247] Example 4

[0248] The DNA template used in this example is the sequence shown in SEQ ID NO: 6 (PIE system DNA template containing Anabeana intron, including EMCV and EGFP). Circular RNA was generated by the intron alternative splicing reaction of the type I intron of Anabeana. The circular RNA contains partial E1 and E2 fragments of Anabeana. The IVT reaction without adding polynucleotide was the same as that in Example 1, and the IVT reaction with adding polynucleotide was the same as that in Example 2. The polynucleotide added to the reaction was A 2MOE pG (that is, the final prepared product #16). After the IVT reaction, DNase I treatment and LiCl precipitation were used for purification and recovery. The product is an RNA precursor, and its electrophoresis pattern is as shown in Figure 6 shown, and the HPLC analysis chart is as shown in Figure 7 shown.

[0249] Preparation of cyclization reaction solution: Mix 1.8 ml of RNA precursor (2 mg), 100 μl of 1 M Tris-HCl pH 7.5, 40 μl of 500 mM MgCl2, and 20 μl of 200 mM GTP, and then add nuclease-free water to make up a 2-ml reaction system.

[0250] Incubate the cyclization reaction solution at 55 °C for 8 min.

[0251] Using the same method, but with the polynucleotides and T7 promoter shown in the following table, products #14, #15, #17, and #18 were prepared. The cyclization rates were tested by the method described above, and the results are shown in Table 3 below.

[0252] Table 3

[0253]

[0254]

[0255] Example 5

[0256] The DNA template used in this example was SEQ ID NO: 7 (containing the C.te.l1 intron structural element, the template contains the T7 promoter, C.te.l1 D1, C.te.l1 D2, C.te.l1 D3, C.te.l1 D4, C.te.l1 D5, C.te.l1 D6, EMCV, and EGFP). Using the reaction characteristics of the type II intron of C.te.l1, circular RNA can be generated through alternative splicing between introns and does not contain a scar. The IVT reaction was the same as in Example 2, and the polynucleotides and T7 promoter used are shown in the following table. After the IVT reaction, it was treated with DNase I and purified by LiCl precipitation to recover the product, which was the RNA precursor.

[0257] Preparation of cyclization reaction solution: Mix 1.5 ml of RNA precursor (2 mg), 80 μl of 1 M Tris-HCl pH 7.5, 40 μl of 50 mM MgCl2, and 200 μl of 5 M NH4Cl, and then add nuclease-free water to make up a 2-ml reaction system. After mixing, react at 53 °C for 5 min.

[0258] The cyclization rate was tested by the method described above, and the results are shown in Table 4 below.

[0259] Table 4

[0260]

[0261]

[0262] Example 6

[0263] RNase R is Mg 2+ -dependent 3'→5' ribonuclease exonuclease that can digest single-stranded RNA but not circular RNA. Treating the circularized product with RNase R can purify circular RNA and remove linear RNA.

[0264] Add 200 U of RNase R to the reaction after the reaction in the above example and react at 37 °C for 2 hours.

[0265] After the RNase R reaction is completed, add 5 mL of LiCl solution to the tube for precipitation. After mixing, place it in a -20 °C refrigerator for 30 min, then centrifuge at 12,000 rpm and 4 °C for 25 min. Wash the precipitate twice with 75% ethanol. After drying, add 1 mL of water to each to dissolve the precipitate to obtain the purified product.

[0266] Example 7

[0267] To obtain high-quality circular RNA, ion exchange liquid chromatography (IEX-HPLC) was used for purification. Take 50 mg of RNA and dissolve it in 10 mM Tris, 1 mM EDTA, 75 mM PB, pH 7.4 buffer. Perform HPLC purification at room temperature of 25 °C. Mobile phase A: 50 mM HEPES + 0.5 M Gdn.HCl + 0.1 M NaCl pH 7.5; Mobile phase B: 50 mM HEPES + 0.5 M Gdn.HCl + 1.0 M NaCl pH 7.5. Control the flow rate at 5 ml / min and collect the fractions as indicated. Perform gel electrophoresis detection on different collection tubes.

[0268] The purified product, as Figure 10 shown.

[0269] Example 8

[0270] A549 cells were seeded in a 6-well plate at a density of 4×10 5 / well, and transfection was performed when the cell density was approximately 80%. Transfect 2 μg of mRNA per well, and the transfection reagent is Lipofectamine MessengerMAX Transfection Reagent (Invitrogen). The transfection procedure was carried out according to the instructions. After 24 hours, collect the cells, extract RNA using TRIzol, and reverse transcribe the RNA into cDNA. Finally, use real-time quantitative fluorescence PCR to detect the expression of inflammatory factors in the cells. The internal reference gene is β-ACTIN. Design qPCR primers for different genes to detect the changes in the expression levels of RIG-1, IFNβ, TNF, and IL-6 genes. The results are as Figure 11 and Table 5 shown.

[0271] Example 9

[0272] HEK293T cells were seeded in 6-well plates at a density of 2×10 5 / well, and transfection was performed when the cell density was approximately 80%. 2 μg of mRNA was transfected into each well using Lipofectamine MessengerMAX Transfection Reagent (Invitrogen), and the transfection procedure was carried out according to the instructions. After 24 hours, the cells were digested with trypsin and collected, and the GFP positive expression intensity was detected by flow cytometry. The results are as Figure 12 shown in Table 5.

[0273] Table 5

[0274]

[0275]

[0276] Example 11

[0277] The methods in Example 4 and polynucleotides were used to prepare #24 and #25 FLuc mRNAs. Among them, the preparation of Fluc mRNA used the polynucleotides in Table 6 below and their corresponding promoter sequences. The DNA template was the PIE system DNA template containing Anabaena intron, including EMCV and FLuc (SEQ ID NO: 13).

[0278] Table 6

[0279]

[0280] Cationic lipid (ALC-0315), DSPC, cholesterol, and PEG-lipid (ALC-0159) were dissolved in an ethanol solution at a molar ratio of 50:10:38.5:1.5. The lipid mixture was mixed with 25 mM acetate buffer (pH 4.0) containing #24 Fluc-mRNA or #25 Fluc-mRNA by microfluidics. In the final mixture, the ethanol and mRNA concentrations were 25% and 0.15 mg / ml, respectively.

[0281] The mixture was dialyzed in PBS (pH 7.4), followed by ultrafiltration centrifugation using 10K MWCO, and filtered through a 0.22 μm filter for intramuscular injection in mice.

[0282] Each mouse was injected with 0.1 μg of the mRNA preparation, and in vivo fluorescence imaging was performed at 6 hours, 3 days, and 10 days after injection. The results are as Figure 13 shown.

Claims

1. Method for preparing circular RNA precursor, the method comprising the step of performing in vitro transcription on a transcription reaction system containing a buffer, an enzyme required for the transcription reaction, NTP, a polynucleotide, and a DNA template, characterized in that, The polynucleotide has a structure represented by the following formula (I): In the formula: N1 is 0 or 1; B1 to B n Each is independently a nucleobase; H1 to H n-2 is 0 or 1; M1 to M n-1 Each independently is -O-, -S-, -NH-, -CH2-, -C(halogen group)2- or -CH(halogen group)-; M n is a hydroxyl group; R1 to R n each independently represents hydrogen, hydroxy, alkyl, alkoxy, alkoxy-substituted alkyl, -S-alkyl, -NH-alkyl, -O-aryl, -S-aryl, -NH-aryl, halogenated group, and locked nucleoside modification; X1 to X n Each independently is -O-, -S-, -NH-, -CH2-, -C(halogen group)2- or -CH(halogen group)-; Y1 to Y n each independently is oxygen, sulfur, selenium or boranyl; Z1 to Z n Each independently is a hydroxyl group, a mercapto group, a boranyl group, an aryl group, an alkyl group, an alkoxy group or -O-aryl; n is 5; The polynucleotide satisfies at least one of the following characteristics a) to e): a) The last two bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream +1 and +2 positions of the promoter of the transcription template strand respectively; b) The last two bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream -1 and +1 positions of the promoter of the transcription template strand respectively; c) The last three bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream +1, +2 and +3 positions of the promoter of the transcription template strand respectively; d) The last three bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream -1, +1 and +2 positions of the promoter of the transcription template strand respectively; e) The last four bases at the 3'-end of the polynucleotide are complementary to the bases at the downstream -1, +1, +2 and +3 positions of the promoter of the transcription template strand respectively; and At least one of the first, second and third nucleosides at the 5'-end of the polynucleotide has a modification selected from glycosyl modification and base modification.

2. The method according to claim 1, characterized in that, The polynucleotide is a dinucleotide, and its structure is as shown in the following formula I-1: Wherein: N1 is 0 or 1; R1 and R2 are each independently hydrogen, hydroxyl, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl, halogenated group and locked nucleoside modification; X1 and X2 are each independently -O-, -S-, -NH-, -CH2-, -C(halogenated group)2- or -CH(halogenated group)-; Y1 and Y2 are each independently oxygen, sulfur, selenium or boranyl; Z1 and Z2 are each independently hydroxyl, mercapto, boranyl, aryl, alkyl, alkoxy or O-aryl; M1 is -O-, -S-, -NH-, -CH2-, -C(halogenated group)2- or -CH(halogenated group)-; M2 is hydroxyl; B1 and B2 are each independently a natural or modified nucleoside base.

3. The method according to claim 1, characterized in that, The polynucleotide is a trinucleotide, and its structure is as shown in the following formula (I-2): In the formula: N1 is 0 or 1; R1, R2 and R3 are each independently hydrogen, hydroxyl, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl, halogenated group and locked nucleoside modification; X1, X2 and X3 are each independently -O-, -S-, -NH-, -CH2-, -C(halogenated group)2- or -CH(halogenated group)-; Y1, Y2 and Y3 are each independently oxygen, sulfur, selenium or boranyl; Z1, Z2 and Z3 are each independently hydroxyl, mercapto, boranyl, aryl, alkyl, alkoxy or O-aryl; M1 and M2 are -O-, -S-, -NH-, -CH2-, -C(halogenated group)2- or -CH(halogenated group)-; M3 is hydroxyl; B1, B2 and B3 are each independently a natural or modified nucleoside base.

4. The method according to claim 1, wherein, The polynucleotide is a tetranucleotide, and its structure is as shown in the following formula I-3: Wherein: N1 is 0 or 1; R1, R2, R3, and R4 are each independently hydrogen, hydroxyl, alkyl, alkoxy, S-alkyl, NH-alkyl, O-aryl, S-aryl, NH-aryl, halogenated group, and locked nucleoside modification; X1, X2, X3, and X4 are each independently -O-, -S-, -NH-, -CH2-, -C(halogenated group)2-, or -CH(halogenated group)-; Y1, Y2, Y3, and Y4 are each independently oxygen, sulfur, selenium, or boranyl; Z1, Z2, Z3, and Z4 are each independently hydroxyl, mercapto, boranyl, aryl, alkyl, alkoxy, or O-aryl; M1, M2, and M3 are -O-, -S-, -NH-, -CH2-, -C(halogenated group)2-, or -CH(halogenated group)-; M4 is hydroxyl; B1, B2, B3, and B4 are each independently a natural or modified nucleobase.

5. The method according to any one of claims 1 to 4, wherein, The polynucleotide has one or more of the following characteristics: B1 to B n Each independently selected from: cytosine, N1-methyluracil, uracil; M1 to M n-1 Each independently is -O-; R1 to R n-1 each independently selected from: hydroxyl, methyl, ethyl, methoxy, ethoxy and methoxyethyl; R n is a hydroxyl group; X1 to X n Each independently represents -O-; Y1 to Y n each independently is selected from O and S; and Z1 to Z n Each independently selected from hydroxy groups.

6. The method according to claim 1, wherein, The polynucleotide is selected from: Dinucleotides, selected from: pApG, pN6mApG, pA 2’Ome pG, pN6mA 2’Ome pG, pA 2’OET pG, pN6mA 2’OET pG, pApU, pN6meApU, pA 2’Ome pU, pN6mA 2’Ome pU, pA 2’OET pU, pN6mA 2’OET pU, pApC, pN6meApC, pA 2’Ome pC, pN6mA 2’Ome pC, pA 2’OET pC, pN6mA 2’OET pC, pApA, pN6meApA, pN6meApN6meA, pA 2’Ome pA, pA 2’OET pA, pN6meA 2’Ome pA, pN6meA 2’OET pA, pA 2’Ome pN6meA, pA 2’OET pN6meA, pN6meA 2’Ome pN6meA, pN6meA 2’ OET pN6meA, ApG, N6mApG, A 2’Ome pG, N6mA 2’Ome pG, A 2’OET pG, N6mA 2’OET pG, ApU, N6meApU, A 2’Ome pU, N6mA 2’Ome pU, A 2’ OET pU, N6mA 2’OET pU, ApC, N6meApC, pA 2’Ome pC, N6mA 2’Ome pC, A 2’OET pC, N6mA 2’OET pC, ApA, N6meApA, N6meApN6meA, A 2’Ome pA, A 2’OET pA, N6meA 2’Ome pA, N6meA 2’OET pA, A 2’Ome pN6meA, A 2’OET pN6meA, N6meA 2’Ome pN6meA, N6meA 2’OET pN6meA, pGpG, pG 2‘Ome pG,, pG 2’OET pG, pGpU, pG 2’Ome pU, pG 2’ OET pU, pGpC, pG 2’Ome pC, pG 2’OET pC, pGpA, pG 2Ome pA, pGpN6mA, pG 2’Ome pN6mA, pUpU, pU 2’Ome pU, pUpA, pU 2’Om pA, pUpN6meA, pU 2’Om pN6meA, pN6meA, GpG, G 2’Ome pG, G 2’OET pG, GpU, G 2’Ome pU, G 2’OET pU, GpC, G 2’Ome pC, G 2’OET pC, GpA, G 2’Ome pA, GpN6mA, G 2’ Ome pN6mA, U 2’Ome pU, UpA, U 2’Ome pA, UpN6meA, U 2’Ome pN6meA and one or more of; Trinucleotides, selected from: pApApG, pA 2’Ome pApG, pApA 2’Ome pG, pA 2’Ome pA 2’Ome pG, pN6mApApG, pN6mA 2’Ome pApG, pN6mApA 2’Ome pG, pN6mA 2’Ome pA 2’Ome pG, pN6mApN6mApG, pN6mA 2’Ome pN6mApG, pN6mA 2’Ome pN6mA 2’Ome pG, pN6mApN6mA 2’Ome pG, pApN6mApG, pA 2’Ome N6mApG, pA 2’Ome pN6mA 2’Ome pG, pAN6mA 2’Ome pG, pApUpG, pA 2’Ome pUpG, pApU 2’Ome pG, pA 2’Ome pU 2’Ome pG, ApApG, A 2’Ome pApG, ApA 2’Ome pG, A 2’Ome pA 2’Ome pG, N6mApApG, N6mA 2’Ome pApG, N6mApA 2’Ome pG, N6mA 2’Ome pA 2’Ome pG, N6mApN6mApG, N6mA 2’Ome pN6mApG, N6mA 2’ Ome pN6mA 2’Ome pG, N6mApN6mA 2’Ome pG, ApN6mApG, A 2’Ome N6mApG, A 2’Ome pN6mA 2’Ome pG, AN6mA 2’Ome pG, pApUpG, A 2’Ome pUpG, ApU 2’Ome pG, A 2’Ome pU 2’Ome pG, and one or more of; Tetranucleotides selected from: pApApApG, and one or more thereof.

7. The method according to any one of claims 1 to 6, wherein, The buffer is a buffer containing magnesium ions; Preferably, the buffer contains Tris-HCl, magnesium chloride, spermidine, and DTT; preferably, in this buffer, the concentration of Tris-HCl is 20 - 500 mM, the concentration of magnesium chloride is 30 - 500 mM, the concentration of spermidine is 0 - 50 mM, and the concentration of DTT is 5 - 500 mM; preferably, the pH of the Tris-HCl is between 7.0 and 8.0; Preferably, in the transcription reaction system, by volume, the content of the buffer is 5 - 20%.

8. The method according to any one of claims 1 to 6, wherein, The enzymes required for the transcription reaction include RNA polymerase and inorganic pyrophosphatase; Preferably, the RNA polymerase is T7 RNA polymerase; Preferably, in the transcription reaction system, the concentration of RNA polymerase is 5 - 50 U / μL, such as 30 - 50 U / μL; Preferably, in the transcription reaction system, the concentration of the inorganic pyrophosphatase is 0.00005 - 0.05 U / μL, such as 0.003 - 0.015 U / μL; Preferably, the transcription reaction system further contains a nuclease inhibitor; preferably, the concentration of the nuclease inhibitor in the transcription reaction system is 0.5 - 3 U / μL.

9. The method according to any one of claims 1 to 6, characterized in that In the transcription reaction system, the concentration of each NTP is 1 - 15 mM, such as 1 - 10 mM.

10. The method according to any one of claims 1 to 6, characterized in that In the transcription reaction system, the concentration of the polynucleotide is 1 - 15 mM, such as 1 - 10 mM or 3 - 8 mM.

11. The method according to any one of claims 1 to 6, characterized in that In the transcription reaction system, the concentration of the DNA template is 10 - 100 μg / mL.

12. The method according to any one of claims 1 to 11, characterized in that The DNA template may sequentially contain, from the 5' end to the 3' end: a promoter, the 5' sequence of the Anabeana intron splicing system, an IRES sequence, a coding sequence containing a start codon and a stop codon, and the 3' sequence of the Anabeana intron splicing system.

13. The method according to any one of claims 1 to 12, characterized in that Transcription is carried out at a temperature of 30 - 40 °C, preferably, the transcription time is 0.5 - 5 h; Preferably, the method further includes adding an appropriate amount of DNase I enzyme after the transcription reaction to digest the original DNA template; After digestion, the transcription product is separated, and then an appropriate amount of LiCl is added to the transcription product. After mixing, it is placed at a temperature below -15 °C for a period of time, and then centrifuged. After separating the precipitate, it is washed with ethanol to obtain the transcription product.

14. A circular RNA precursor, which is a linear RNA, characterized in that The 5'-end of the circular RNA precursor is the polynucleotide represented by the formula (I) described in any one of claims 1 to 3; preferably, the circular RNA precursor is prepared by the method described in any one of claims 1-10.

15. A method for preparing circular RNA, characterized in that The method includes the step of performing a cyclization reaction on the circular RNA precursor described in claim 14; preferably, the reaction system of the cyclization reaction contains the circular RNA precursor described in claim 14 and a buffer.

16. The method according to claim 15, characterized in that: (1) The method includes: performing cyclization using a ligase, and the cyclization reaction system contains a buffer, ATP, the circular RNA precursor described in claim 14, and a ligase; or (2) The method is based on the self-splicing and cyclization of group I intron ribozyme, and its cyclization reaction system contains the circular RNA precursor described in claim 14, a buffer, a magnesium salt, and GTP; or (3) The method is based on the self-splicing and cyclization of group II intron ribozyme, and its cyclization reaction system contains the circular RNA precursor described in claim 14 and a buffer.

17. The method according to claim 16, characterized in that: (1) In this case, the buffer is a buffer containing magnesium ions; preferably, the buffer contains Tris-HCl, magnesium chloride, and DTT; preferably, in this buffer, the concentration of Tris-HCl is 400-600 mM, the concentration of magnesium chloride is 50-150 mM, and the concentration of DTT is 5-15 mM; preferably, the pH of Tris-HCl is between 7.0 and 8.5, such as between 7.3 and 7.7; preferably, in the cyclization reaction system, the content of ATP is 0.01-10 mM, such as 0.5-2 mM; preferably, in the cyclization reaction system, the content of the circular RNA precursor is 0.1-1.0 mg / mL, such as 0.2-0.6 mg / mL; preferably, in the cyclization reaction system, the content of the ligase is 100-500 U / mL; (2) In this case, the concentration of the circular RNA precursor is 0.5-1.5 mg / mL, the concentration of the buffer is 20-200 mM and the pH is 7.0-8.5, the concentration of the magnesium salt is 1-20 mM, such as 5-20 mM, and the concentration of GTP is 0.1-10 mM, such as 1-10 mM; (3) In this case, in the reaction system, the concentration of the circular RNA precursor is 0.5-1.5 mg / mL, the concentration of the buffer is 20-80 mM and the pH is 7.0-8.5; preferably, the reaction system further contains a magnesium salt and / or an ammonium salt, wherein the concentration of the magnesium salt is 0.1-2 mM, such as 0.5-2 mM, and the concentration of the ammonium salt is 300-600 mM.

18. The method according to claim 13, characterized in that (1) In this case, the cyclization reaction system is placed at 18-37 °C for a period of time, such as 0.5-8 h, and after the reaction is completed, circular RNA is separated; In (2) and (3), the cyclization reaction system is placed under reaction at 50 - 57 °C for 1 - 10 minutes, and after the reaction ends, circular RNA is obtained by separation.

19. The method according to any one of claims 15 to 18, characterized in that The method further includes a purification step after the cyclization reaction; preferably, the purification is carried out using RNase R enzyme for purification, ion exchange liquid chromatography for purification, and / or PAGE gel for purification.

20. A circular RNA molecule, characterized in that The circular RNA molecule comprises the polynucleotide shown by formula (I) described in any one of claims 1 - 6 or the circular RNA precursor described in claim 14; preferably, the circular RNA molecule is prepared by the method described in any one of claims 15 - 19.

21. An application selected from the following: The application of the polynucleotide of formula (I) according to any one of claims 1 to 6 in the preparation of a circular RNA precursor or circular RNA; The application of the circular RNA precursor according to claim 14 in the preparation of circular RNA; The application of the circular RNA molecule according to claim 20 in the preparation of a lipid nanoparticle or pharmaceutical composition for treating or preventing a disease or disorder of a subject; Use of the polynucleotide of formula (I) according to any one of claims 1 to 6 in reducing the immunogenicity of a circular RNA molecule and / or enhancing the protein translation ability of a circular RNA molecule; Use of the circular RNA precursor according to claim 14 in reducing the immunogenicity of a circular RNA molecule and / or enhancing the protein translation ability of a circular RNA molecule.

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