Compositions and methods for producing circular RNA

Abstract

Compositions and methods for generating circular RNAs (circRNAs) are provided. Nucleic acid molecules (e.g., RNA molecules) are provided that include a ribozyme and an insert sequence. In some embodiments, the ribozyme within the RNA molecules of the present disclosure is cleaved. In some embodiments, the cleaved RNA molecule is circularized by a ligase. The present disclosure further provides methods and systems for generating circRNAs, as well as methods for using circRNAs (including therapeutic and preventative methods).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 435,767, filed December 28, 2022, and U.S. Provisional Patent Application No. 63 / 501,318, filed May 10, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains an ST.26 compliant Sequence Listing, which was submitted in XML format via the Patent Center and is incorporated herein by reference in its entirety. The XML Sequence Listing is a 216,644 byte file with the file name 153908_8001_WO00_SL.xml, created on December 20, 2023.

[0003] The present disclosure relates to compositions and methods for generating circular RNAs (circRNAs). Nucleic acid molecules (e.g., RNA molecules) are provided that include a ribozyme and an insert sequence. In some embodiments, the ribozyme within the RNA molecules of the present disclosure is cleaved. In some embodiments, the cleaved RNA molecule is circularized by a ligase. The present disclosure further provides methods and systems for generating circRNAs, as well as methods for using circRNAs (including therapeutic and preventive methods). [Background technology]

[0004] Circular RNA (circRNA) is a single-stranded RNA that can resist degradation by exonucleases. However, constructing and producing circRNA is difficult. There remains a need for a method that can efficiently circularize RNA (especially large RNA molecules) while reducing contamination with linear RNA, which can cause immunogenicity and reduce expression of inserted gene products. The present specification provides an embodiment that meets this need. Summary of the Invention [Means for solving the problem]

[0005] Provided herein are ribonucleic acid (RNA) molecules, combinations thereof, and reaction intermediates or reaction products thereof, including linear RNA, truncated linear RNA, and circular RNA (circRNA). In some embodiments, the RNA molecules and various reaction intermediates or reaction products provided herein are for producing circRNA. Additionally, provided are methods for producing the above-described RNA molecules, combinations thereof, reaction intermediates or reaction products thereof, or compositions comprising any of these. Additionally, provided are methods and uses of the above-described RNA molecules, combinations thereof, reaction intermediates or reaction products thereof, or compositions thereof, including therapeutic and prophylactic uses.

[0006] As used herein, a ribonucleic acid (RNA) molecule is defined as having, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme The present invention provides an RNA molecule comprising:

[0007] In some embodiments, the 5' substrate sequence comprises a 5' overhang sequence and the 3' substrate sequence comprises a 3' overhang sequence. In some embodiments, the 5' ribozyme and the 3' ribozyme can cooperate to cleave the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence, the insert sequence, and the 3' overhang sequence.

[0008] In some embodiments, the RNA molecule further comprises a 5' homology region and a 3' homology region. In some embodiments, the 5' homology region is located 3' to the 5' ribozyme. In some embodiments, the 3' homology region is located 3' to the insert sequence.

[0009] Further, as used herein, a truncated RNA molecule is defined as comprising, in order from the 5' end to the 3' end: 5' overhang sequence of the Twister-Sister ribozyme, a 5' homologous region; an insert sequence; a 3' homologous region; 3' overhang sequence of Twister ribozyme The present invention provides an RNA molecule comprising: In some embodiments, at least a portion of the 5' homologous region and at least a portion of the 3' homologous region are complementary and can form a stem structure.

[0010] In some embodiments, the cleaved RNA molecule comprises a hydroxyl group at the 5'-terminus and a 2',3'-cyclic phosphate at the 3'-terminus. In some embodiments, the hydroxyl group at the 5'-terminus of the cleaved RNA molecule and the 2',3'-cyclic phosphate at the 3'-terminus can be ligated in the presence of an RNA ligase to generate a circular RNA molecule.

[0011] In some embodiments, the RNA ligase is a tRNA splicing ligase. In some embodiments, the RNA ligase is an RtcB ligase. In some embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO: 43, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 43.

[0012] Further, as used herein, there is provided a circular RNA molecule comprising: 5' overhang sequence of the Twister-Sister ribozyme, a 5' homologous region; an insert sequence; a 3' homologous region; 3' overhang sequence of Twister ribozyme The present invention provides an RNA molecule comprising:

[0013] In some embodiments, the Twister-Sister ribozyme comprises a Twister-Sister-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme. In some embodiments, the Twister-Sister ribozyme comprises a TS-1 ribozyme.

[0014] In some embodiments, the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO:6. In some embodiments, the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6.

[0015] In some embodiments, the catalytic sequence of the Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO:4, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO:4. In some embodiments, the catalytic sequence of the Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO:4.

[0016] In some embodiments, the Twister ribozyme comprises a P1-type Twister ribozyme.In some embodiments, the Twister ribozyme comprises a P1-type Twister ribozyme derived from Parasitoid wasp.

[0017] In some embodiments, the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO:21. In some embodiments, the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21.

[0018] In some embodiments, the catalytic sequence of the twister ribozyme comprises the sequence set forth in SEQ ID NO: 23, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 23. In some embodiments, the catalytic sequence of the twister ribozyme comprises the sequence set forth in SEQ ID NO: 23.

[0019] In some embodiments, the 5' homology region comprises the sequence set forth in SEQ ID NO: 10, or a sequence having at least, or at least about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the 5' homology region comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the 3' homology region comprises the sequence set forth in SEQ ID NO: 19, or a sequence having at least, or at least about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 19. In some embodiments, the 3' homology region comprises the sequence set forth in SEQ ID NO:19.

[0020] In some embodiments, the insert sequence comprises a translation initiation element. In some embodiments, the translation initiation element is an internal ribosome entry site (IRES) or a translation initiator of short 5' UTR (TISU) element. In some embodiments, the translation initiation element comprises the sequence set forth in SEQ ID NO: 14, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 14. In some embodiments, the translation initiation element comprises the sequence set forth in SEQ ID NO: 14.

[0021] In some embodiments, the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules, in some embodiments, the one or more exogenous molecules are selected from a vaccine antigen, a cancer antigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, and a reporter molecule.

[0022] In some embodiments, the one or more exogenous molecules comprise a vaccine antigen. In some embodiments, the vaccine antigen comprises a viral vaccine antigen. In some embodiments, the vaccine antigen comprises a cancer antigen.

[0023] In some embodiments, the one or more exogenous molecules comprise a sequence-specific nuclease. In some embodiments, the sequence-specific nuclease is a Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas12 nuclease, or a Cas13 nuclease.

[0024] In some embodiments, the one or more exogenous molecules comprise an antibody or antigen-binding fragment thereof.

[0025] In some embodiments, the one or more exogenous molecules comprise an immunomodulatory polypeptide. In some embodiments, the immunomodulatory polypeptide comprises a cytokine.

[0026] In some embodiments, the one or more exogenous molecules comprise a transcription factor.

[0027] In some embodiments, the one or more exogenous molecules comprise a reporter molecule. In some embodiments, the reporter molecule comprises firefly luciferase, enhanced green fluorescent protein (eGFP), or red fluorescent protein (RFP). In some embodiments, the reporter molecule comprises firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 16.

[0028] In some embodiments, the length of the insert sequence is at least about 500 nucleotides (nt), about 750 nt, about 1000 nt, about 1250 nt, about 1500 nt, about 2000 nt, about 2500 nt, about 3000 nt, about 3500 nt, about 4000 nt, about 4500 nt, about 5000 nt, about 6000 nt, about 7000 nt, about 8000 nt, about 9000 nt, or about 10000 nt.

[0029] In some embodiments, in the population of RNA molecules, cleavage of the 5' substrate sequence and / or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population, hi some embodiments, in the population of RNA molecules, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are cleaved RNA molecules.

[0030] In some embodiments, in the population of RNA molecules, ligation of the 5' end to the 3' end occurs in the presence of an RNA ligase in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population. In some embodiments, in the population of RNA molecules, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are circular RNA molecules.

[0031] In some embodiments, the RNA molecule comprises a modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine or N 1 -Contains methyl methylpseudouridine.

[0032] In some embodiments, the RNA molecule reduces activation of or avoids detection by one or more Toll-like receptors (TLRs) when incubated with cells containing TLRs.

[0033] Further provided herein is a combination of RNA molecules, a first RNA molecule and a second RNA molecule; The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; Offer a combination.

[0034] In some embodiments, the 5' substrate sequence comprises a 5' overhang sequence. In some embodiments, the 5' ribozyme of a first RNA molecule and the trans-acting ribozyme of a second RNA molecule can cooperate to cleave the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence, an insert sequence, and the 3' overhang sequence.

[0035] In some embodiments, the cleaved RNA molecule further comprises a 5' homology region and a 3' homology region. In some embodiments, the 5' homology region is located 3' to the 5' ribozyme. In some embodiments, the 3' homology region is located 3' to the insert sequence. In some embodiments, at least a portion of the 5' homology region and at least a portion of the 3' homology region are complementary and can form a stem structure.

[0036] In some embodiments, the cleaved RNA molecule comprises a hydroxyl group at the 5'-terminus and a 2',3'-cyclic phosphate at the 3'-terminus. In some embodiments, the hydroxyl group at the 5'-terminus of the cleaved RNA molecule and the 2',3'-cyclic phosphate at the 3'-terminus can be ligated in the presence of an RNA ligase to generate a circular RNA molecule.

[0037] In some embodiments, the RNA ligase is a tRNA splicing ligase. In some embodiments, the RNA ligase is an RtcB ligase. In some embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO: 43, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 43.

[0038] In some embodiments, the Twister-Sister ribozyme comprises a Twister-Sister-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme. In some embodiments, the Twister-Sister ribozyme comprises a TS-1 ribozyme.

[0039] In some embodiments, the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO:6. In some embodiments, the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6.

[0040] In some embodiments, the catalytic sequence of the Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO:4, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO:4. In some embodiments, the catalytic sequence of the Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO:4.

[0041] In some embodiments, the Twister ribozyme comprises a P1-type Twister ribozyme.In some embodiments, the Twister ribozyme comprises a P1-type Twister ribozyme derived from Parasitoid wasp.

[0042] In some embodiments, the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO:21. In some embodiments, the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21.

[0043] In some embodiments, the catalytic sequence of the twister ribozyme comprises the sequence set forth in SEQ ID NO: 23, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 23. In some embodiments, the catalytic sequence of the twister ribozyme comprises the sequence set forth in SEQ ID NO: 23.

[0044] In some embodiments, the 5' homology region comprises the sequence set forth in SEQ ID NO: 10, or a sequence having at least, or at least about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 10. In some embodiments, the 5' homology region comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the 3' homology region comprises the sequence set forth in SEQ ID NO: 19, or a sequence having at least, or at least about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 19. In some embodiments, the 3' homology region comprises the sequence set forth in SEQ ID NO:19.

[0045] In some embodiments, the insert sequence comprises a translation initiation element. In some embodiments, the translation initiation element is an internal ribosome entry site (IRES) or a translation initiator of short 5' UTR (TISU) element. In some embodiments, the translation initiation element comprises the sequence set forth in SEQ ID NO: 14, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 14. In some embodiments, the translation initiation element comprises the sequence set forth in SEQ ID NO: 14.

[0046] In some embodiments, the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules, in some embodiments, the one or more exogenous molecules are selected from a vaccine antigen, a cancer antigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, and a reporter molecule.

[0047] In some embodiments, the one or more exogenous molecules comprise a vaccine antigen. In some embodiments, the vaccine antigen comprises a viral vaccine antigen. In some embodiments, the vaccine antigen comprises a cancer antigen.

[0048] In some embodiments, the one or more exogenous molecules comprise a sequence-specific nuclease. In some embodiments, the sequence-specific nuclease is a Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas12 nuclease, or a Cas13 nuclease.

[0049] In some embodiments, the one or more exogenous molecules comprise an antibody or antigen-binding fragment thereof.

[0050] In some embodiments, the one or more exogenous molecules comprise an immunomodulatory polypeptide. In some embodiments, the immunomodulatory polypeptide comprises a cytokine.

[0051] In some embodiments, the one or more exogenous molecules comprise a transcription factor.

[0052] In some embodiments, the one or more exogenous molecules comprise a reporter molecule. In some embodiments, the reporter molecule comprises firefly luciferase, enhanced green fluorescent protein (eGFP), or red fluorescent protein (RFP). In some embodiments, the reporter molecule comprises firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 16.

[0053] In some embodiments, the length of the insert sequence is at least about 500 nucleotides (nt), about 750 nt, about 1000 nt, about 1250 nt, about 1500 nt, about 2000 nt, about 2500 nt, about 3000 nt, about 3500 nt, about 4000 nt, about 4500 nt, about 5000 nt, about 6000 nt, about 7000 nt, about 8000 nt, about 9000 nt, or about 10000 nt.

[0054] In some embodiments, in the population of RNA molecules, cleavage of the 5' substrate sequence and / or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population, hi some embodiments, in the population of RNA molecules, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are cleaved RNA molecules.

[0055] In some embodiments, in the population of RNA molecules, ligation of the 5' end to the 3' end occurs in the presence of an RNA ligase in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population. In some embodiments, in the population of RNA molecules, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are circular RNA molecules.

[0056] In some embodiments, the RNA molecule comprises a modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine or N 1 -Contains methyl methylpseudouridine.

[0057] In some embodiments, the RNA molecule reduces activation of or avoids detection by one or more Toll-like receptors (TLRs) when incubated with cells containing TLRs.

[0058] Further provided herein are deoxyribonucleic acid (DNA) molecules. In some embodiments, the DNA molecules encode an RNA molecule provided herein, a first RNA molecule of a combination provided herein, a second RNA molecule of a combination provided herein, or the first and second RNA molecules of a combination provided herein.

[0059] Further provided herein are systems for producing circular RNA molecules. In some embodiments, the systems include the RNA molecules provided herein, or combinations provided herein.

[0060] Further provided herein are systems for producing circular RNA molecules, in some embodiments, the systems comprise the DNA molecules provided herein and reagents for in vitro transcription.

[0061] In some embodiments, the system further comprises an RNA ligase. In some embodiments, the RNA ligase is a tRNA splicing ligase. In some embodiments, the RNA ligase is an RtcB ligase. In some embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO: 43, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 43.

[0062] Further provided herein is a method of making a ribonucleic acid (RNA) molecule, the method comprising making an RNA molecule provided herein.

[0063] Further provided herein is a method for making a ribonucleic acid (RNA) molecule, comprising: From the 5' end to the 3' end, a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme The present invention provides a method for producing an RNA molecule comprising:

[0064] Further provided herein is a method of making a combination of ribonucleic acid (RNA) molecules, the method comprising making a combination provided herein.

[0065] Further provided herein is a method for making a combination of ribonucleic acid (RNA) molecules, comprising: generating a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; A method is provided.

[0066] In some embodiments, the method further comprises generating a cleaved RNA molecule by incubating the RNA molecule in solution.

[0067] Further provided herein is a method for producing a truncated RNA molecule, comprising: (1) producing an RNA molecule provided herein; and (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; The present invention provides a method comprising:

[0068] Further provided herein is a method for producing a truncated RNA molecule, comprising: (1) In the order from the 5' end to the 3' end, a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme producing an RNA molecule comprising: (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; The present invention provides a method comprising:

[0069] Further provided herein is a method for producing a truncated RNA molecule, comprising: (1) making a combination provided herein; and (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; The present invention provides a method comprising:

[0070] Further provided herein is a method for producing a truncated RNA molecule, comprising: (1) generating a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; and (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; Including, The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; A method is provided.

[0071] In some embodiments, the method further comprises generating a circular RNA molecule by incubating the RNA molecule with an RNA ligase.

[0072] Further provided herein is a method for producing a circular RNA molecule, comprising: (1) producing an RNA molecule provided herein; (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; The present invention provides a method comprising:

[0073] Further provided herein is a method for producing a circular RNA molecule, comprising: (1) In the order from the 5' end to the 3' end, a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme producing an RNA molecule comprising: (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; The present invention provides a method comprising:

[0074] Further provided herein is a method for producing a circular RNA molecule, comprising: (1) making a combination provided herein; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; The present invention provides a method comprising:

[0075] Further provided herein is a method for producing a circular RNA molecule, comprising: (1) preparing a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; Including, The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; A method is provided.

[0076] Further provided herein is a method for producing a circular RNA molecule, comprising: (1) producing an RNA molecule provided herein; (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. The present invention provides a method comprising:

[0077] Further provided herein is a method for producing a circular RNA molecule, comprising: (1) In the order from the 5' end to the 3' end, a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme producing an RNA molecule comprising: (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. The present invention provides a method comprising:

[0078] Further provided herein is a method for producing a circular RNA molecule, comprising: (1) making a combination provided herein; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. The present invention provides a method comprising:

[0079] Further provided herein is a method for producing a circular RNA molecule, comprising: (1) preparing a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. Including, The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; A method is provided.

[0080] In some embodiments, the circular RNA is generated by an RNA ligase that is endogenously present in the subject.

[0081] In some embodiments, the RNA molecule, or the first or second RNA molecule of the combination, is produced by in vitro transcription.

[0082] In some embodiments, the RNA molecule, or the first or second RNA molecule of the combination, is produced by RNA synthesis.

[0083] In some embodiments, the incubation step of step (2) generates a cleaved RNA molecule after the 5' substrate sequence and the 3' substrate sequence are cleaved by the catalytic sequence. In some embodiments, the incubation step of step (2) generates a hydroxyl group at the 5' end and a 2',3'-cyclic phosphate at the 3' end of the cleaved RNA molecule.

[0084] In some embodiments, the solution includes sodium acetate (NaOAc), magnesium acetate (MgOAc), or both. In some embodiments, the solution does not include potassium chloride (KCl) or magnesium chloride (MgCl).

[0085] In some embodiments, the solution contains cyclic diguanosine monophosphate (c-di-GMP). In some embodiments, the solution contains c-di-GMP at a concentration of about 0.5 mM to about 10 mM. In some embodiments, the solution contains c-di-GMP at a concentration of about 5 mM.

[0086] In some embodiments, the solution comprises distilled water (DW). In some embodiments, the solution consists of distilled water (DW).

[0087] In some embodiments, the solution comprises a Tris-EDTA (TE) buffer, and may comprise a TE buffer of pH 7.0 or a TE buffer of pH 8.0.

[0088] In some embodiments, the method further comprises a denaturation step and a renaturation step. In some embodiments, the denaturation step is carried out at about 60°C to about 85°C, about 65°C to about 80°C, about 65°C, or about 80°C. In some embodiments, the renaturation step comprises incubating at ambient temperature or 4°C after the denaturation step.

[0089] In some embodiments, the denaturing step and the renaturing step are performed in the solution for producing the cleaved RNA molecule, which in some embodiments comprises a Tris-EDTA (TE) buffer, and may comprise a TE buffer at pH 7.0 or a TE buffer at pH 8.0.

[0090] In some embodiments, in a population of RNA molecules produced by the method, cleavage of the 5' substrate sequence and / or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population. In some embodiments, in a population of RNA molecules produced by the method, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are cleaved RNA molecules.

[0091] In some embodiments, a circular RNA molecule can be generated by ligating the hydroxyl group at the 5' end of the cleaved RNA molecule to the 2',3'-cyclic phosphate at the 3' end in the presence of an RNA ligase.

[0092] In some embodiments, the incubation with the RNA ligase in step (3) is carried out for about 5 to about 60 minutes, about 10 to about 30 minutes, about 15 to about 25 minutes, or about 10 to about 20 minutes. In some embodiments, the incubation with the RNA ligase in step (3) is carried out for about 20 minutes.

[0093] In some embodiments, the incubation with RNA ligase in step (3) is carried out at about 30°C to about 40°C, about 35°C to about 39°C, or about 36°C to about 38°C. In some embodiments, the incubation with RNA ligase in step (3) is carried out at about 37°C.

[0094] In some embodiments, the incubation with RNA ligase in step (3) is carried out in the presence of Mg 2+ In some embodiments, the incubation with RNA ligase in step (3) is carried out in a buffer containing Tris-HCl, KCl, MgCl, and DTT. In some embodiments, the incubation with RNA ligase in step (3) is carried out in a buffer containing 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl, and 10 mM DTT.

[0095] In some embodiments, the incubation with RNA ligase in step (3) is carried out in the presence of Mg 2+ The procedure is performed in a buffer that does not contain

[0096] In some embodiments, the RNA ligase is a tRNA splicing ligase. In some embodiments, the RNA ligase is an RtcB ligase. In some embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO: 43, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 43.

[0097] In some embodiments, the method further comprises purifying the cleaved linear or circular RNA molecule. In some embodiments, the purification is performed by chromatography. In some embodiments, the chromatography is high-performance liquid chromatography (HPLC), size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), or size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC).

[0098] In some embodiments, the method further comprises concentrating the circular RNA molecules. In some embodiments, the concentrating step is carried out by incubation with a kinase. In some embodiments, the kinase comprises polynucleotide kinase (PNK). In some embodiments, the concentrating step is carried out by incubation with a phosphatase.

[0099] In some embodiments, the method further comprises analyzing the circular RNA molecule. In some embodiments, part of the analyzing step is performed by running the circular RNA on a gel (e.g., an agarose gel). In some embodiments, the gel containing the circular RNA is run at 4°C. In some embodiments, after running the gel, the gel is cooled on ice. As used herein, "running" a gel includes applying an electrophoretic field to the gel to move charged particles loaded into wells, as is well known to those skilled in the art.

[0100] In some embodiments, the enrichment step is performed by incubation with one or more ribonucleases, which in some embodiments include RNase R and / or a 5' phosphate-dependent exonuclease.

[0101] In some embodiments, in the population of RNA molecules produced by the method, ligation of the 5' end to the 3' end occurs in the presence of an RNA ligase in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population. In some embodiments, in the population of RNA molecules produced by the method, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are circular RNA molecules.

[0102] In some embodiments, the RNA molecule comprises a modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine or N 1 -Contains methyl methylpseudouridine.

[0103] Further provided herein are RNA molecules produced by the methods provided herein.

[0104] Further provided herein are truncated RNA molecules produced by the methods provided herein. In some embodiments, the RNA molecules are linear RNA molecules.

[0105] Further provided herein are circular RNA molecules produced by the methods provided herein.

[0106] Further provided herein are compositions comprising the RNA molecules provided herein.

[0107] Further provided herein are compositions comprising the truncated RNA molecules provided herein.

[0108] Further provided herein are compositions comprising the circular RNA molecules provided herein.

[0109] Further provided herein are compositions comprising the combinations provided herein.

[0110] In some embodiments, the composition is a pharmaceutical composition, hi some embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0111] In some embodiments, the composition comprises a lipid nanoparticle (LNP).

[0112] Further provided herein are methods of vaccinating a subject, the method comprising administering an RNA molecule provided herein, a combination provided herein, an RNA molecule produced by a method provided herein, or a composition provided herein.

[0113] Further provided herein are methods of treating a disease or disorder in a subject, the method comprising administering an RNA molecule provided herein, a combination provided herein, an RNA molecule produced by a method provided herein, or a composition provided herein.

[0114] Further provided herein is an RNA molecule provided herein, a combination provided herein, an RNA molecule produced by a method provided herein, or a composition provided herein for use in vaccinating a subject, wherein the RNA molecule or composition is administered to the subject.

[0115] Further provided herein is an RNA molecule provided herein, a combination provided herein, an RNA molecule produced by a method provided herein, or a composition provided herein for use in treating a disease or disorder in a subject, wherein the RNA molecule or composition is administered to the subject.

[0116] Further provided herein is the use of an RNA molecule provided herein, a combination provided herein, an RNA molecule produced by a method provided herein, or a composition provided herein in the manufacture of a medicament for vaccinating a subject, wherein the medicament is administered to the subject.

[0117] Further provided herein is the use of an RNA molecule provided herein, a combination provided herein, an RNA molecule produced by a method provided herein, or a composition provided herein in the manufacture of a medicament for the treatment of a disease or disorder in a subject, wherein the medicament is administered to the subject. [Brief explanation of the drawings]

[0118] [Figures 1A-1D]Figure 1A shows a schematic diagram illustrating the synthesis of exemplary circular RNAs (circRNAs) by unimolecular synthesis using a 5′ Twister-Sister ribozyme and a 3′ Twister ribozyme (Figure 1A) or by bimolecular synthesis using a 5′ Twister-Sister ribozyme and a trans-acting Twister ribozyme (Figure 1B), followed by ligation / circularization using RNA ligase. Figure 1A shows the design of an exemplary construct U1, which contains a 5′ Twister-Sister-1 (TS-1) ribozyme and a 3′ Twister ribozyme derived from N. vitripennis, with an internal ribosome entry site (IRES) sequence and an exemplary insert (the coding sequence for firefly luciferase) between the 5′ TS-1 ribozyme and the 3′ Twister ribozyme. Figure 1B shows the design of an exemplary construct B1, which contains a substrate moiety for a 3' twister ribozyme at its 3' end, which can be cleaved by construct B2, which contains a trans-acting twister ribozyme. Cleavage of the 5' and 3' ends yields a linear pre-circRNA containing a 5' hydroxyl group and a 2',3'-cyclic phosphate at the 3' end. This linear pre-circRNA is then circularized by ligation with a tRNA splicing ligase, such as RtcB ligase, to generate a circRNA.

[0119] Figure 1C shows the RNA folding predictions of the secondary structure of the precursor RNA of construct U1 (SEQ ID NO: 29) before and after cleavage by the 5' ribozyme and 3' ribozyme. The arrows indicate the cleavage sites of the 5' Twister-Sister ribozyme and the 3' Twister ribozyme, respectively. The insets, from left to right, show the portions of construct U1 corresponding to SEQ ID NO: 119, SEQ ID NO: 116-118, and SEQ ID NO: 120-121, respectively. Figure 1D shows the RNA folding predictions of the secondary structure of the precursor RNA of construct B1 (SEQ ID NO: 37) and construct B2 (SEQ ID NO: 35) before and after cleavage by the 5' ribozyme and 3' cleavage by the trans-acting ribozyme. The arrows indicate the cleavage sites of the 5' TS-1 and the trans-acting Twister ribozyme, respectively. The small dotted box shows the RNA folding prediction of the secondary structure of Construct B2, a trans-acting Twister ribozyme specific for the 3' substrate contained in Construct B1. The insets show, from left to right, the portions of Construct B1 and Construct B2 corresponding to SEQ ID NOs: 122-123, 35, and 120-121, respectively.

[0120] [Figures 2A-2C]Electrophoretic gel profiles showing the circularization efficiency of various constructs using various 5' and 3' ribozymes are shown. Figure 2A shows electrophoretic profiles of RNA preparations, showing cleavage intermediates of construct 3 containing the 5' P3 Twister U2A ribozyme and the 3' P1 Twister ribozyme after in vitro transcription with and without RtcB circularization and with and without RNase R enrichment. Band a: Linear RNA, including uncleaved precursor RNA, RNA cleaved at only one end (single cleavage), and pre-circ RNA cleaved at both the 5' and 3' ends; band b: Circular RNA; band c: Cleaved 5' ribozyme; band d: Cleaved 3' ribozyme. Figure 2B shows electrophoretic profiles showing the cleavage activity of various constructs containing the 5' Twister-Sister ribozyme or Twister ribozyme and various 3' ribozymes. Lanes 1, 3, 5, 7, and 9: RNA samples before the cleavage reaction. Lanes 2, 4, 6, 8, and 10: RNA samples after the cleavage reaction in cleavage buffer 1. Figure 2C shows a bar graph plotting the relative intensities of the cleaved 5' ribozyme bands after 1 hour of cleavage reaction.

[0121] [Figures 3A-3F]Electrophoretic profiles showing circularization efficiency under various cleavage and ligase buffer conditions are shown. The circularization rate was quantified by measuring the bands densitometrically. Figure 3A shows the percentage of circRNA generated after cleavage under various cleavage buffer conditions. Lanes 2–6 show the results of in vitro transcription of precursor RNA cleaved in commercially available T7 RNA polymerase buffers from Thermo Fisher Scientific (lane 2), Promega Biosciences (lane 3), Roche Biosciences (lane 4), or MEGAscript Biosciences (lane 6), or in cleavage buffer 1 (lane 5). Lane 1: No cleavage reaction (negative control). Figure 3B shows the percentage of circRNA generated after cleavage in cleavage buffers containing various additives or various buffer compositions. Lane 1: Cleavage buffer 1. Lanes 2 and 3: Cleavage buffer 1 supplemented with low (0.5 mM) or high (5 mM) concentrations of cyclic di-GMP (c-di-GMP), respectively. Lanes 4 and 5: Cleavage buffer 2 and cleavage buffer 3. Figure 3C shows a comparison of the percentage of circRNA generated after cleavage in cleavage buffer 1 or cleavage buffer 2 with (+) or without (-) c-di-GMP (5 mM). Figure 3D shows the effect of magnesium ions (Mg2+) on the stability of generated circRNA and the generation of nicked circular RNA at various temperatures. Figure 3E shows the effect of the amount of RtcB ligase in the ligation reaction on circularization efficiency. Figure 3F shows the effect of the length of the RtcB ligation reaction time on circularization efficiency.

[0122] [Figures 4A-4D]We demonstrate the function of circRNAs and linear intermediate RNAs generated using an exemplary unimolecular construct U1, introduced into mammalian cells, and expressed the encoded gene products. Figure 4A shows electrophoretic profiles of linear RNA in vitro transcribed from construct U1 (lane 1; "IVT"), cleaved linear RNA in vitro transcribed from construct U1 (lane 2; "CLV"), and cleaved linear RNA in vitro transcribed from construct U1, circularized with RtcB, and enriched by RNase R treatment (lane 3; "RtcB+RR") in transfected HEK 293T cells. Figure 4B shows circRNA production measured by qPCR using primers targeting the circRNA junction site 24 hours after transfection. Average expression levels were calculated as the mean. P values ​​were calculated by one-way ANOVA. NS: not significant; **P<0.005. Figure 4C shows luciferase activity in HEK 293T cells transfected with various RNA preparations (IVT, CLV, RtcB+RR) derived from construct U1 after 24, 48, 72, 96, and 120 hours. Figure 4D shows the kinetics of luciferase activity over 120 hours in HEK 293T cells transfected with various RNA preparations (IVT, CLV, RtcB+RR) derived from construct U1, compared with HEK 293T cells transfected with linear mRNA encoding firefly luciferase containing unmodified uridine (U) or modified pseudouridine (pU; N1-methylpseudouridine).

[0123] [Figure 5] Cleavage activity of construct B1 by the trans-acting Twister ribozyme of construct B2 in cleavage buffer 1 or MEGAscript T7 RNA transcription buffer for the cleavage reaction in the presence (+) or absence (-) of ligation reaction with RtcB and / or in the presence (+) or absence (-) of enrichment with RNase R. Circularization efficiency was measured by measuring each band by densitometry.

[0124] [Figures 6A-6B] Figure 6A shows gel electrophoresis of RT-PCR products generated using the exemplary unimolecular construct U1 or the exemplary bimolecular construct B1 / B2 pair in the presence (+) or absence (-) of RtcB ligase treatment, using a junction site-specific primer (upper panel) or a non-junction site-specific (internal) primer (lower panel). Figure 6B shows Sanger sequencing results of the circRNA junction site sequence, which perfectly matches the prediction based on the ribozyme cleavage and ligation sites. A diagram showing the relative positions and orientations of RT-PCR primer pairs for amplifying the internal region or circRNA junction site is shown. This figure discloses SEQ ID NO: 124.

[0125] [Figures 7A-7D] Figure 1 shows the function of circRNAs and linear intermediate RNAs generated using an exemplary bimolecular construct B1 / B2 pair, the conversion of linear RNAs to circRNAs in mammalian cells, and the expression of the encoded gene products.

[0126] Figure 7A shows the results of gel electrophoresis of each RT-PCR product using junction-specific primers (upper panel) or non-junction-specific (internal) primers (lower panel). HEK 293T cells or control cells were transfected with linear RNA in vitro transcribed from construct B1 ("IVT"), construct B1 cleaved by incubation with the trans-acting ribozyme of construct B2 ("CLV"), or in vitro transcribed linear RNA from construct B1 cleaved by incubation with construct B2 followed by circularization with RtcB and enrichment by RNase R treatment ("RtcB+RR"). 24 hours later, RNA was extracted and subjected to RT-PCR and gel electrophoresis. Figure 7B (semi-quantitative PCR) and Figure 7C (quantitative PCR) show the relative amounts of junction sites (indicating circRNA formation) amplified from HEK 293T cells transfected with construct B1 / B2 CLV or RtcB+RR preparations, plotted as bar graphs. Figure 7D shows luciferase activity after 24 hours in HEK 293T cells transfected with various RNA preparations (IVT, CLV, RtcB+RR) derived from construct B1 / B2.

[0127] [Figure 8] Electrophoretic profiles showing the efficiency of circRNA generation using the exemplary unimolecular construct U1 or group I intron PIE system. Each RNA molecule contained either an unmodified uridine residue (unmodified U) or a modified pseudouridine (pseudo-U; N1-methylpseudouridine) and was run in the presence (+) or absence (+) of incubation with RtcB ligase and / or enrichment with RNase R.

[0128] [Figures 9A-9D]Figure 9A shows the HPLC profiles of cleaved linear RNA preparations (Figure 9A shows RNA containing unmodified uridine (U), and Figure 9C shows RNA containing N1-methylpseudouridine (m1Ψ)), or RNA preparations containing a mixture of circRNA and linear RNA obtained after ligation with RtcB treatment (Figure 9B shows RNA containing unmodified uridine, and Figure 9D shows RNA containing m1Ψ). Both RNA preparations were made using the exemplary unimolecular construct U1. Figure 9E shows the gel electrophoresis profile of each RNA preparation after HPLC (Figure 9E).

[0129] [Figures 10A-10C] We incubated HEK-Blue TLR-3 (Figure 10A), HEK-Blue TLR-7 (Figure 10B), or HEK-Blue TLR-8 (Figure 10C) reporter cells with truncated linear RNA preparations made using the exemplary unimolecular construct U1, or RNA preparations containing a mixture of circRNA and linear RNA obtained after ligation with RtcB treatment, and measured SEAP reporter activity in the culture supernatants. Each RNA preparation contained unmodified uridine (U) or N1-methylpseudouridine (m1Ψ). Linear mRNA encoding firefly luciferase, polyI:C (TLR3 ligand), or R848 (TLR7 / 8 ligand) was used as controls. Averages were calculated as the mean. P values ​​were calculated by one-way ANOVA. ***P<0.001, and ****P<0.0001.

[0130] [Figures 11A-11B]A schematic diagram showing the various purification and enrichment steps used to purify and enrich circRNAs in the preparation is shown. These steps include sequential treatment with T4 polynucleotide kinase (PNK), 5' phosphate-dependent exonuclease, and RNase R. Figure 11A shows a schematic diagram showing the reactions catalyzed by PNK, 5' phosphate-dependent exonuclease, and RNase R, to which circRNAs are resistant. After sequential treatment, the resulting preparation is enriched in circRNAs. Figure 11B shows the electrophoretic profile and percentage of circRNAs after enrichment by treatment with various enzymes. The cleaved RNA was purified by HPLC and sequentially treated with the following enzymes: Lane 1: treatment with RtcB only; Lane 2: treatment with RtcB followed by treatment with PNK and then treatment with 5' phosphate-dependent exonuclease; Lane 3: treatment with RtcB followed by treatment with PNK and then treatment with RNase R; Lane 4: treatment with RtcB followed by treatment with PNK and then treatment with 5' phosphate-dependent exonuclease and RNase R.

[0131] [Figures 12A-12B] Schematic diagram showing the conserved features of twister ribozymes and twister-sister ribozymes. Figure 12A shows the consensus sequence and secondary structure model of twister ribozymes (left panel, including secondary structure diagrams of P1-type twister ribozymes, P3-type twister ribozymes and P5-type twister ribozymes) and twister-sister ribozymes (right panel). Figure 12B shows the structure of a bimolecular construct derived from TS-1 ribozyme (see Roth et al. Nat Chem Biol, 10(1), 56-60 (2014); Weinberg et al., Nat Chem Bio, 11(8):606-610 (2015)). This figure shows SEQ ID NO: 125.

[0132] [Figures 13A-13B]Figure 13A shows the cleavage efficiency of the 3' twister ribozyme in generating circRNA. Figure 13A shows a schematic diagram of an exemplary construct U1 modified with 3' extension sequences of various lengths. These constructs are designated construct U2, construct U3, and construct U4. Figure 13B shows the electrophoretic profiles of RNA species after cleavage and ligation of various constructs with 3' extensions of various lengths.

[0133] [Figures 14A-14F] Figure 14 shows gel electrophoresis profiles indicating the circRNA production efficiency under various cleavage and ligation conditions. Figure 14A shows the gel electrophoresis profiles and circRNA production percentage (circularization efficiency) after no separate cleavage reaction ("No cleavage (DW)") or after a separate cleavage reaction in cleavage buffer ("Cleaving"). Figure 14B shows the gel electrophoresis profiles and circRNA percentage after ligation in buffers with various MnCl2 concentrations. Figure 14C shows the gel electrophoresis profiles and circRNA percentage after ligation in RtcB buffers with various pHs. Figure 14D shows the gel electrophoresis profiles and circRNA percentage after ligation in solutions containing individual components of RtcB buffer. Figure 14E shows the gel electrophoresis profiles and circRNA percentage after denaturation / renaturation in TE buffers with various pHs. Figure 14F shows the gel electrophoresis profiles of various exemplary constructs (Construct U1, Construct U2, Construct U3, and Construct U4) after denaturation and renaturation in TE buffer and ligation in RtcB reaction buffer, as well as the percentage of circRNAs produced from these constructs.

[0134] [Figures 15A-15C]Figure 15A shows that circular RNA (+RtcB) is cleaved when gel electrophoresis is performed at room temperature. Figure 15B shows that the same circular RNA (+RtcB) used in Figure 15A is not cleaved when gel electrophoresis is performed at 4°C. Linear RNA (-RtcB) was used as a control to determine whether or not circular RNA was cleaved. Figure 15C shows that circular RNA (+RtcB) is cleaved when exposed to denaturing conditions (65°C for 3 minutes) and then gel electrophoresed at 4°C. In contrast, circular RNA not exposed to these denaturing conditions is not cleaved and remains circular.

[0135] [Figures 16A-16B] Figure 16A shows that the heat generated at 4°C cleaves the circular RNA when the circular RNA (+RtcB) is gel-run for a relatively long time (10 min) at 4°C. Figure 16B shows that gel-run at 4°C for 3 min, followed by cooling on ice for 10 min, repeating this procedure at least three times, prevents the circular RNA from being cleaved.

[0136] [Figures 17A-17B] Figure 17A shows the circularization efficiency of genes of various lengths. RNAs expressing EGFP (720 bp), human erythropoietin (hEPO, 582 bp), firefly luciferase (FLuc, 1653 bp), or Cas9 (3270 bp) in the LVT14 format were circularized by RtcB. Following the circularization reaction, gel electrophoresis of the circularized RNA (+RtcB) or linear RNA (-RtcB) was performed using the cooling conditions described in Figures 16A and 16B. Figure 17B shows the circularization efficiency of each gene of the indicated gene size, quantified using Image Lab software (Bio-Rad).

[0137] [Figures 18A-18C]The results of a comparison of the functions of IRES from various viruses based on the IRES from Coxsackievirus (CVB3) are shown. Figure 18A shows a schematic diagram illustrating the structure of the CVB3 IRES that induces FLuc expression and the domains to which the key translation factors eIF4G and eIF4A bind. Figure 18B shows a schematic diagram illustrating the CVB3 IRES that expresses FLuc and the IRESs of other viruses. Figure 18C shows the results of measuring firefly luciferase (FLuc) activity 24 hours after transfection of HEK293T cells with circRNAs containing the IRESs of CVB3 or other viruses indicated in the graph. FLuc activity was normalized by dividing the FLuc activity of each sample by the Renilla luciferase activity used in the cotransfection. The relative fold FLuc activity was calculated by dividing the normalized FLuc activity of each IRES by the FLuc activity of the CVB3 IRES. Data are means ± sem for n = 4 biological replicates.

[0138] [Figures 19A-19B] Figure 19 shows the enhancement of FLuc activity by recombinant CVB3 IRES with aptamers that bind to poly(A)-binding protein (PABP) and / or eIF4G. Figure 19A shows a schematic diagram of recombinant CVB3 IRES, showing the location of aptamers that bind to PABP and eIF4G in LVT14. Figure 19B shows the relative FLuc activity expressed by each recombinant CVB3 IRES, calculated by comparison with the activity of CVB3 IRES (LVT14). Data are means ± sem, with n = 3 biological replicates.

[0139] [Figures 20A-20G]These results demonstrate that circRNAs can evade detection by TLRs, RIG-I, and MDA5. Figure 20A shows linear or circular RNAs purified by HPLC and confirmed by gel electrophoresis using the cooling conditions described in Figure 16. Figures 20B-20G show the results of transfecting HEK reporter cell lines expressing human TLR-3 (Figure 20B), human TLR-7 (Figure 20C), human TLR-8 (Figure 20D), or RIG1 (Figure 20E), or A549 cells with RIG1 (Figure 20F) or MDA5 (Figure 20G) knockout (KO), with purified linear RNA, circular RNA, mRNA containing 5-methylpseudouridine (5MoU), or the corresponding receptor agonists (3-phpRNA, Poly I:C, or R848), respectively. Twenty-four hours after stimulation, secreted alkaline phosphatase (SEAP) or expressed lucia luciferase activity was measured in the corresponding cell lines following each stimulus. Data are means ± sem of n = 4 biological replicates.

[0140] [Figures 21A-21C] Evaluation of the immunogenicity of circular RNA. Figures 21A-21C show the results of ELISA measurement of IL-6 (Figure 21A), IFN-β (Figure 21B), or RANTES (Figure 21C) in supernatants from cell lines treated with purified linear RNA, circular RNA, or mRNA containing 5-methylpseudouridine (5MoU), or the corresponding receptor agonists (3-phpRNA, Poly I:C, or R848) 24 hours after stimulation of A549 cells. Data are mean ± sem for n = 3 biological replicates.

[0141] [Figures 22A-22C]Figure 22 shows that ex vivo, circular RNA exhibits a threefold longer half-life and 45-fold higher FLuc activity than mRNA (5MoU). 293T cells were treated with FLuc mRNA (5MoU) or circular RNA encapsulated in lipid nanoparticles (LNPs), and FLuc activity was measured 7 days after treatment. Figure 22A shows normalized FLuc activity by dividing the FLuc activity measured at each time point by the FLuc activity 24 hours after transfection. Half-life was calculated based on the normalized FLuc activity. Figure 22B shows absolute FLuc activity from mRNA or circular RNA measured daily over 7 days after transfection. Figure 22C shows cumulative FLuc activity from mRNA or circular RNA over 7 days.

[0142] [Figures 23A-23C] Figure 23A shows that circular RNA exhibits 7.6-fold higher FLuc activity in vivo than mRNA (5MoU). Figure 23A shows the results of treating mice with FLuc mRNA (5MoU) or circRNA FLuc encapsulated in lipid nanoparticles (LNPs), and measuring FLuc activity by in vivo luminescence imaging at the indicated time points over a 9-day period. Figure 23B shows the results of injecting FLuc mRNA (5MoU) or FLuc circRNA into each group (n=5), and measuring FLuc activity by in vivo luminescence imaging at the indicated time points over a 9-day period. Figure 23C shows the cumulative FLuc activity of mRNA or circular RNA measured in each group over a 9-day period. DETAILED DESCRIPTION OF THE INVENTION

[0143] The present invention provides compositions and methods for producing circular RNA (circRNA). In some embodiments, the compositions of the present invention comprise nucleic acid molecules such as RNA molecules, such as linear RNA molecules, truncated linear RNA molecules, and circRNA molecules. In some embodiments, the RNA molecules provided herein comprise a ribozyme, such as a self-cleaving ribozyme, at one or both ends of the nucleic acid molecule. In some embodiments, a combination of nucleic acid molecules is further provided, such as a combination of RNA molecules comprising a first RNA molecule comprising a ribozyme and a second RNA molecule comprising a trans-acting ribozyme. In some embodiments, the ribozyme contained in the RNA molecules provided herein or the combination thereof is cleaved to generate a truncated linear RNA molecule. In some embodiments, the cleaved linear RNA molecule is circularized in the presence of a ligase, such as an RNA ligase, to generate a circRNA. In some embodiments, the cleaved RNA molecule is circularized in the presence of an endogenous ligase, such as an endogenous ligase of a mammalian cell. In some embodiments, the generated circRNA molecules can be used to deliver gene products encoded by insert sequences within the circRNA molecules to cells, such as mammalian cells, and subjects, such as mammals or humans, to express the gene products in these cells and subjects. In some embodiments, the embodiments provided herein enable efficient generation of circRNAs, particularly for large RNA molecules, to consistently and stably express the encoded gene products in cells or subjects.

[0144] Further provided herein are methods for producing the RNA molecules, combinations, intermediates, reaction products, and circRNAs provided herein. In some situations, the methods provided herein, including specific reaction conditions and components, can improve the efficiency and yield of the generated RNA molecules, combinations, intermediates, reaction products, and circRNAs, resulting in improved circRNA production efficiency, improved purity and stability of the generated circRNA compositions, improved gene product expression, and / or reduced immunogenicity. Further provided are methods and uses of the RNA molecules, combinations, intermediates, reaction products, and circRNAs provided herein, for example, in therapeutic and prophylactic uses, such as treating diseases or disorders and vaccinating subjects. The methods and uses provided herein may be applicable to the treatment of various patients with various diseases, including infectious diseases and cancer, the development of therapeutics, and / or the development of vaccines for various diseases. Further provided are generated RNA molecules, combinations, intermediates, reaction products, circRNAs, and kits for use in producing the RNA molecules provided herein and / or in the methods provided herein.

[0145] In some embodiments, the reduced immunogenicity includes reduced detection by pattern recognition receptors, hi some embodiments, the reduced immunogenicity includes reduced detection by TLR, RIG-1, or MDA5.

[0146] In some aspects, the embodiments provided herein provide various advantages and improvements with respect to generating RNA molecules for delivery of genes and gene products (e.g., therapeutic and / or vaccinal gene products) to cells or subjects. In some aspects, the methods and compositions for generating circRNAs provided herein enable stable protein expression in cells or subjects. CircRNAs are single-stranded RNAs that are resistant to degradation by exonucleases. In some aspects, the structure of circRNAs confers numerous advantages compared to linear mRNAs, including extended half-life, enhanced gene product expression, functional stability, and reduced immunogenicity. In some embodiments provided herein, circRNAs are generated using self-cleaving ribozymes, such as twister ribozymes and twister-sister ribozymes.

[0147] In some embodiments, circRNAs exhibit higher protein expression than corresponding non-circular RNAs or are capable of higher protein expression than corresponding non-circular RNAs. As used herein, a "corresponding non-circular RNA" refers to a non-circular RNA molecule that has substantially the same components as the compared circRNA (i.e., substantially the same promoter, coding region, and regulatory elements). The corresponding non-circular RNA may also be an mRNA molecule. In some embodiments, circRNAs exhibit higher protein expression than corresponding non-circular mRNAs.

[0148] Existing circRNA production methods have various limitations. First, methods such as permuted intron-exon (PIE) splicing have significant limitations in terms of circRNA purification due to contamination with nicked circRNAs consistently generated during the circularization reaction. Another issue with existing methods is that the efficiency of circRNA production decreases as the insert size increases, indicating an inverse correlation between insert size and circRNA production efficiency. Furthermore, existing methods such as PIE splicing cannot use modified nucleosides, such as modified uridines, within RNA molecules without significantly affecting circRNA production efficiency. Therefore, there is a need for methods and compositions that can efficiently generate circRNAs, produce pure circRNA compositions without contamination by RNA species such as nicked circRNAs, and improve circRNA production efficiency, particularly for large inserts. As demonstrated in the examples described herein, the embodiments provided herein meet this need.

[0149] As observed and described in the examples provided herein, exemplary molecules generated according to the embodiments provided herein can efficiently cleave RNA molecules, efficiently ligate the cleaved RNA molecules, and stably express gene products in cells into which various forms of RNA molecules, such as cleaved linear RNAs and circRNAs, have been introduced. In some aspects, the embodiments provided herein can directly deliver cleaved linear RNA molecules into cells, where they can be circularized by endogenous ligases present in the cells to produce circRNAs within the cells. In some situations, the embodiments provided herein can reduce the need for additional ligation reactions to produce circRNAs. In some aspects, the embodiments provided herein are based on the observation that exemplary molecules generated according to the embodiments provided herein can improve the cleavage, circularization, and gene product expression steps, particularly for RNA molecules containing large inserts, and can reduce the immunogenicity of the RNA molecules, thereby improving gene product expression.

[0150] Furthermore, the embodiments provided herein can improve the purity of the resulting circRNA-containing composition, thereby reducing the proportion of undesired RNA species, such as linear RNA or nicked circRNA, and increasing the yield of circRNA. In various aspects, the RNA molecules provided herein have been observed to reduce or eliminate undesired immune responses, as evidenced, for example, by reduced activation of one or more Toll-like receptors (TLRs) or avoidance of detection by one or more TLRs when incubated with TLR-expressing cells. Furthermore, the embodiments provided herein have improved the purity and efficiency of large inserts, thereby improving the efficient translation and durable expression of gene products in cells, such as mammalian cells. Furthermore, as described in the Examples, the embodiments provided herein enable the use of modified nucleosides, such as pseudouridine, within RNA molecules without significantly affecting the efficiency of circRNA production. RNA molecules obtained by the present invention, such as circRNA molecules and compositions containing the circRNA molecules, can be used in a variety of therapeutic and prophylactic applications, including, for example, infectious disease vaccines, cancer vaccines, immunotherapy, gene therapy, and gene editing.

[0151] All publications mentioned in this application, including patent documents, scientific articles, databases, etc., are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were incorporated by reference. To the extent that a definition set forth herein is contrary to or inconsistent with a definition in a patent, application, published application, or other publication incorporated by reference herein, the definition set forth herein shall take precedence over the definition incorporated by reference herein.

[0152] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein.

[0153] I. Nucleic acid molecules for the generation of circular RNA (circRNA) Provided are nucleic acid molecules, e.g., RNA molecules, combinations thereof, and their reaction intermediates or reaction products, including linear RNA, truncated linear RNA, and circular RNA (circRNA), as well as methods, kits, and systems for producing circRNA. In some aspects, the embodiments provided herein can improve the efficiency of circRNA production and the purity and stability of compositions containing circRNA, thereby improving the expression of gene products (e.g., encoded by inserts contained in nucleic acid molecules) in cells or subjects.

[0154] In some aspects, embodiments provided herein include utilizing a unimolecular method to generate circRNAs using, for example, a single RNA molecule containing two ribozymes at the 5' and 3' ends.

[0155] In some aspects, embodiments provided herein include utilizing a bimolecular method to generate circRNAs using two separate RNA molecules, for example, an RNA molecule comprising a 5' ribozyme and a 3' substrate sequence, and a separate RNA molecule comprising a trans-acting ribozyme.

[0156] In some embodiments, methods are further provided, such as methods for generating circRNAs by specific cleavage and ligation / circularization reactions, and methods for improving the efficiency of the process for generating circRNAs.

[0157] A. Circular RNA (circRNA) This paper provides compositions, systems, and methods for generating circular RNAs (circRNAs). CircRNAs are continuous loops of single-stranded RNAs covalently closed. CircRNAs can be classified into four types: exonic circRNAs (ecircRNAs), circular intronic RNAs (ciRNAs), exon-intron circRNAs (ElciRNAs), and intergenic circRNAs.

[0158] When delivering RNA-encoded genes into cells to express gene products, RNA stability is one of the crucial factors in enhancing gene product expression. To overcome the problems of linear mRNA, which is unstable and potentially immunogenic, various strategies have been developed. These include the use of untranslated regions (UTRs), such as those present in native β-globin mRNA, the use of methylguanosine cap analogs to protect mRNA from decapping enzymes, nucleoside modifications, and codon optimization. However, only modest improvements have been observed.

[0159] The structure of circRNAs offers several advantages over linear mRNAs, including resistance to exonuclease degradation, improved stability, prolonged half-life, increased protein expression, and reduced immunogenicity (Chen, RNA Biol, 12(4):381-388 (2015); Wesselhoeft et al., Nat Commun, 9(1):2629 (2018)). CircRNAs typically have a longer half-life compared to their linear counterparts (Wesselhoeft et al., Nat Commun, 9(1):2629 (2018)). Therefore, circRNAs enhance protein (e.g., encoded gene product) expression during their lifespan compared to linear mRNAs.

[0160] Existing methods for synthesizing circRNAs involve creating linear RNA precursors and then ligating their 5' and 3' ends to obtain covalently closed loops. CircRNAs can be synthesized in vitro using chemical, enzymatic, or ribozyme-based methods. One such method is permutation intron-exon (PIE) splicing, which involves circularizing RNA and expressing gene products from circRNAs. The PIE splicing system, based on group I introns naturally found in rRNA, tRNA, and mRNA genes in non-metazoan eukaryotes and bacteria, can generate circRNAs by self-splicing. However, this method has a significant limitation in terms of circRNA purification due to contamination with nicked circRNAs, which are consistently generated during the circularization reaction. Because nicked and unnicked circRNAs have the same molecular weight, it is extremely difficult to separate these two RNA species using methods such as high-performance liquid chromatography (HPLC). Furthermore, when introduced into a subject, nicked circRNAs stimulate the innate immune system and induce immunogenicity, often resulting in reduced translation and production of the desired gene product in the subject. Another problem with existing methods is that the larger the insert size, the lower the circRNA production efficiency, indicating an inverse correlation between insert size and circRNA production efficiency. Furthermore, existing methods, such as PIE splicing, cannot use modified nucleosides, such as modified uridines, within RNA molecules without significantly affecting circRNA production efficiency.

[0161] Embodiments provided herein include unimolecular methods using, for example, one RNA molecule containing two ribozymes at its 5' and 3' ends. Additionally, embodiments provided herein include bimolecular methods using two separate RNA molecules to generate circRNAs, for example, an RNA molecule containing a 5' ribozyme and a 3' substrate sequence, and a separate RNA molecule containing a trans-acting ribozyme.

[0162] B. Single-molecule ribonucleic acid (RNA) method Provided herein are ribonucleic acid (RNA) molecules comprising one or more ribozyme sequences, for example, at the 5'-end and 3'-end. In some embodiments, a single RNA molecule comprising two ribozymes, one at the 5'-end and one at the 3'-end, is provided. In some embodiments, this single RNA molecule is used to generate circular RNA (circRNA).

[0163] In some embodiments, a single-molecule method for generating circRNAs includes the steps of generating a linear RNA molecule containing two ribozymes, one at the 5' end and one at the 3' end (in some embodiments, also referred to as a "precursor RNA" before cleavage), cleaving the 5'-end ribozyme and the 3'-end ribozyme to generate a cleaved RNA molecule (in some embodiments, also referred to as a "pre-circRNA"), and ligating the ends of the cleaved RNA molecule to generate a circular RNA (circRNA) (see, e.g., Figure 1A).

[0164] In some embodiments, the ribozyme sequence at the 5' end of the linear RNA molecule is different from the ribozyme sequence at the 3' end. In some embodiments, the single RNA molecule comprises a 5' twister-sister ribozyme. In some embodiments, the single RNA molecule comprises a 3' twister ribozyme. In some embodiments, the RNA molecule comprises an insert sequence encoding one or more exogenous molecules. In some embodiments, the RNA molecule comprises other sequences, such as one or more homologous regions and / or one or more spacer sequences.

[0165] In some embodiments, an RNA molecule (e.g., a unimolecular RNA molecule) comprises, from the 5' end to the 3' end, a 5' ribozyme comprising a Twister-Sister ribozyme, an insert sequence, and a 3' ribozyme comprising a Twister ribozyme. In some embodiments, an RNA molecule (e.g., a unimolecular RNA molecule) comprises, from the 5' end to the 3' end, a 5' ribozyme comprising a Twister-Sister ribozyme comprising a 5' substrate sequence, an insert sequence, and a 3' ribozyme comprising a Twister ribozyme comprising a 3' substrate sequence. In some embodiments, the 5' ribozyme comprises a 5' substrate sequence. In some embodiments, the 5' substrate sequence comprises a 5' overhang sequence. In some embodiments, the 3' ribozyme comprises a 3' substrate sequence. In some embodiments, the 3' substrate sequence comprises a 3' overhang sequence.

[0166] In some embodiments, a 5' ribozyme can undergo a cleavage reaction to cleave a 5' substrate sequence to generate a cleaved RNA molecule comprising a 5' overhang sequence. In some embodiments, a 3' ribozyme can undergo a cleavage reaction to cleave a 3' substrate sequence to generate a cleaved RNA molecule comprising a 3' overhang sequence. In some embodiments, a 5' ribozyme and a 3' ribozyme can undergo a cleavage reaction to cleave a 5' substrate sequence and a 3' substrate sequence to generate a cleaved RNA molecule comprising a 5' overhang sequence, an insert sequence, and a 3' cleavage sequence.

[0167] 1. Twister-Sister Ribozymes In some embodiments, the RNA molecules provided herein comprise a Twister-Sister ribozyme at the 5' end. In some embodiments provided herein, the 5' ribozyme comprises a Twister-Sister ribozyme. In some embodiments provided herein, the 5' ribozyme is a Twister-Sister ribozyme. In some embodiments, the RNA molecules provided herein comprise a 5' ribozyme, including a Twister-Sister ribozyme comprising a 5' substrate sequence. In some embodiments, the RNA molecules provided herein comprise a Twister-Sister ribozyme at the 5' end. In some embodiments, the RNA molecules provided herein comprise a Twister-Sister ribozyme substrate sequence at the 5' end. In some embodiments, the RNA molecules provided herein comprise a 5' overhang sequence of a Twister-Sister ribozyme at the 5' end. In some embodiments, the RNA molecules provided herein comprise a TS-1 ribozyme at the 5' end. In some embodiments, the RNA molecules provided herein comprise a substrate sequence for a TS-1 ribozyme at the 5' end. In some embodiments, the RNA molecules provided herein comprise a 5' overhang sequence for a TS-1 ribozyme. In some embodiments, the circRNA molecules provided herein comprise a 5' overhang sequence for a TS-1 ribozyme.

[0168] In some embodiments, Twister-Sister ribozyme (also known as RNAs Associated with Genes Associated with Twister and Hammerhead-3 or RAGATH-3) is another type of self-cleaving catalytic RNA that shares some similarity in sequence and secondary structure with Twister ribozyme (see Figure 12A; Roth et al. Nat Chem Biol, 10(1), 56-60 (2014); Weinberg et al., Nat Chem Bio, 11(8):606-610 (2015)). Twister-Sister ribozyme was identified based on conserved RNA structure sequence search and has been identified to occur near the gene elements listed in pfam06252 and pfam09299 (Weinberg et al., (2015) Nat Chem Biol, 11(8), 606-610). Twister-Sister ribozymes have a P1-P5 stem with a similar arrangement to Twister ribozyme species, and similar nucleotides in the P4 terminal loop are observed. However, while pseudoknot formation via Watson-Crick base pairing is observed in all known Twister ribozymes, pseudoknots are sometimes absent in Twister-Sister ribozymes. Furthermore, there is low identity between Twister and Twister-Sister ribozymes at many of the most highly conserved nucleotides. Cleavage by Twister-Sister ribozymes is dependent on the catalytic RNA sequence and Mg. 2+ The cleavage site of the Twister-Sister ribozyme is between the C13 and A14 nucleotides, but it is on the opposite side of the internal loop cleaved by the Twister ribozyme (Weinberg et al., (2015) Nat Chem Biol, 11(8), 606-610).

[0169] The Twister-Sister ribozyme primarily comprises two helical segments exhibiting coaxial stacking, linked by a three-way junction and two tertiary contacts (Liu et al., Nat Chem Biol, (2017), 13(5):508-513). Five divalent metal ions are directly coordinated to the RNA substrate, and a pseudohelical loop region constructed by a hydrogen-bonding network contains the scissile phosphate. The divalent metal ion directly binds the scissile phosphate to the 5' position of the nucleobase, and a water molecule located in the inner sphere interacts with the O2' nucleophile. This divalent metal ion is thought to act as a general base for the cleavage reaction. The cleavage rate of the ribozyme correlates log-linearly with the pKa of the divalent metal ion, consistent with a transition-state proton transfer (Liu et al., Nat Chem Biol, (2017), 13(5):508-513). In some situations, the catalytic activity of Twister-Sister complexes increases with pH, ​​and Mg 2+ These ribozymes rely on divalent metal ions, such as ribozymes. In some circumstances, nucleolytic ribozymes cleave specific phosphodiester bonds via an SN2 mechanism. O2' acts as a nucleophile attacking the adjacent P, resulting in the elimination of O5' as a leaving group. The resulting catalytic product contains a cyclic 2',3'-phosphate and a 5'-hydroxyl group (Ren et al., Curr Opin Chem Biol. 2017 Dec; 41: 71-83). In some embodiments, the RNA molecule contains a 5'-hydroxyl group after cleavage of the 5'-terminal Twister-Sister substrate sequence.

[0170] Exemplary twister-sister ribozymes used in any of the embodiments provided herein include twister-sister-1 (TS-1) ribozyme, TS-2 ribozyme, TS-3 ribozyme and TS-4 ribozyme.Further, exemplary twister-sister ribozymes include those described, for example, in Weinberg et al., Nat Chem Biol, 11(8):606-610 (2015), Liu et al., Nat Chem Biol, (2017), 13(5):508-513, and Ren et al., Curr Opin Chem Biol. 2017 Dec; 41: 71-83. The TS-1 ribozyme was generated from a microbial metagenomic DNA source and comprises a biomolecule strand including a 5' enzyme strand, a 3' substrate strand, and a P1-P5 stem region (Figure 12B; see Weinberg et al., Nat Chem Bio, 11(8):606-610 (2015)). In some embodiments, the Twister-Sister ribozyme comprises TS-1. In some embodiments, the Twister-Sister ribozyme comprises TS-2. In some embodiments, the Twister-Sister ribozyme comprises TS-3. In some embodiments, the Twister-Sister ribozyme comprises TS-4.

[0171] In some embodiments, the RNA molecules provided herein comprise a Twister-Sister ribozyme overhang sequence. In some embodiments, the 5' ribozyme comprises a Twister-Sister ribozyme overhang sequence. In some embodiments, the RNA molecules provided herein comprise a TS-1 overhang sequence. In some embodiments, the 5' ribozyme comprises a TS-1 overhang sequence.

[0172] In some embodiments, an "overhang sequence" (sometimes referred to as a "cleavage sequence") refers to a portion of a substrate sequence that remains attached to an RNA molecule containing an insert sequence (the "cleaved RNA" or "pre-circRNA" after cleavage by a ribozyme) after cleavage by a ribozyme. In some embodiments, as used herein, an overhang sequence is a portion of a substrate sequence, and a substrate sequence comprises an overhang sequence.

[0173] In some embodiments, the RNA molecules provided herein comprise a 5' overhang sequence and a 3' overhang sequence. In some embodiments, the length of the 5' overhang sequence is 2 to 15 nucleotides (nt), e.g., 3 to 10 nt, or 4 to 8 nt, or about 3 nt, about 4 nt, about 5 nt, about 6 nt, about 7 nt, about 8 nt, about 9 nt, or about 10 nt. In some embodiments, the length of the 5' overhang sequence is less than 10 nt. In some embodiments, the length of the 5' overhang sequence is 6 nt. In some embodiments, the 5' overhang sequence and the 3' overhang sequence can together form a loop structure that is recognized by an RNA ligase, such as RtcB.

[0174] In some embodiments, the 5' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:6. In some embodiments, the 5' ribozyme comprises SEQ ID NO:6. In some embodiments, the 5' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:5. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO:5.

[0175] In some embodiments, the 5' overhang sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:6. In some embodiments, the 5' overhang sequence comprises SEQ ID NO:6. In some embodiments, the 5' overhang sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:5. In some embodiments, the 5' overhang sequence comprises a sequence encoded by SEQ ID NO:5.

[0176] In some embodiments, the RNA molecules provided herein comprise a substrate sequence for a 5' ribozyme. In some embodiments, the 5' ribozyme comprises a substrate sequence for a 5' ribozyme. In some embodiments, the RNA molecules provided herein comprise a substrate sequence for TS-1. In some embodiments, the 5' ribozyme comprises a substrate sequence for TS-1.

[0177] In some embodiments, "substrate sequence" refers to a portion of a ribozyme sequence that is cleaved by the catalytic portion of the ribozyme. In some embodiments, the substrate sequence of the ribozyme before cleavage may be contained in the same RNA molecule as the RNA molecule containing the catalytic sequence of the ribozyme (unimolecular ribozyme, i.e., cis-acting ribozyme). In some embodiments, the substrate sequence of the ribozyme before cleavage may be contained in an RNA molecule different from the RNA molecule containing the catalytic sequence of the ribozyme (binamolecular ribozyme, i.e., trans-acting ribozyme). In some embodiments, the substrate sequence comprises an overhang sequence that remains attached to the cleaved RNA molecule containing the insert sequence after cleavage by the ribozyme.

[0178] In some embodiments, the 5' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 45. In some embodiments, the 5' ribozyme comprises SEQ ID NO: 45. In some embodiments, the 5' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 44. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO: 44.

[0179] In some embodiments, the 5' substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 45. In some embodiments, the 5' substrate sequence comprises SEQ ID NO: 45. In some embodiments, the 5' substrate sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 44. In some embodiments, the 5' substrate sequence comprises a sequence encoded by SEQ ID NO: 44.

[0180] In some embodiments, the 5' ribozyme comprises the catalytic sequence of a 5' ribozyme. In some embodiments, the 5' ribozyme comprises the catalytic sequence of TS-1.

[0181] In some embodiments, "catalytic sequence" refers to a portion of a ribozyme sequence that catalyzes the cleavage of a substrate sequence. In some embodiments, the catalytic sequence cleaves the substrate sequence at or after the overhang sequence, leaving the overhang sequence attached to the cleaved RNA molecule containing the insert sequence after cleavage by the ribozyme catalytic sequence.

[0182] In some embodiments, the 5' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:4. In some embodiments, the 5' ribozyme comprises SEQ ID NO:4. In some embodiments, the 5' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:3. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO:3.

[0183] In some embodiments, the 5′ Twister-Sister catalytic sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:4. In some embodiments, the 5′ Twister-Sister catalytic sequence comprises SEQ ID NO:4. In some embodiments, the 5′ Twister-Sister catalytic sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:3. In some embodiments, the 5′ Twister-Sister catalytic sequence comprises a sequence encoded by SEQ ID NO:3.

[0184] In some embodiments, the Twister-Sister ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 4. In some embodiments, the Twister-Sister ribozyme comprises SEQ ID NO: 4. In some embodiments, the Twister-Sister ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 3. In some embodiments, the Twister-Sister ribozyme comprises a sequence encoded by SEQ ID NO: 3.

[0185] In some embodiments, the 5' ribozyme comprises the catalytic and substrate sequences of a Twister-Sister ribozyme, hi some embodiments, the 5' ribozyme comprises the catalytic and substrate sequences of TS-1.

[0186] In some embodiments, the 5' ribozyme (comprising the catalytic sequence and the substrate sequence) comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 47. In some embodiments, the 5' ribozyme (comprising the catalytic sequence and the substrate sequence) comprises SEQ ID NO: 47. In some embodiments, the 5' ribozyme (comprising the catalytic sequence and the substrate sequence) is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 46. In some embodiments, the 5' ribozyme (including the catalytic and substrate sequences) comprises the sequence encoded by SEQ ID NO:46.

[0187] 2. Twister Ribozyme In some embodiments, the RNA molecules provided herein (e.g., unimolecular RNA molecules) comprise a Twister ribozyme at their 3' ends. In some embodiments provided herein, the 3' ribozyme comprises a Twister ribozyme. In some embodiments provided herein, the 3' ribozyme is a Twister ribozyme. In some embodiments, the RNA molecules provided herein comprise a 3' ribozyme comprising a Twister ribozyme comprising a 3' substrate sequence. In some embodiments, the RNA molecules provided herein comprise a Twister ribozyme at their 3' ends. In some embodiments, the RNA molecules provided herein comprise a Twister ribozyme substrate sequence at their 3' ends. In some embodiments, the RNA molecules provided herein comprise a 3' overhang sequence of a Twister ribozyme at their 3' ends. In some embodiments, the RNA molecules provided herein comprise a P1-type Twister ribozyme from Parasitoid wasp at their 3' ends. In some embodiments, the RNA molecules provided herein comprise a substrate sequence for a P1-type Twister ribozyme derived from Parasitoid wasp at the 3' end. In some embodiments, the RNA molecules provided herein comprise a 3' overhang sequence of a P1-type Twister ribozyme derived from Parasitoid wasp. In some embodiments, the circRNA molecules provided herein comprise a 3' overhang sequence of a P1-type Twister ribozyme derived from Parasitoid wasp.

[0188] In some embodiments, the twister ribozyme is a type of self-cleaving catalytic RNA with two pseudoknot RNA structures (see Figure 12A; Roth et al. Nat Chem Biol, 10(1), 56-60 (2014); and Weinberg et al., Nat Chem Bio, 11(8):606-610 (2015)). Twister ribozymes were identified from a highly conserved small RNA motif found in Clostridium bacteria and various eukaryotes (Roth et al. Nat Chem Biol, 10(1), 56-60 (2014)), and have a consensus secondary structure in which three stem structures are connected by internal and terminal loops. In certain instances, the twister motif is circularly permuted, and in representative instances, both ends are contained within the P1 stem (P1 type), the P3 stem (P3 type), or the P5 stem (P5 type) (see Figure 12A). Cleavage of the twister ribozyme is mediated by the catalytic RNA sequence and the Mg 2+ The catalytic activity of Twister depends on the presence of Mg (Roth et al. Nat Chem Biol, 10(1), 56-60 (2014)), and cleavage occurs via an internal phosphate transfer in which the 2′ oxygen of U5 attacks the adjacent phosphorus atom, resulting in the loss of the 5′ oxygen of A6 (Roth et al. Nat Chem Biol, 10(1), 56-60 (2014)). In some situations, the catalytic activity of Twister increases with pH and is enhanced by the presence of Mg. 2+These ribozymes rely on divalent metal ions, such as ATP, to cleave specific phosphodiester bonds. In some circumstances, nucleolytic ribozymes cleave specific phosphodiester bonds via an SN2 mechanism. Twister ribozyme cleavage is based on general acid-base catalysis involving the highly conserved adenine (A1) and guanine (G33) bases, with N3 of A1 acting as a proton donor and G33 acting as a general base. The resulting catalytic product contains a cyclic 2',3'-phosphate and a 5'-hydroxyl group (Roth et al. Nat Chem Biol, 10(1), 56-60 (2014)). In some embodiments, after cleavage of the 3'-terminal Twister substrate sequence, the RNA molecule contains a cyclic 2',3'-phosphate at the 3' end.

[0189] Exemplary twister ribozymes for use in any of the embodiments provided herein include those described, for example, in Roth et al. Nat Chem Biol, 10(1), 56-60 (2014), Liu et al., Nat Chem Biol. 10 (9): 739-744 (2014), and Eiler et al., Proceedings of the National Academy of Sciences. 111 (36): 13028-13033 (2014). Nearly 2,700 twister ribozymes have been identified, and these twister ribozymes share a consensus secondary structure in which three stems are connected by internal and terminal loops. Exemplary twister ribozymes include P1-type twister ribozymes, P3-type twister ribozymes, and P5-type twister ribozymes. Further exemplary Twister ribozymes include the Twister ribozyme from Nasonia vitripennis, the Twister ribozyme from Clostridium bolteae, the Twister ribozyme from rice (Oryza sativa), the Twister ribozyme from Schistosoma mansoni, Twister env22, and Twister env9. In some embodiments, the Twister ribozyme comprises a P1-type Twister ribozyme from Nasonia vitripennis. In some embodiments, the Twister ribozyme comprises a Twister ribozyme from C. bolteae. In some embodiments, the Twister ribozyme comprises a Twister ribozyme from rice. In some embodiments, the Twister ribozyme comprises a Twister ribozyme from Schistosoma mansoni.

[0190] In some embodiments, the RNA molecules provided herein comprise an overhang sequence of a Twister ribozyme. In some embodiments, the 3' ribozyme comprises an overhang sequence of a Twister ribozyme. In some embodiments, the RNA molecules provided herein comprise an overhang sequence of a P1-type Twister ribozyme from Parasitoid wasp. In some embodiments, the 3' ribozyme comprises an overhang sequence of a P1-type Twister ribozyme from Parasitoid wasp.

[0191] In some embodiments, the RNA molecules provided herein comprise a 5' overhang sequence and a 3' overhang sequence. In some embodiments, the length of the 3' overhang sequence is 2 to 15 nucleotides (nt), e.g., 3 to 10 nt, or 4 to 8 nt, or about 3 nt, about 4 nt, about 5 nt, about 6 nt, about 7 nt, about 8 nt, about 9 nt, or about 10 nt. In some embodiments, the length of the 3' overhang sequence is less than 10 nt. In some embodiments, the length of the 3' overhang sequence is 4 nt. In some embodiments, the 5' overhang sequence and the 3' overhang sequence can together form a loop structure that is recognized by an RNA ligase, such as RtcB.

[0192] In some embodiments, the 3' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 21. In some embodiments, the 3' ribozyme comprises SEQ ID NO: 21. In some embodiments, the 3' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 20. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO: 20.

[0193] In some embodiments, the 3' overhang sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 21. In some embodiments, the 3' overhang sequence comprises SEQ ID NO: 21. In some embodiments, the 3' overhang sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 20. In some embodiments, the 3' overhang sequence comprises a sequence encoded by SEQ ID NO: 20.

[0194] In some embodiments, the 3' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 52. In some embodiments, the 3' ribozyme comprises SEQ ID NO: 52. In some embodiments, the 3' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 51. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO: 51.

[0195] In some embodiments, the 3' overhang sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 52. In some embodiments, the 3' overhang sequence comprises SEQ ID NO: 52. In some embodiments, the 3' overhang sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 51. In some embodiments, the 3' overhang sequence comprises a sequence encoded by SEQ ID NO: 51.

[0196] In some embodiments, the RNA molecules provided herein comprise a substrate sequence for a 3' ribozyme. In some embodiments, the 3' ribozyme comprises a substrate sequence for a 3' ribozyme. In some embodiments, the RNA molecules provided herein comprise a substrate sequence for a P1-type Twister ribozyme from Parasitoid wasp. In some embodiments, the 3' ribozyme comprises a substrate sequence for a P1-type Twister ribozyme from Parasitoid wasp.

[0197] In some embodiments, the substrate sequence of the ribozyme before cleavage may be contained in the same RNA molecule as the RNA molecule containing the catalytic sequence of the ribozyme (unimolecular ribozyme, i.e., cis-acting ribozyme). In some embodiments, the substrate sequence of the ribozyme before cleavage may be contained in an RNA molecule different from the RNA molecule containing the catalytic sequence of the ribozyme (binamolecular ribozyme, i.e., trans-acting ribozyme). In some embodiments, the substrate sequence contains an overhang sequence that remains attached to the cleaved RNA molecule containing the insert sequence after cleavage by the ribozyme.

[0198] In some embodiments, the 3' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 33. In some embodiments, the 3' ribozyme comprises SEQ ID NO: 33. In some embodiments, the 3' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 32. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO: 32.

[0199] In some embodiments, the 3' substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 33. In some embodiments, the 3' substrate sequence comprises SEQ ID NO: 33. In some embodiments, the 3' substrate sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 32. In some embodiments, the 3' substrate sequence comprises a sequence encoded by SEQ ID NO: 32.

[0200] In some embodiments, the 3' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 31. In some embodiments, the 3' ribozyme comprises SEQ ID NO: 31. In some embodiments, the 3' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 30. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO: 30.

[0201] In some embodiments, the 3' substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 31. In some embodiments, the 3' substrate sequence comprises SEQ ID NO: 31. In some embodiments, the 3' substrate sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 30. In some embodiments, the 3' substrate sequence comprises a sequence encoded by SEQ ID NO: 30.

[0202] In some embodiments, the 3' ribozyme comprises the catalytic sequence of a twister ribozyme. In some embodiments, the 3' ribozyme comprises the catalytic sequence of a P1-type twister ribozyme from Parasitic wasp. In some embodiments, in unimolecular methods (e.g., the unimolecular methods described herein in Section IB), the catalytic sequence of the 3' ribozyme is contained within the same molecule (e.g., within a unimolecular RNA molecule) as a substrate sequence for the same ribozyme. In some embodiments, in bimolecular methods (e.g., the bimolecular methods described herein in Section IC), the catalytic sequence of the 3' ribozyme is contained within a separate RNA molecule from the substrate sequence for that ribozyme.

[0203] In some embodiments, the 3' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 23. In some embodiments, the 3' ribozyme comprises SEQ ID NO: 23. In some embodiments, the 3' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 22. In some embodiments, the 3' ribozyme comprises a sequence encoded by SEQ ID NO: 22.

[0204] In some embodiments, the 3'Twister catalytic sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 23. In some embodiments, the 3'Twister catalytic sequence comprises SEQ ID NO: 23. In some embodiments, the 3'Twister catalytic sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 22. In some embodiments, the 3'Twister catalytic sequence comprises a sequence encoded by SEQ ID NO: 22.

[0205] In some embodiments, the twister ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 23. In some embodiments, the twister ribozyme comprises SEQ ID NO: 23. In some embodiments, the twister ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 22. In some embodiments, the twister ribozyme comprises a sequence encoded by SEQ ID NO: 22.

[0206] In some embodiments, the 3' ribozyme comprises the catalytic and substrate sequences of a Twister ribozyme. In some embodiments, the 3' ribozyme comprises the catalytic and substrate sequences of a P1-type Twister ribozyme from Parasitoid wasp.

[0207] In some embodiments, the 3' ribozyme (comprising a catalytic sequence and a substrate sequence) comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 47. In some embodiments, the 3' ribozyme (comprising a catalytic sequence and a substrate sequence) comprises SEQ ID NO: 47. In some embodiments, the 3' ribozyme (comprising a catalytic sequence and a substrate sequence) is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 46. In some embodiments, the 3' ribozyme (including the catalytic and substrate sequences) comprises the sequence encoded by SEQ ID NO:46.

[0208] 3. Exemplary Molecules In some embodiments, an exemplary unimolecular RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises, from the 5' end to the 3' end, a 5' coverage sequence, a catalytic sequence of a 5' twister-sister ribozyme, a substrate sequence of a 5' twister-sister ribozyme (including a 5' overhang sequence), a 5' homology region, a 5' spacer sequence, an IRES sequence, a coding sequence of an exogenous molecule, a polyAC sequence, a 3' homology region, a substrate sequence of a 3' twister ribozyme (including a 3' overhang sequence), a catalytic sequence of a 3' twister ribozyme, and a 3' coverage sequence.

[0209] In some embodiments, an exemplary unimolecular RNA molecule (e.g., precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises, from the 5' end to the 3' end, a 5' coverage sequence, a 5' TS-1 catalytic sequence, a 5' TS-1 substrate sequence (including a 5' overhang sequence), a 5' homology region, a 5' spacer sequence, an IRES sequence derived from Coxsackievirus type B3, a coding sequence for firefly luciferase (FLuc), a polyAC sequence, a 3' homology region, a 3' P1-type Twister substrate sequence (including a 3' overhang sequence) derived from Parasitoid wasp, a 3' P1-type Twister catalytic sequence derived from Parasitoid wasp, and a 3' coverage sequence.

[0210] In some embodiments, a unimolecular RNA molecule provided herein (e.g., a precursor RNA molecule) before cleavage of a ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 29. In some embodiments, a unimolecular RNA molecule before cleavage of a ribozyme substrate sequence comprises SEQ ID NO: 29. In some aspects, an exemplary RNA molecule designated Construct U1 in the Examples provided herein is provided. In some embodiments, the unimolecular RNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 28. In some embodiments, the unimolecular RNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 28.

[0211] C. Bimolecular Ribonucleic Acid (RNA) Methods and Combinations The present invention provides ribonucleic acid (RNA) molecules for bimolecular method for producing circRNA, as well as combinations and systems of RNA molecules for producing circRNA. In some embodiments, the bimolecular RNA molecules comprise two different RNA molecules, and these two RNA molecules cooperate to cleave the 5'-end and 3'-end of one RNA molecule to produce circRNA. In some embodiments, the combination or system of RNA molecules for producing circRNA by bimolecular method comprises a first RNA molecule comprising a 5' ribozyme and a ribozyme substrate sequence at its 3'-end, and a second RNA molecule comprising a trans-acting ribozyme that can cleave the substrate sequence present at the 3'-end of the first RNA molecule. In some embodiments of the bimolecular method, the catalytic sequence of the ribozyme that cleaves the 3' substrate sequence on the first RNA molecule exists as a separate second RNA molecule comprising a trans-acting ribozyme.

[0212] In some embodiments, the bimolecular method for generating circRNA involves generating a first RNA molecule, which is a linear RNA molecule (in some embodiments, also referred to as "precursor RNA" before cleavage) that contains a cleavage sequence and a substrate sequence for one ribozyme at its 5' end and only the substrate sequence for the ribozyme at its 3' end. The second RNA molecule contains a trans-acting ribozyme that can cleave the 3' substrate sequence of the first RNA molecule. The 5' ribozyme at the 5' end of the first RNA molecule is also cleaved to generate a cleaved RNA molecule (in some embodiments, also referred to as "pre-circRNA"), and then the two ends of the cleaved RNA molecule are ligated to generate a circular RNA (circRNA) (see, for example, Figure 1B).

[0213] In some embodiments, the ribozyme sequence at the 5' end of the first RNA molecule and the ribozyme sequence of the trans-acting ribozyme are different. In some embodiments, the first RNA molecule comprises a 5' Twister-Sister ribozyme, e.g., a Twister-Sister ribozyme described in Section IB1 herein. In some embodiments, the second RNA molecule comprises a catalytic sequence of a trans-acting Twister ribozyme, e.g., a Twister ribozyme described in Section IB2 herein. In some embodiments, the first RNA molecule comprises an insert sequence encoding one or more exogenous molecules. In some embodiments, the first RNA molecule comprises other sequences, such as one or more homologous regions and / or one or more spacer sequences.

[0214] In some embodiments, the bimolecular method for generating circRNA utilizes a trans-acting ribozyme comprising a catalytic sequence, which cleaves a substrate sequence at the 3' end of a first RNA molecule at or after the overhang sequence, leaving the overhang sequence attached to the cleaved RNA molecule containing the insert sequence after cleavage by the trans-acting ribozyme.

[0215] In some embodiments, the second RNA molecule comprises a trans-acting ribozyme. In some embodiments, the trans-acting ribozyme is or comprises a twister ribozyme.

[0216] Further provided herein are combinations of RNA molecules. In some embodiments, the combination of RNA molecules comprises a first RNA molecule, such as a first RNA molecule described herein, for example, as described in Section IC1, and a second RNA molecule, such as a second RNA molecule described herein, for example, as described in Section IC2. In some embodiments, the combination of RNA molecules comprises a plurality of first RNA molecules and a plurality of second RNA molecules.

[0217] 1. First RNA molecule - 5' ribozyme and 3' substrate sequence In some embodiments, the first RNA molecule comprises a 5' ribozyme comprising a 5' substrate sequence near its 5' end and a 3' substrate sequence for the ribozyme near its 3' end.

[0218] In some embodiments provided herein, the 5' ribozyme of the first RNA molecule comprises a Twister-Sister ribozyme. In some embodiments provided herein, the 5' ribozyme of the first RNA molecule is a Twister-Sister ribozyme. In some embodiments provided herein, the 3' substrate sequence of the first RNA molecule comprises a Twister ribozyme substrate sequence. In some embodiments provided herein, the 3' substrate sequence of the first RNA molecule is a Twister ribozyme substrate sequence.

[0219] In some embodiments, the first RNA molecule comprises a 5' Twister-Sister ribozyme near its 5' end and a 3' substrate sequence for the Twister ribozyme near its 3' end. In some embodiments, the first RNA molecule comprises an insert sequence adjacent to the 5' ribozyme on the 5' side and adjacent to the 3' substrate sequence on the 3' side. In some embodiments, the 3' substrate sequence of the first RNA molecule is cleaved in the presence of a trans-acting ribozyme, such as a trans-acting ribozyme of a second RNA molecule. In some embodiments, the trans-acting ribozyme is a Twister ribozyme. In some embodiments, the combination of the first RNA molecule and the second RNA molecule generates a cleaved RNA molecule by cleaving the 5' substrate sequence and the 3' substrate sequence. In some embodiments, after cleavage, the cleaved RNA molecule comprises a 5' overhang sequence, an insert, and a 3' overhang sequence.

[0220] In some embodiments, the 5' twister-sister ribozyme is or includes a twister-sister ribozyme described herein, for example, as described in Section IB1. In some embodiments, the 5' twister-sister ribozyme is or includes a TS-1 ribozyme. In some embodiments, the first RNA molecule is generally similar in overall structure to the unimolecular precursor RNA molecule, except that it contains only the 3' ribozyme substrate sequence at its 3' end and the 3' ribozyme catalytic sequence is not present in the same molecule. In bimolecular methods, the catalytic sequence of the ribozyme that cleaves the 3' substrate is present in the second RNA molecule as a trans-acting ribozyme.

[0221] In some embodiments, the 5' ribozyme comprises an overhang sequence. In some embodiments, the 5' ribozyme comprises the 5' overhang sequence of a TS-1 ribozyme. In some embodiments, the 5' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:6. In some embodiments, the 5' ribozyme comprises SEQ ID NO:6. In some embodiments, the 5' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 5. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO: 5.

[0222] In some embodiments, the 5' ribozyme comprises a substrate sequence. In some embodiments, the 5' ribozyme comprises a 5' substrate sequence for a TS-1 ribozyme. In some embodiments, the 5' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:45. In some embodiments, the 5' ribozyme comprises SEQ ID NO:45. In some embodiments, the 5' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 44. In some embodiments, the 5' ribozyme comprises a sequence encoded by SEQ ID NO:44.

[0223] In some embodiments, the 5' ribozyme comprises a catalytic sequence. In some embodiments, the 5' ribozyme comprises the catalytic sequence of a TS-1 ribozyme. In some embodiments, the 5' ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:4. In some embodiments, the 5' ribozyme comprises SEQ ID NO:4. In some embodiments, the 5' ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO:3. In some embodiments, the 5' ribozyme comprises the sequence encoded by SEQ ID NO:3.

[0224] In some embodiments, the 5' ribozyme (comprising the catalytic sequence and the substrate sequence) comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 47. In some embodiments, the 5' ribozyme (comprising the catalytic sequence and the substrate sequence) comprises SEQ ID NO: 47. In some embodiments, the 5' ribozyme (comprising the catalytic sequence and the substrate sequence) is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 46. In some embodiments, the 5' ribozyme (including the catalytic and substrate sequences) comprises the sequence encoded by SEQ ID NO:46.

[0225] In some embodiments, the first RNA molecule comprises a 3' overhang sequence of a ribozyme. In some embodiments, the 3' overhang sequence is or comprises an overhang sequence of a Twister ribozyme. In some embodiments, the Twister ribozyme is, for example, any of the Twister ribozymes described in Section IB2 of this specification, or comprises any of these Twister ribozymes. In some embodiments, the 3' overhang sequence is a Twister overhang sequence of a P1-type Twister ribozyme from Parasitoid wasp. In some embodiments, the first RNA molecule comprises a 3' overhang sequence of a P1-type Twister ribozyme from Parasitoid wasp. In some embodiments, after the 3' substrate sequence in the first RNA molecule is cleaved by the trans-acting ribozyme of the second RNA molecule, the 3' overhang sequence remains as part of the cleaved RNA molecule containing the insert sequence.

[0226] In some embodiments, the 3' overhang sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 21. In some embodiments, the 3' overhang sequence comprises SEQ ID NO: 21. In some embodiments, the 3' overhang sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 20. In some embodiments, the 3' overhang sequence comprises a sequence encoded by SEQ ID NO: 20.

[0227] In some embodiments, the 3' overhang sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 52. In some embodiments, the 3' overhang sequence comprises SEQ ID NO: 52. In some embodiments, the 3' overhang sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 51. In some embodiments, the 3' overhang sequence comprises a sequence encoded by SEQ ID NO: 51.

[0228] In some embodiments, the first RNA molecule comprises a 3' substrate sequence for a ribozyme. In some embodiments, the 3' substrate sequence is or comprises a substrate sequence for a Twister ribozyme. In some embodiments, the Twister ribozyme is or comprises any of the Twister ribozymes described, for example, in Section IB2 of this specification. In some embodiments, the 3' substrate sequence is a Twister substrate sequence for a P1-type Twister ribozyme from Parasitoid wasp. In some embodiments, the first RNA molecule comprises a 3' substrate sequence for a P1-type Twister ribozyme from Parasitoid wasp. In some embodiments, the 3' overhang sequence is part of the 3' substrate sequence, and after the 3' substrate sequence in the first RNA molecule is cleaved by the trans-acting ribozyme of the second RNA molecule, the 3' overhang sequence remains as part of the cleaved RNA molecule containing the insert sequence.

[0229] In some embodiments, the first RNA molecule comprises a 3' substrate sequence at its 3' end. In some embodiments, the 3' substrate sequence is or comprises a substrate sequence for a Twister ribozyme. In some embodiments, the first RNA molecule comprises a 3' substrate sequence for a P1-type Twister ribozyme derived from Parasitoid wasp.

[0230] In some embodiments, the 3' substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 33. In some embodiments, the 3' substrate sequence comprises SEQ ID NO: 33. In some embodiments, the 3' substrate sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 32. In some embodiments, the 3' substrate sequence comprises a sequence encoded by SEQ ID NO: 32.

[0231] In some embodiments, the 3' substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 31. In some embodiments, the 3' substrate sequence comprises SEQ ID NO: 31. In some embodiments, the 3' substrate sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 30. In some embodiments, the 3' substrate sequence comprises a sequence encoded by SEQ ID NO: 30.

[0232] 2. Second RNA molecule—trans-acting ribozyme In some embodiments, the second RNA molecule comprises a trans-acting ribozyme comprising a catalytic sequence of the ribozyme. In some embodiments, the trans-acting ribozyme of the second RNA molecule comprises a twister ribozyme. In some embodiments, the trans-acting ribozyme of the second RNA molecule is a twister ribozyme. In some embodiments, the second RNA molecule comprises a catalytic sequence of a twister ribozyme. In some embodiments, the 3' substrate sequence of the first RNA molecule is cleaved in the presence of the trans-acting ribozyme of the second RNA molecule.

[0233] In some embodiments, the second RNA molecule comprises a trans-acting ribozyme. In some embodiments, the trans-acting ribozyme comprises a twister ribozyme, such as the twister ribozyme described in Section IB2 of this specification. In some embodiments, the trans-acting ribozyme comprises a catalytic sequence derived from a twister ribozyme.

[0234] In some embodiments, the Twister catalytic sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 23. In some embodiments, the Twister catalytic sequence comprises SEQ ID NO: 23. In some embodiments, the Twister catalytic sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 22. In some embodiments, the Twister catalytic sequence comprises a sequence encoded by SEQ ID NO: 22.

[0235] In some embodiments, the Twister catalytic sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 35. In some embodiments, the Twister catalytic sequence comprises SEQ ID NO: 35. In some embodiments, the Twister catalytic sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 34. In some embodiments, the Twister catalytic sequence comprises a sequence encoded by SEQ ID NO: 34.

[0236] In some embodiments, the trans-acting ribozyme comprises the catalytic sequence of a Twister ribozyme. In some embodiments, the trans-acting ribozyme comprises the catalytic sequence of a P1-type Twister ribozyme from Parasitic wasp. In some embodiments, in bimolecular methods (e.g., those described in Section IC herein), the catalytic sequence of the trans-acting ribozyme is contained in a molecule separate from the substrate sequence for that ribozyme (e.g., in a second RNA molecule), and the substrate sequence is contained at the 3' end of the first RNA molecule.

[0237] In some embodiments, the trans-acting ribozyme comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 23. In some embodiments, the trans-acting ribozyme comprises SEQ ID NO: 23. In some embodiments, the trans-acting ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 22. In some embodiments, the trans-acting ribozyme comprises a sequence encoded by SEQ ID NO: 22.

[0238] In some embodiments, the trans-acting ribozyme comprises one or more unnatural guanosine residues at its 5'-terminus. In some embodiments, the trans-acting ribozyme comprises two unnatural guanosine residues at its 5'-terminus. In some embodiments, the trans-acting ribozyme comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 35. In some embodiments, the trans-acting ribozyme comprises SEQ ID NO: 35. In some embodiments, the trans-acting ribozyme is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 34. In some embodiments, the trans-acting ribozyme comprises a sequence encoded by SEQ ID NO: 34. In some embodiments, a triphosphate is present at the 5' end of the trans-acting ribozyme or the second RNA molecule.

[0239] 3. Exemplary Molecules In some embodiments, an exemplary combination of RNA molecules for producing circRNAs using a bimolecular method includes a first RNA molecule comprising a 5' ribozyme and a 3'-terminal ribozyme substrate sequence, and a second RNA molecule comprising a trans-acting ribozyme capable of cleaving the substrate sequence located at the 3' end of the first RNA molecule. In some embodiments, an exemplary first RNA molecule (e.g., a precursor RNA molecule) before the ribozyme substrate sequence is cleaved comprises, from the 5' end to the 3' end, a 5' coverage sequence, a catalytic sequence for a 5' twister-sister ribozyme, a substrate sequence for a 5' twister-sister ribozyme (including a 5' overhang sequence), a 5' homology region, a 5' spacer sequence, an IRES sequence, a coding sequence for an exogenous molecule, a polyAC sequence, a 3' homology region, and a substrate sequence for a 3' twister ribozyme (including a 3' overhang sequence). In some embodiments, an exemplary second RNA molecule comprises a catalytic sequence for a twister ribozyme.

[0240] In some embodiments, the exemplary first RNA molecule (e.g., precursor RNA molecule) before the ribozyme substrate sequence is cleaved comprises, in order from the 5' end to the 3' end, a 5' coverage sequence, a 5' TS-1 catalytic sequence, a 5' TS-1 substrate sequence (including a 5' overhang sequence), a 5' homology region, a 5' spacer sequence, an IRES sequence derived from Coxsackievirus B3, a coding sequence for firefly luciferase (FLuc), a polyAC sequence, a 3' homology region, and a 3' P1-type Twister substrate sequence (including a 3' overhang sequence) derived from Parasitoid wasp. In some embodiments, the exemplary second RNA molecule comprises a P1-type Twister catalytic sequence derived from Parasitoid wasp.

[0241] In some embodiments, an exemplary first RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 37. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequence comprises SEQ ID NO: 37. In some embodiments, an exemplary first RNA molecule designated as Construct B1 in the Examples provided herein is provided. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 36. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 36.

[0242] In some embodiments, an exemplary first RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 109. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequence comprises SEQ ID NO: 109. In some embodiments, an exemplary first RNA molecule is provided, designated LVT-18 in the examples provided herein. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 108. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 108.

[0243] In some embodiments, an exemplary first RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 111. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequence comprises SEQ ID NO: 111. In some embodiments, an exemplary first RNA molecule is provided, designated LVT-20 in the examples provided herein. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 110. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 110.

[0244] In some embodiments, an exemplary first RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 113. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequence comprises SEQ ID NO: 113. In some embodiments, an exemplary first RNA molecule is provided, designated LVT-22 in the examples provided herein. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 112. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 112.

[0245] In some embodiments, an exemplary first RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 115. In some embodiments, the first RNA molecule before cleavage of the ribozyme substrate sequence comprises SEQ ID NO: 115. In some embodiments, an exemplary first RNA molecule is provided that is designated LVT14 / eIF4G in the examples provided herein. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 114. In some embodiments, the first DNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 114.

[0246] In some embodiments, an exemplary second RNA molecule comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 23. In some embodiments, the second RNA molecule comprises SEQ ID NO: 23. In some embodiments, an exemplary second RNA molecule designated as Construct B1 in the Examples provided herein is provided. In some embodiments, the second RNA molecule comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 22. In some embodiments, the second RNA molecule comprises a sequence encoded by SEQ ID NO:22.

[0247] In some embodiments, an exemplary second RNA molecule comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 35. In some embodiments, the second RNA molecule comprises SEQ ID NO: 35. In some embodiments, an exemplary second RNA molecule designated as Construct B1 in the Examples provided herein is provided. In some embodiments, the second RNA molecule comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 34. In some embodiments, the second RNA molecule comprises a sequence encoded by SEQ ID NO:34.

[0248] In some embodiments, an exemplary second RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 109. In some embodiments, the second RNA molecule before cleavage of the ribozyme substrate sequence comprises SEQ ID NO: 109. In some embodiments, an exemplary second RNA molecule is provided, designated LVT-18 in the examples provided herein. In some embodiments, the second DNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 108. In some embodiments, the second DNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 108.

[0249] In some embodiments, an exemplary second RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 111. In some embodiments, the second RNA molecule before cleavage of the ribozyme substrate sequence comprises SEQ ID NO: 111. In some embodiments, an exemplary second RNA molecule is provided, designated LVT-20 in the examples provided herein. In some embodiments, the second DNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 110. In some embodiments, the second DNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 110.

[0250] In some embodiments, an exemplary second RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 113. In some embodiments, the second RNA molecule before cleavage of the ribozyme substrate sequence comprises SEQ ID NO: 113. In some embodiments, an exemplary second RNA molecule is provided, designated LVT-22 in the examples provided herein. In some embodiments, the second DNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 112. In some embodiments, the second DNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 112.

[0251] In some embodiments, an exemplary second RNA molecule (e.g., a precursor RNA molecule) before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 115. In some embodiments, the second RNA molecule before cleavage of the ribozyme substrate sequence comprises SEQ ID NO: 115. In some embodiments, an exemplary second RNA molecule is provided that is designated LVT14 / eIF4G in the examples provided herein. In some embodiments, the second DNA molecule before cleavage of the ribozyme substrate sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 114. In some embodiments, the second DNA molecule before cleavage of the ribozyme substrate sequence comprises the sequence encoded by SEQ ID NO: 114.

[0252] D. Other sequences In some embodiments, the RNA molecules provided herein, such as single-molecule RNA precursor molecules, first RNA molecules, second RNA molecules, truncated RNA molecules (pre-circRNAs), and circRNA molecules, further comprise additional sequences and / or additional components. In some embodiments, exemplary configurations and names of sequences contained in the RNA molecules provided herein are shown in Figure 1A (single-molecule method) and Figure 1B (binary-molecule method).

[0253] 1. Homologous regions, coverage sequences, and extension sequences In some embodiments, the RNA molecules provided herein further comprise a 5' homologous region and a 3' homologous region. In some embodiments, these 5' homologous region and 3' homologous region can bind or hybridize to each other to form a stem structure (see Figures 1A and 1B). In some embodiments, the sequences of the 5' homologous region and the 3' homologous region are at least partially complementary to each other. In some embodiments, the sequences of the 5' homologous region and the 3' homologous region comprise sequences complementary to each other.

[0254] In some embodiments, in the cleaved RNA molecule, the 5' homologous region and the 3' homologous region can form a stem structure. In some situations, in the cleaved RNA molecule (i.e., pre-circRNA), the 5' homologous region and the 3' homologous region bind or hybridize to each other (e.g., by forming a stem structure), bringing the 5'-terminal hydroxyl group and the 3'-terminal 2',3'-cyclic phosphate of the cleaved RNA molecule into close proximity to each other, and the two ends are ligated by a ligase to generate a circRNA. In some embodiments, in the precursor RNA molecule (e.g., the single RNA or the first RNA molecule) or the cleaved RNA molecule, the 5' homologous region is located 3' to the 5' ribozyme. In some embodiments, in the precursor RNA molecule or the cleaved RNA molecule, the 3' homologous region is located 3' to the insert sequence. In some embodiments, the 5' homologous region and the 3' homologous region remain in the circRNA molecule. In some embodiments, the length of the homologous region is 5 to 50 nucleotides (nt), e.g., 8 to 45 nt, 10 to 30 nt, 14 to 25 nt, or 15 to 20 nt, or about 8 nt, about 9 nt, about 10 nt, about 11 nt, about 12 nt, about 13 nt, about 14 nt, about 15 nt, about 16 nt, about 17 nt, about 18 nt, about 19 nt, about 20 nt, about 21 nt, about 22 nt, about 23 nt, about 24 nt, about 25 nt, about 26 nt, about 27 nt, about 28 nt, about 29 nt, about 30 nt, about 31 nt, about 32 nt, about 33 nt, about 34 nt, about 35 nt, about 36 nt, about 37 nt, about 38 nt, about 39 nt, about 40 nt, about 41 nt, about 42 nt, about 43 nt, about 44 nt, or about 45 nt. In some embodiments, the homology region is 19 nt in length.

[0255] In some embodiments, the 5' homology region comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 10. In some embodiments, the 5' homology region comprises SEQ ID NO: 10. In some embodiments, the 5' homology region is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 9. In some embodiments, the 5' homology region comprises a sequence encoded by SEQ ID NO: 9.

[0256] In some embodiments, the 3' homology region comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 19. In some embodiments, the 3' homology region comprises SEQ ID NO: 19. In some embodiments, the 3' homology region is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 18. In some embodiments, the 3' homology region comprises a sequence encoded by SEQ ID NO: 18.

[0257] In some embodiments, the precursor RNA molecule (e.g., a single RNA or a first RNA molecule) comprises a 5' coverage sequence and / or a 3' coverage sequence. In some embodiments, the precursor RNA molecule (e.g., a single RNA or a first RNA molecule) comprises a 5' coverage sequence. In some embodiments, the precursor RNA molecule (e.g., a single RNA or a first RNA molecule) comprises a 3' coverage sequence. In some embodiments, the 5' coverage sequence can bind or hybridize to a 5' homologous sequence before cleavage by a 5' ribozyme. In some embodiments, the 3' coverage sequence can bind or hybridize to a 3' homologous sequence before cleavage by a 3' ribozyme or a trans-acting ribozyme. In some embodiments, in the precursor RNA molecule before cleavage, the 5' coverage sequence is located at or near the 5' end of the precursor RNA molecule. In some embodiments, in the precursor RNA molecule before cleavage, the 3' coverage sequence is located at or near the 3' end of the precursor RNA molecule. In some embodiments, the first RNA molecule in the bimolecular combination does not include a 3' coverage sequence.

[0258] In some embodiments, the length of the coverage sequence is 5 to 50 nucleotides (nt), e.g., 8 to 45 nt, 10 to 30 nt, 14 to 25 nt, or 15 to 20 nt, or about 8 nt, about 9 nt, about 10 nt, about 11 nt, about 12 nt, about 13 nt, about 14 nt, about 15 nt, about 16 nt, about 17 nt, about 18 nt, about 19 nt, about 20 nt, about 21 nt, about 22 nt, about 23 nt, about 24 nt, about 25 nt, about 26 nt, about 27 nt, about 28 nt, about 29 nt, about 30 nt, about 31 nt, about 32 nt, about 33 nt, about 34 nt, about 35 nt, about 36 nt, about 37 nt, about 38 nt, about 39 nt, about 40 nt, about 41 nt, about 42 nt, about 43 nt, about 44 nt, or about 45 nt. In some embodiments, the coverage sequence is 8 nt in length. In some embodiments, the coverage sequence is 14 nt in length.

[0259] In some embodiments, the 5' coverage sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 2. In some embodiments, the 5' coverage sequence comprises SEQ ID NO: 2. In some embodiments, the 5' coverage sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 1. In some embodiments, the 5' coverage sequence comprises a sequence encoded by SEQ ID NO: 1.

[0260] In some embodiments, the 3' coverage sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 27. In some embodiments, the 3' coverage sequence comprises SEQ ID NO: 27. In some embodiments, the 3' coverage sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 26. In some embodiments, the 3' coverage sequence comprises a sequence encoded by SEQ ID NO: 26.

[0261] In some embodiments, the RNA molecules provided herein further comprise a spacer sequence. In some embodiments, the spacer sequence is located near the 5' end of the RNA molecule (i.e., a 5' spacer sequence). In some embodiments, the spacer sequence is located near the 3' end of the RNA molecule (i.e., a 3' spacer sequence). In some embodiments, the spacer sequence can be located between different sequence elements within the RNA molecule, for example, between the 5' homologous region and the translation initiation element, and / or between the coding sequence of the exogenous molecule and the 3' homologous region.

[0262] In some embodiments, the 5' spacer sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 12. In some embodiments, the 5' spacer sequence comprises the sequence set forth in SEQ ID NO: 12. In some embodiments, the 5' spacer sequence is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 11. In some embodiments, the 5' spacer sequence comprises the sequence encoded by SEQ ID NO: 11.

[0263] In some embodiments, the precursor RNA molecule (e.g., a single RNA or a first RNA molecule) comprises a 5' extension sequence and / or a 3' extension sequence. In some embodiments, the precursor RNA molecule (e.g., a single RNA or a first RNA molecule) comprises a 5' extension sequence. In some embodiments, the precursor RNA molecule (e.g., a single RNA or a first RNA molecule) comprises a 3' extension sequence. In some embodiments, in the precursor RNA molecule before cleavage, the 5' extension sequence is located at or near the 5' end of the precursor RNA molecule. In some embodiments, in the precursor RNA molecule before cleavage, the 3' extension sequence is located at or near the 3' end of the precursor RNA molecule.

[0264] In some embodiments, the 3' extension sequence is located 3' to the substrate sequence of the 3' ribozyme. In some embodiments, the extension sequence of the ribozyme before cleavage may be contained in the same RNA molecule as the RNA molecule containing the catalytic sequence of the ribozyme (unimolecular ribozyme, i.e., cis-acting ribozyme). In some embodiments, the extension sequence of the ribozyme before cleavage may be contained in a different RNA molecule from the RNA molecule containing the catalytic sequence of the ribozyme (binamolecular ribozyme, i.e., trans-acting ribozyme). In some embodiments, the RNA molecule comprises a 3' extension sequence located 3' to the cleavage site of the 3' ribozyme.

[0265] In some embodiments, the length of the extension sequence is 50 to 5000 nucleotides (nt), e.g., 80 to 4000 nt, 100 to 3000 nt, 200 to 2000 nt, or 300 to 1000 nt, or about 100 nt, about 200 nt, about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, about 800 nt, about 900 nt, about 1000 nt, about 1250 nt, about 1500 nt, about 2000 nt, or about 3000 nt. In some embodiments, the length of the extension sequence is 312 nt. In some embodiments, the length of the extension sequence is 544 nt. In some embodiments, the length of the extension sequence is about 1000 nt.

[0266] In some embodiments, the 3' extension sequence comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 61-63. In some embodiments, the 3' extension sequence comprises SEQ ID NO: 61. In some embodiments, the 3' extension sequence comprises SEQ ID NO: 62. In some embodiments, the 3' extension sequence comprises SEQ ID NO: 63.

[0267] 2. Insert In some embodiments, RNA molecules provided herein, such as truncated RNA molecules (i.e., pre-circRNA) and circRNA molecules, contain an insert sequence. In some embodiments, the insert sequence comprises a sequence encoding one or more exogenous molecules. In some embodiments, the insert sequence is retained after cleavage and circularization. In some embodiments, the insert sequence comprises a coding sequence for a gene product (e.g., a protein), and the RNA molecules and / or circRNA molecules provided herein comprising the insert sequence are delivered to a cell or subject. In some embodiments, the RNA molecules and / or circRNA molecules are used to deliver the insert sequence to a cell or subject and express the exogenous molecule (e.g., a gene product encoded by a coding sequence within the insert) in the cell or subject. In some embodiments, the insert sequence comprises a coding sequence for a gene and / or other components that may be required for the transcription, translation, and / or expression of the encoded gene product.

[0268] In some embodiments, the insert sequence may refer to a sequence located between the 5' overhang sequence and the 3' overhang sequence in the RNA molecules and / or circRNA molecules provided herein. In some embodiments, the insert sequence may refer to a sequence located between the 5' homologous region and the 3' homologous region in the RNA molecules and / or circRNA molecules provided herein.

[0269] In some embodiments, the length of the insert sequence is at least about 50 nucleotides (nt), about 750 nt, about 1000 nt, about 1250 nt, about 1500 nt, about 2000 nt, about 2500 nt, about 3000 nt, about 3500 nt, about 4000 nt, about 4500 nt, about 5000 nt, about 6000 nt, about 7000 nt, about 8000 nt, about 9000 nt, or about 10000 nt. In some embodiments, the length of the insert sequence is at least about 2000 nt. In some embodiments, the length of the insert sequence is at least about 3000 nt. In some embodiments, the length of the insert sequence is at least about 4000 nt. In some embodiments, the length of the insert sequence is at least about 5000 nt.

[0270] a. Exogenous molecules In some embodiments, the RNA molecules provided herein, such as truncated RNA molecules (i.e., pre-circRNA) and circRNA molecules, contain an insert sequence that includes a sequence encoding one or more exogenous molecules. In some embodiments, the one or more exogenous molecules (e.g., gene products encoded by the coding sequence in the insert) include one or more of a vaccine antigen, a cancer antigen, a CRISPR system including a nuclease and / or guide RNA (gRNA), a nuclease, a therapeutic polypeptide, an antibody or antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, and a reporter molecule.

[0271] In some embodiments, the exogenous molecule comprises a vaccine antigen. In some embodiments, the vaccine antigen comprises a viral vaccine antigen. In some embodiments, the viral vaccine antigen comprises an antigen encoded by a virus. In some embodiments, the viral vaccine antigen is derived from a virus that is a member of the Coronaviridae family. In some embodiments, the viral vaccine antigen is derived from an alphacoronavirus, a betacoronavirus, a deltacoronavirus, or a gammacoronavirus. In some embodiments, the virus is human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), Middle East respiratory syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the virus is SARS-CoV-2. In some embodiments, the viral vaccine antigen is derived from a SARS-CoV-2 virus.

[0272] In some embodiments, vaccine antigen comprises cancer antigen.In some embodiments, cancer antigen is tumor-associated antigen (TAA) or tumor-specific antigen (TSA).Exemplary cancer antigens include but are not limited to TAA or TSA, for example, Liu et al., Journal of Hematology & Oncology 15:28 (2022); Buonaguro et al., Vaccines (Basel).2020 December;8(4):615; and Zhao et al., Vaccines (Basel).2021 February;9(2):85.

[0273] In some embodiments, the exogenous molecule comprises one or more components of a CRISPR system. In some embodiments, the term "CRISPR system" is a general term referring to transcripts and other factors involved in inducing the expression or activity of CRISPR-associated ("Cas") genes, and includes sequences encoding Cas genes, various nucleic acid sequences associated with Cas nucleases, such as guide RNAs (gRNAs), and / or other sequences and transcripts derived from the CRISPR locus. In some embodiments, the CRISPR system comprises a sequence-specific nuclease. In some embodiments, the sequence-specific nuclease is a Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas12 nuclease, or a Cas13 nuclease. In some embodiments, the CRISPR system comprises a non-coding guide RNA (gRNA) that binds to DNA in a sequence-specific manner and a Cas nuclease (e.g., Cas9, CasX, Cas12, or Cas13) that has sequence-specific nuclease function. In some embodiments, the gRNA is a nucleic acid that facilitates specific targeting or homing of the gRNA molecule / Cas nuclease complex to a target nucleic acid, such as a locus on the genomic DNA of a cell. In some embodiments, the exogenous molecule comprises a guide RNA (gRNA). In some embodiments, the exogenous molecule encoded by a sequence within the insert sequence comprises a Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease. In some embodiments, the Cas nuclease is a CasX nuclease. In some embodiments, the Cas nuclease is a Cas12 nuclease. In some embodiments, the Cas nuclease is a Cas13 nuclease.Exemplary Cas nucleases that can be encoded in RNA molecules, such as the circRNA molecules provided herein, include those described, for example, in Jinek et al., Science, 343(6176):1247997, 2014; Nishimasu et al., Cell, 156:935-949, 2014; Cong et al., Science 2013, 399(6121):819-823; Wang et al., Cell 2013, 153(4):910-918; Mali et al., Science 2013, 399(6121):823-826; Cebrian-Serrano et al., Mamm Genome. 2017; 28(7): 247-261; Collias et al., Nature Communications 12:555 (2021); and Chen et al., Innovation (Camb). 2022 Jul 12; 3(4): 100264.

[0274] In some embodiments, the exogenous molecule comprises an antibody or an antigen-binding fragment thereof. In some embodiments, exemplary antibodies or antigen-binding fragments thereof that can be encoded in RNA molecules such as the circRNA molecules provided herein include known therapeutic antibodies, such as those described in The Therapeutic Structural Antibody Database (Thera-SAbDab); Raybould et al., Nucleic Acids Research, 2020, 48(D1): D383-D388; and http: / / opig.stats.ox.ac.uk / webapps / therasabdab.

[0275] In some embodiments, the exogenous molecule comprises an exogenous molecule comprising an immunomodulatory polypeptide. In some aspects, the immunomodulatory polypeptide is selected from an adjuvant, an immune checkpoint inhibitor, a cytokine, or any combination thereof. In some embodiments, the immunomodulatory polypeptide comprises a cytokine. In some embodiments, the immunomodulatory polypeptide is selected from the group consisting of IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, interferon (IFN)-α, IFN-β, IFN-γ, tumor necrosis factor (TNF)-α, TNF-β, human growth hormone, N-methionyl human growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor, fibroblast growth factor (FGF), prolactin, placental lactate, and the like. The antibody or antibody compounds are selected from the group consisting of agonists, tumor necrosis factor alpha, tumor necrosis factor beta, Mullerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor (VEGF), integrins, thrombopoietin (TPO), nerve growth factor (NGF)-beta, platelet-derived growth factor, transforming growth factor (TGF)-alpha, TGF-beta, insulin-like growth factor (IGF)-1, IGF-2, erythropoietin (EPO), bone morphogenetic factor, macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), leukemia inhibitory factor (LIF), kit ligand (KL), and / or portions thereof and / or combinations thereof.

[0276] In some embodiments, the exogenous molecule comprises a transcription factor. In some embodiments, exemplary transcription factors that can be encoded in RNA molecules, such as the circRNA molecules provided herein, include those described in, for example, Becskei et al., Molecules. 2020 Apr; 25(8): 1902 or Pandelakis et al., Cell Systems (2020) 10(1): 1-14, and / or those described in, for example, U.S. Patent Publication No. 6,140,081; U.S. Patent Publication No. 6,453,242; U.S. Patent Publication No. 6,534,261; WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536; WO03 / 016496; U.S. Patent Publication No. 20110301073; and Gaj et al., Trends in Biotechnology, 2013, 31(7), Examples of such proteins include zinc finger proteins (ZFPs) or transcription activator-like effector (TALE) proteins that can be recombined with target-specific sequences as described in U.S. Pat. No. 3,977-405.

[0277] In some embodiments, the exogenous molecule comprises a reporter molecule. In some embodiments, the reporter molecule is a detectable protein, for example, a fluorescent protein, such as green fluorescent protein (GFP); enhanced green fluorescent protein (eGFP) such as super-fold GFP (sfGFP); red fluorescent protein (RFP) such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed, or DsRed2; cyan fluorescent protein (CFP); blue-green fluorescent protein (BFP); enhanced blue fluorescent protein (EBFP); yellow fluorescent protein (YFP), or variants thereof, including chemical variants, monomeric variants, and codon-optimized stabilized and / or highly sensitive variants of fluorescent proteins. In some embodiments, the reporter molecule is an enzyme, for example, a luciferase such as firefly luciferase, the lacZ gene from Escherichia coli, alkaline phosphatase, secreted placental alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter molecules include luciferase (luc), firefly luciferase, β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof. In some embodiments, enzyme expression can be detected by adding a substrate that can detect the expression and functional activity of the enzyme. In some embodiments, the reporter molecule comprises eGFP. In some embodiments, the reporter molecule comprises RFP.

[0278] In some embodiments, the reporter molecule comprises firefly luciferase. In some embodiments, the RNA sequence of the firefly luciferase comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 16. In some embodiments, the RNA sequence of the firefly luciferase comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, the firefly luciferase is encoded by a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the firefly luciferase is encoded by the sequence set forth in SEQ ID NO:15.

[0279] b. Regulatory sequences and translation initiation elements In some embodiments, the RNA molecules provided herein comprise one or more translation initiation elements. In some embodiments, the insert sequence comprises a translation initiation element. In some embodiments, the insert sequence comprises a translation initiation element, for example, located 5' to the sequence encoding the exogenous molecule, which directs translation of the coding sequence of the exogenous molecule.

[0280] In some embodiments, the translation initiation element is or comprises an internal ribosome entry site (IRES). In some embodiments, the IRES is located near the 5' end of the RNA molecule. In some embodiments, the IRES is located 3' to a 5' overhang sequence, a 5' homology region, and / or a 5' spacer sequence.

[0281] In some embodiments, the IRES sequence is selected from the group consisting of an IRES sequence derived from Coxsackievirus type B3 (SEQ ID NO: 14), an IRES sequence derived from Coxsackievirus type B1 (CVB1) (SEQ ID NO: 71), an IRES sequence derived from Encephalomyocarditis virus (EMCV) (SEQ ID NO: 73), an IRES sequence derived from Epstein-Barr virus nuclear antigen 1 (EBNA1) (SEQ ID NO: 75), an IRES sequence derived from Enterovirus serotype EV-B107 (SEQ ID NO: 77), an IRES sequence derived from Enterovirus serotype EV-D94 (SEQ ID NO: 79), an IRES sequence derived from Echovirus type E11 (EchoV11) (SEQ ID NO: 81), an IRES sequence derived from Coronavirus 19 (Covid19) (SEQ ID NO: 83), an IRES sequence derived from Coxsackievirus type A20 (CVA20) (SEQ ID NO: 85), an IRES sequence derived from Poliovirus serotype 3 (PV3) (SEQ ID NO: 87), and an IRES sequence derived from Simian V4 The IRES sequence includes an IRES sequence derived from a virus (SEQ ID NO: 89), an IRES sequence derived from human rhinovirus type A1 (HRV-A1) (SEQ ID NO: 91), an IRES sequence derived from hepatitis C virus (HCV) (SEQ ID NO: 93), an IRES sequence derived from human rhinovirus type A21 (HRV-A21) (SEQ ID NO: 95), an IRES sequence derived from human rhinovirus type B17 (HRV-B17) (SEQ ID NO: 97), an IRES sequence derived from human rhinovirus (HRV-A100) (SEQ ID NO: 99), an IRES sequence derived from human rhinovirus type B37 (HRV-B37) (SEQ ID NO: 101), an IRES sequence derived from human rhinovirus type B92 (HRV-B92) (SEQ ID NO: 103), an IRES sequence derived from human rhinovirus type B3 (HRV-B3) (SEQ ID NO: 105), or an IRES sequence derived from human rhinovirus type C54 (HRV-C54) (SEQ ID NO: 107).

[0282] In some embodiments, the IRES comprises an internal ribosome entry site from Coxsackievirus type B3 (CVB3 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 14. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 14. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 13. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 13.

[0283] In some embodiments, the IRES comprises an internal ribosome entry site from Coxsackievirus type B1 (CVB1 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 71. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 71. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 70. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:70.

[0284] In some embodiments, the IRES comprises an internal ribosome entry site from encephalomyocarditis virus (EMCV IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 73. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 72. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:72.

[0285] In some embodiments, the IRES comprises an internal ribosome entry site derived from Epstein-Barr virus nuclear antigen 1 (EBNA1 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 75. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 75. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 74. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 74.

[0286] In some embodiments, the IRES comprises an internal ribosome entry site (ERS) sequence from enterovirus serotype EV-B107 (EV-B107 IRES). In some embodiments, the IRES RNA sequence comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 77. In some embodiments, the IRES RNA sequence comprises the sequence set forth in SEQ ID NO: 77. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 76. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:76.

[0287] In some embodiments, the IRES comprises an internal ribosome entry site (ERS) sequence from enterovirus serotype EV-D94 (EV-D94 IRES). In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 79. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 79. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 78. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:78.

[0288] In some embodiments, the IRES comprises an internal ribosome entry site from echovirus type E11 (EchoV11 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 81. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 81. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 80. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:80.

[0289] In some embodiments, the IRES comprises an internal ribosome entry site from coronavirus 19 (Covid 19 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 96%, 97%, 98%, 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 83. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 82. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:82.

[0290] In some embodiments, the IRES comprises an internal ribosome entry site from Coxsackievirus type A20 (CVA20 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 85. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 85. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 84. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:84.

[0291] In some embodiments, the IRES comprises an internal ribosome entry site from poliovirus serotype 3 (PV3 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 96%, 97%, 98%, 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 87. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 86. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:86.

[0292] In some embodiments, the IRES comprises an internal ribosome entry site from simian V4 virus (Simian V4 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 89. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 89. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 88. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:88.

[0293] In some embodiments, the IRES comprises an internal ribosome entry site from human rhinovirus type A1 (HRV-A1 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 91. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 91. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 90. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:90.

[0294] In some embodiments, the IRES comprises an internal ribosome entry site from hepatitis C virus (HCV IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 96%, 97%, 98%, 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 93. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 92. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:92.

[0295] In some embodiments, the IRES comprises an internal ribosome entry site from human rhinovirus type A21 (HRV-A21 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 95. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 95. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 94. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:94.

[0296] In some embodiments, the IRES comprises an internal ribosome entry site from human rhinovirus type B17 (HRV-B17 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 97. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 97. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 96. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:96.

[0297] In some embodiments, the IRES comprises an internal ribosome entry site from human rhinovirus type A100 (HRV-A100 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 99. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 99. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 98. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO:98.

[0298] In some embodiments, the IRES comprises an internal ribosome entry site from human rhinovirus type B37 (HRV-B37 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 101. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 101. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 100. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 100.

[0299] In some embodiments, the IRES comprises an internal ribosome entry site from human rhinovirus type B92 (HRV-B92 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 103. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 103. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 102. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 102.

[0300] In some embodiments, the IRES comprises an internal ribosome entry site from human rhinovirus type B3 (HRV-B3 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 105. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 105. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 104. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 104.

[0301] In some embodiments, the IRES comprises an internal ribosome entry site from human rhinovirus type C54 (HRV-C54 IRES) sequence. In some embodiments, the RNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 107. In some embodiments, the RNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 107. In some embodiments, the DNA sequence of the IRES comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 106. In some embodiments, the DNA sequence of the IRES comprises the sequence set forth in SEQ ID NO: 106.

[0302] In some embodiments, the translation initiation element comprises a Translation Initiator of Short 5' UTR (TISU) element, a regulatory element that controls both transcription and translation initiation of genes with essential cellular functions.

[0303] In some cases, when the insert sequence contains sequences encoding multiple exogenous molecules, a multicistronic element can be used to express multiple polypeptide gene products, and this multicistronic element can usually be placed between different types of coding sequences. An exemplary multicistronic element is a 2A element that separates the end of the 2A sequence from the next peptide downstream (see, e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)).

[0304] In some embodiments, the RNA molecules provided herein comprise a polyadenine-cytosine (polyAC) sequence. In some embodiments, the polyAC sequence can be located 3' to the coding sequence of the exogenous molecule. In some embodiments, the polyAC RNA sequence comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO:50. In some embodiments, the polyAC RNA sequence comprises the sequence set forth in SEQ ID NO:50. In some embodiments, the DNA sequence of polyAC comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 17. In some embodiments, the DNA sequence of polyAC comprises the sequence set forth in SEQ ID NO: 17.

[0305] II. Methods for generating circRNAs Provided herein are methods for producing or manufacturing circular RNA (circRNA). In some embodiments, the methods provided herein include using the RNA molecules provided herein, combinations thereof, and any of their reaction intermediates or reaction products, including linear RNA, truncated linear RNA, and circular RNA (circRNA). In some embodiments, the methods provided herein can improve the efficiency of circRNA production and the purity and stability of compositions containing circRNA, thereby improving the expression of gene products (e.g., encoded by inserts contained in nucleic acid molecules) in cells or subjects.

[0306] In some embodiments, the methods provided herein include generating an RNA molecule described herein, such as a precursor RNA (including a single precursor RNA molecule or a first RNA molecule). In some embodiments, the methods provided herein include generating a cleaved RNA molecule (sometimes referred to as a "pre-circRNA") by incubating the precursor RNA molecule under conditions suitable for cleavage of a ribozyme substrate by a ribozyme (e.g., a cleavage reaction). In some cases, the methods provided herein include generating a circRNA by incubating the cleaved RNA molecule under conditions suitable for ligation and circularization of the cleaved RNA molecule (e.g., a ligation reaction in the presence of an RNA ligase). In some embodiments, the methods provided herein do not include a separate ligation reaction, and the cleaved RNA molecule is circularized in a cell or in a subject in vivo. In some embodiments, the methods provided herein further include purifying and concentrating a preparation or reaction sample containing the RNA molecule to obtain a highly purified composition of the RNA molecule provided herein, such as a circRNA molecule or a cleaved RNA molecule.

[0307] In some embodiments, there is provided a method of generating a truncated RNA molecule, comprising: (1) producing a precursor RNA molecule provided herein; and (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; A method is provided, comprising:

[0308] In some embodiments, a method for producing a combination of RNA molecules is provided. In some embodiments, the method further comprises incubating the RNA molecules in a solution to produce cleaved RNA molecules.

[0309] In some embodiments, there is provided a method of generating a truncated RNA molecule, comprising: (1) producing a precursor RNA molecule described herein; and (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; The present invention provides a method comprising:

[0310] In some embodiments, there is provided a method of generating a truncated RNA molecule, comprising: (1) generating a combination of RNA molecules provided herein; and (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; The present invention provides a method comprising:

[0311] In some embodiments, a method for producing a circRNA molecule is further provided. In some embodiments, the method further comprises incubating the cleaved RNA molecule with an RNA ligase to generate the circRNA.

[0312] In some embodiments, there is provided a method for generating a circRNA molecule, comprising: (1) producing a precursor RNA molecule provided herein; (2) generating cleaved RNA molecules by incubating the precursor RNA molecules in solution; and (3) generating circRNA molecules by incubating the cleaved RNA molecules with RNA ligase; The present invention provides a method comprising:

[0313] In some embodiments, there is provided a method for generating a circRNA molecule, comprising: (1) generating a combination of RNA molecules provided herein; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) generating circRNA molecules by incubating the cleaved RNA molecules with RNA ligase; The present invention provides a method comprising:

[0314] In some embodiments, a method for generating circRNA comprises: (1) generating a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule described herein; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) generating circRNA molecules by incubating the cleaved RNA molecules with RNA ligase; The present invention provides a method comprising:

[0315] In some embodiments, the methods provided herein further comprise a purification step. In some embodiments, the methods provided herein further comprise a concentration step for the circular RNA.

[0316] A. Creation of RNA molecules In some embodiments, the RNA molecules provided herein, particularly single precursor RNA molecules, or combinations of a first RNA molecule and a second RNA molecule, are produced. In some embodiments, precursor RNA molecules capable of cleaving one or both of the 5' and 3' ends, and methods for producing the precursor RNA molecules are provided. In some embodiments, the methods include producing the RNA molecules provided herein, including known methods such as in vitro transcription, chemical synthesis, RNA synthesis, oligonucleotide synthesis, and oligonucleotide assembly. In some embodiments, large RNA molecules, such as single precursor RNA molecules and the first RNA molecule included in the combination, can contain large insert sequences and can be produced by in vitro transcription. In some embodiments, small RNA molecules, such as the second RNA molecule included in the combination, can be produced by chemical synthesis. Any known method for producing RNA sequences and RNA molecules can be used.

[0317] In some embodiments, the RNA molecule is produced by in vitro transcription. In some embodiments, the RNA molecule is produced by RNA synthesis.

[0318] In some embodiments, when making the RNA molecule, for example, pseudouridine, e.g., N 1 Modified nucleosides, such as -methylpseudouridine (m1Ψ), can be used to generate precursor RNA molecules containing modified nucleosides.

[0319] B. Cleavage of RNA molecules In some embodiments, the method for producing circRNA provided herein comprises incubating an RNA molecule under conditions that specifically promote cleavage of a ribozyme substrate within the RNA molecule, thereby producing a cleaved RNA molecule. In some embodiments, the method comprises a separate cleavage reaction step. In some embodiments, the method comprises incubating the RNA molecule in a solution.

[0320] 1. Cleavage reaction In some embodiments, the methods provided herein include a cleavage reaction, which is carried out, for example, by incubating a precursor RNA molecule in solution after the precursor RNA molecule is produced. In some embodiments, the cleavage reaction is carried out on multiple ribozymes in a precursor RNA molecule. In some embodiments, the cleavage reaction is a spontaneous cleavage reaction. In some embodiments, the cleavage reaction is an autocleavage reaction. In some embodiments, the cleavage reaction occurs in solution. In some embodiments, the cleavage reaction is carried out in a solution different from the solution used to produce the RNA molecule (e.g., by in vitro transcription). In some embodiments, the cleavage reaction is carried out in the same solution as the solution used to produce the RNA molecule (e.g., by in vitro transcription), but under different conditions or in a different container.

[0321] In some embodiments, the solution comprises sodium acetate (Na2OAc), magnesium acetate (MgOAc2), or both.

[0322] In some embodiments, the solution does not contain potassium chloride (KCl). In some embodiments, the solution does not contain magnesium chloride (MgCl). In some embodiments, the solution does not contain potassium chloride (KCl) or magnesium chloride (MgCl). In some embodiments, the solution contains potassium chloride (KCl) and magnesium chloride (MgCl). In some embodiments, the solution is a solution used for in vitro transcription. In some embodiments, the solution contains 40 mM HEPES pH 7.5, 40 mM DTT, 10 mM NaOAc, 75 mM MgOAc, and 0.2 mM spermidine. In some embodiments, the solution contains 30 mM HEPES pH 7.5, 100 mM KCl, and 20 mM MgCl. In some embodiments, the solution comprises 100 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM spermidine, 40 mM DTT, and 0.1 mg / ml BSA.

[0323] In some embodiments, the solution contains cyclic diguanosine monophosphate (c-di-GMP). In some embodiments, the solution contains c-di-GMP at a concentration of about 0.5 mM to about 10 mM. In some embodiments, the solution contains c-di-GMP at a concentration of about 5 mM. In some aspects, addition of c-di-GMP increases the picomolar or nanomolar dissociation constant (K) for the riboswitch. D ), which is more than three orders of magnitude higher in binding affinity than guanine analogs (Lee et al. Science, 329(5993), 845-848, Sudarsan et al., Science, 321(5887), 411-413). Such strong binding affinity may allow c-di-GMP to access the catalytic site of the ribozyme, potentially enhancing cleavage activity by providing a highly nucleophilic environment at the catalytic site of the ribozyme. In some embodiments, the cleavage reaction is carried out in the presence of c-di-GMP.

[0324] In some embodiments, the solution comprises distilled water (DW). In some embodiments, the solution consists of distilled water (DW).

[0325] In some embodiments, the solution comprises a Tris-EDTA (TE) buffer, and may comprise a TE buffer of pH 7.0 or a TE buffer of pH 8.0.

[0326] In some embodiments, in the population of RNA molecules, the cleavage reaction occurs in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population. In some embodiments, in the population of RNA molecules, a 5' cleavage reaction occurs in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population. In some embodiments, in the population of RNA molecules, a 3' cleavage reaction occurs in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population. In some embodiments, in the population of RNA molecules, both a 5' cleavage reaction and a 3' cleavage reaction occur in at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the RNA molecules in the population. In some embodiments, in the population of RNA molecules, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are linear RNA molecules truncated at at least the 5' end. In some embodiments, in the population of RNA molecules, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are linear RNA molecules truncated at at least the 3' end. In some embodiments, in the population of RNA molecules, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are linear RNA molecules truncated at both the 5' end and the 3' end.

[0327] 2. Cleaved RNA molecules In some embodiments, a truncated RNA molecule (also referred to as pre-circRNA) is provided, which is generated after cleavage of ribozyme substrates present at the 5' and 3' ends of a precursor RNA molecule. In some embodiments, this cleavage reaction generates the truncated RNA molecule.

[0328] In some embodiments, "truncated RNA molecule" further refers to pre-circRNA (see Figures 1A and 1B). In some embodiments, the truncated RNA molecule is produced by the methods provided herein. In some embodiments, the truncated RNA molecule after cleavage lacks the 5' Twister-Sister catalytic sequence. In some embodiments, the truncated RNA molecule lacks the Twister catalytic sequence. In some embodiments, the truncated RNA molecule lacks the 5' Twister-Sister catalytic sequence and the Twister catalytic sequence.

[0329] In some embodiments, both unimolecular and bimolecular methods can produce identical cleaved RNA molecules after cleavage. In some embodiments, even if the ribozyme cleaving the 3' substrate sequence is located at a different position (either at the 3' end of the precursor RNA molecule in unimolecular methods or as a trans-acting ribozyme in bimolecular methods), the cleaved RNA (pre-circRNA) produced after cleavage can be identical as long as the same ribozyme substrate sequence and catalytic sequence are used and the insert sequence is the same.

[0330] In some embodiments, a cleaved RNA is provided that comprises, in order from the 5' end to the 3' end, a 5' overhang sequence of a Twister-Sister ribozyme, a 5' homology region, an insert sequence, a 3' homology region, and a 3' overhang sequence of a Twister ribozyme.

[0331] In some embodiments, the cleaved RNA molecule comprises one or more overhang sequences, for example, a 5' overhang sequence of a Twister-Sister ribozyme, or a 3' overhang sequence of a Twister ribozyme.

[0332] In some embodiments, the cleaved RNA molecule comprises an insert sequence flanked by a 5' homology region and a 3' homology region. In some embodiments, the cleaved RNA molecule comprises an insert sequence flanked by a 5' overhang sequence and a 5' homology region and a 3' overhang sequence and a 3' homology region.

[0333] In some embodiments, the cleaved RNA molecule after cleavage comprises a hydroxyl group at the 5' end. In some embodiments, the cleaved RNA molecule comprises a 2',3'-cyclic phosphate at the 3' end. In some embodiments, the hydroxyl group at the 5' end and the 2',3'-cyclic phosphate at the 3' end of the cleaved RNA molecule can be ligated in the presence of an RNA ligase, such as RtcB ligase.

[0334] In some embodiments, exemplary cleaved RNA molecules (e.g., pre-circRNAs) comprise, from the 5' to 3' end, a 5' twister-sister ribozyme overhang sequence, a 5' homology region, a 5' spacer sequence, an IRES sequence, a coding sequence for an exogenous molecule, a polyAC sequence, a 3' homology region, and a 3' twister ribozyme overhang sequence. In some embodiments, exemplary cleaved RNA molecules (e.g., pre-circRNAs) comprise a hydroxyl group at the 5' end and a 2',3'-cyclic phosphate at the 3' end.

[0335] In some embodiments, an exemplary cleaved RNA molecule (e.g., pre-circRNA) comprises, from the 5' to 3' end, a 5' TS-1 overhang sequence, a 5' homology region, a 5' spacer sequence, an IRES sequence derived from Coxsackievirus type B3, a coding sequence for firefly luciferase (FLuc), a polyAC sequence, a 3' homology region, and a 3' overhang sequence of the P1-type Twister ribozyme derived from Parasitoid wasp. In some embodiments, an exemplary cleaved RNA molecule (e.g., pre-circRNA) comprises a hydroxyl group at the 5' end and a 2',3'-cyclic phosphate at the 3' end.

[0336] In some embodiments, the truncated RNA molecules (e.g., pre-circRNAs) provided herein comprise a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 54. In some embodiments, the truncated RNA molecules (e.g., pre-circRNAs) provided herein comprise SEQ ID NO: 54. In some embodiments, the truncated RNA molecules (e.g., pre-circRNAs) provided herein comprise a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 53. In some embodiments, the truncated RNA molecule (e.g., pre-circRNA) provided herein comprises a sequence encoded by SEQ ID NO:53.

[0337] C. Degeneration / Regeneration In some embodiments, the methods and uses provided herein further comprise a denaturing step and a renaturing step.In some embodiments, the denaturing step is carried out after incubating the RNA molecule for cleavage reaction.In some embodiments, the renaturing step is carried out after the denaturing step.

[0338] In some embodiments, the denaturing step is carried out in the same solution as that used in the cleavage reaction. In some embodiments, the denaturing step is carried out in a different solution than that used in the cleavage reaction. In some embodiments, the regeneration step is carried out in the same solution as that used in the denaturing step and / or the cleavage reaction. In some embodiments, the regeneration step is carried out in a different solution than that used in the denaturing step and / or the cleavage reaction.

[0339] In some embodiments, the denaturation step is carried out at a temperature high enough to denature and disrupt stem-loop structures within the RNA molecule. In some embodiments, the denaturation step is carried out at about 60°C to about 85°C, or about 65°C to about 80°C. In some embodiments, the denaturation step is carried out at about 65°C or about 80°C. In some embodiments, the denaturation step is carried out at about 65°C.

[0340] In some embodiments, the denaturation step is carried out for a specific time. In some embodiments, the denaturation step is carried out for 1 minute to 20 minutes, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, or 20 minutes. In some embodiments, the denaturation step is carried out for 5 minutes or less.

[0341] In some embodiments, the renaturation step is carried out at a temperature low enough to renature the RNA molecules. In some embodiments, the renaturation step is carried out at about 0°C to about 30°C, or about 4°C to about 25°C. In some embodiments, the renaturation step is carried out at ambient temperature. In some embodiments, the renaturation step is carried out at about 25°C. In some embodiments, the renaturation step is carried out at about 4°C.

[0342] In some embodiments, the regeneration step is carried out for a specific time. In some embodiments, the regeneration step is carried out for 1 minute to 20 minutes, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, or 20 minutes. In some embodiments, the regeneration step is carried out for 5 minutes or less.

[0343] In some embodiments, the denaturation and renaturation steps are performed in a solution for producing cleaved RNA molecules. In some embodiments, the solution comprises Tris-EDTA (TE) buffer. In some embodiments, the solution comprises TE buffer at pH 7.0 or TE buffer at pH 8.0. In some embodiments, the solution comprises TE buffer at pH 7.0.

[0344] D. Ligation / Circularization of RNA Molecules In some embodiments, the methods for producing circRNAs provided herein include generating circular RNA (circRNA) molecules by incubating cleaved RNA molecules (pre-circRNA) under specific conditions that promote ligation of the cleaved 5' and 3' ends. In some embodiments, the methods include a separate ligation reaction step. In some embodiments, the methods include incubating the cleaved RNA molecules with an RNA ligase, such as a tRNA splicing ligase, e.g., RtcB ligase.

[0345] In some embodiments, the method includes a separate in vitro ligation reaction by incubating the generated cleaved RNA molecule with a ligase. In some embodiments, the method does not include a separate in vitro ligation reaction. In some cases, when the cleaved RNA molecule (pre-circRNA) is introduced into a cell or a subject (i.e., in vivo), the 5' and 3' ends of the cleaved RNA molecule (pre-circRNA) are ligated. In some embodiments, an endogenous RNA ligase, such as endogenous RtcB ligase in a cell or a subject, catalyzes ligation to generate circRNA in a cell or in vivo.

[0346] 1. Ligation / Circularization Reaction In some embodiments, the methods provided herein include a ligation reaction. In some embodiments, the ligation reaction comprises incubating the cleaved RNA molecule with an RNA ligase to generate a circular RNA molecule. In some embodiments, the RNA ligase catalyzes ligation between the hydroxyl group at the 5' end of the cleaved RNA molecule and the 2',3'-cyclic phosphate at the 3' end to generate a circRNA. In some embodiments, the ligation reaction comprises incubating the generated cleaved RNA molecule with a tRNA splicing ligase.

[0347] a. tRNA splicing ligase In some embodiments, the ligase is an RNA ligase. In some embodiments, the RNA ligase is a tRNA splicing ligase. In some embodiments, the RNA ligase is an RtcB ligase.

[0348] tRNA splicing ligase (tRNA ligase) is a GTP-dependent enzyme that catalyzes the direct joining of terminal 2',3'-cyclic phosphates and 5'-hydroxyl groups of RNA strands, and plays an essential role in tRNA splicing (the ligation of exons during nascent tRNA synthesis), the unfolded protein response, and RNA repair. tRNA splicing ligases function by incorporating a 2',3'-cyclic phosphate from the substrate into the resulting 3',5'-phosphodiester bond. tRNA splicing ligases are generally conserved across multiple biological domains (see, for example, Tanaka et al., J Biol Chem 2011 Sep 2; 286(35):30253-30257; Chakravarty et al., PNAS (2012) 109 (16) 6072-6077; Kroupova et al., eLife 10:e71656). In mammals, RtcB ligase (also known as HSPC117 or FAAP) is involved in tRNA splicing and is endogenously present in mammalian cells.

[0349] In some embodiments, after the 5' and 3' substrate sequences are cleaved to form a cleaved linear RNA, the 5' and 3' overhang sequences can form a loop structure (see, e.g., the pre-circRNA shown in Figures 1A and 1B and the description in Sections IB1 and IB2). In some embodiments, after cleavage, the 5' and 3' homologous regions can form a stem structure, promoting the generation of a loop structure. The stem-and-loop structure resembles a physiological tRNA substrate for tRNA ligases such as RtcB. In tRNA, the 5' and 3' ends are present at opposite ends of a base-paired stem. The stem-and-loop structure of the cleaved RNA molecule promotes the formation of a structure similar to an endogenous substrate for tRNA ligases such as RtcB, in which the 5'-terminal hydroxyl group and the 3'-terminal 2',3'-cyclic phosphate of the cleaved RNA molecule are in close proximity to each other, allowing the two ends of the cleaved RNA to be ligated to generate a circRNA.

[0350] In some embodiments, the ligation reaction using a ligase comprises incubating the cleaved RNA molecule with an RNA ligase. In some embodiments, the RNA ligase is RtcB ligase. In some embodiments, the 2',3'-cyclic phosphate at the 3' end and the 5' end of the cleaved RNA molecule can be ligated in the presence of the RNA ligase.

[0351] In some embodiments, the RNA ligase comprises a sequence having at least, or about, 90%, 95%, 86%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 43. In some embodiments, the RNA ligase comprises the sequence set forth in SEQ ID NO: 43. In some embodiments, the RNA ligase is encoded by a sequence having at least, or about, 90%, 95%, 86%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 42. In some embodiments, the RNA ligase is encoded by the sequence set forth in SEQ ID NO: 42.

[0352] In some embodiments, ligation occurs in the presence of an endogenous tRNA ligase, such as RtcB ligase, within the cell or subject.

[0353] b. Ligation conditions In some embodiments, the methods provided herein include a ligation reaction (e.g., incubation with a ligase). The ligation reaction can be carried out over a range of temperatures. In some embodiments, the incubation step with RNA ligase is carried out at about 30°C to about 40°C, about 35°C to about 39°C, or about 36°C to about 38°C. In some embodiments, the incubation step with RNA ligase is carried out at about 37°C. In some embodiments, the ligation reaction is carried out at 37°C. In some embodiments, the ligation reaction is carried out at 30°C. In some embodiments, the ligation reaction is carried out at 25°C. In some embodiments, the ligation reaction is carried out at or near room temperature.

[0354] In some embodiments, the incubation step with RNA ligase is performed in the presence of Mg 2+The procedure is carried out in a buffer containing

[0355] In some embodiments, the ligation reaction is carried out for a specific time. In some embodiments, the ligation reaction is carried out for 1 minute to 120 minutes, for example, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 60 minutes, or 90 minutes. In some embodiments, the ligation is carried out for 10 minutes or less. In some embodiments, the ligation is carried out for 20 minutes or less. In some embodiments, the ligation reaction is carried out for 30 minutes or less. In some embodiments, the incubation step with RNA ligase is carried out for about 5 to 60 minutes, about 10 to about 30 minutes, about 15 to about 25 minutes, or about 10 to about 20 minutes. In some embodiments, the incubation step with RNA ligase is carried out for 20 minutes.

[0356] In some embodiments, as described herein, extending the ligation reaction time beyond 30 minutes reduced the efficiency of circRNA production, while incubation for 20 minutes showed the highest efficiency of circRNA production. In some embodiments, the ligation is performed at 37°C for 10 minutes or less. In some embodiments, the ligation is performed at 37°C for 20 minutes or less. In some embodiments, the ligation is performed at 37°C for 30 minutes or less.

[0357] In some embodiments, the 5'-terminal hydroxyl group and the 3'-terminal 2',3'-cyclic phosphate of the cleaved RNA molecule can be ligated in the presence of an RNA ligase.

[0358] 2. Circular RNA (circRNA) molecules In some embodiments, circular RNA (circRNA) molecules are provided. In some embodiments, the circular RNA molecules are produced by the methods or systems provided herein.

[0359] In some embodiments, circRNA molecules are generated after the 5' and 3' ends of cleaved RNA (pre-circRNA) are ligated, for example, by RtcB ligase (see, for example, the circRNA in Figures 1A and 1B). In some embodiments, the circRNA molecule comprises a 5' overhang sequence, a 5' homologous region, a 3' homologous region, and a 3' overhang sequence. In some embodiments, the circular RNA molecule comprises a 5' overhang sequence of a Twister-Sister ribozyme, a 5' homologous region, an insert sequence, a 3' homologous region, and a 3' overhang sequence of a Twister ribozyme.

[0360] In some embodiments, exemplary circRNA molecules are closed circular loops and contain the same sequence as the truncated RNA molecules described herein, except that they lack the 5' and 3' ends.

[0361] In some embodiments, an exemplary circRNA molecule comprises a 5' Twister-Sister ribozyme overhang sequence, a 5' homology region, a 5' spacer sequence, an IRES sequence, a coding sequence for the exogenous molecule, a polyAC sequence, a 3' homology region, and a 3' Twister ribozyme overhang sequence.

[0362] In some embodiments, an exemplary circRNA molecule comprises a 5' TS-1 overhang sequence, a 5' homology region, a 5' spacer sequence, an IRES sequence derived from Coxsackievirus type B3, a coding sequence for firefly luciferase (FLuc), a polyAC sequence, a 3' homology region, and a 3' overhang sequence of P1 type Twister derived from Parasitoid wasp.

[0363] In some embodiments, the circRNA molecules provided herein comprise a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 54, and are closed circular loops. In some embodiments, the circRNA molecules provided herein comprise SEQ ID NO: 54 and are closed circular loops. In some embodiments, the circRNA molecules provided herein comprise a sequence having at least, or about, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence encoded by SEQ ID NO: 53, and are closed circular loops. In some embodiments, the circRNA molecule provided herein comprises a sequence encoded by SEQ ID NO: 53 and is a closed circular loop.

[0364] E. Purification method In some embodiments, methods for purifying RNA molecules described herein, including precursor RNA molecules, truncated RNA molecules, or circRNA molecules, are provided.

[0365] In some embodiments, a purification step can be used to remove unwanted by-products (e.g., ribozyme catalytic sequences, cleaved short sequences, etc.) or undesired contaminating species. In some embodiments, circRNA purity is a crucial factor for maximizing protein production from circRNA and avoiding the innate cellular immune response associated with nicked circRNA contaminants. Contamination of circRNA compositions with nicked circRNAs makes circRNA purification difficult. In some embodiments, nicked circRNAs are resistant to treatment with RNase R and have the same molecular weight as circRNAs, making it difficult to create circRNA compositions with reduced or minimal nicked circRNA contamination.

[0366] In some embodiments, the purification method is performed by chromatography. In some embodiments, the chromatography comprises high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEC), or size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). In some embodiments, the purification method comprises HPLC.

[0367] F. Concentration method In some embodiments, provided herein are methods for enriching circRNA molecules.

[0368] In some embodiments, the composition of RNA molecules can be enriched to obtain desired circRNA molecules by incubating a preparation of RNA molecules with enzymes that regulate and / or degrade various types of undesired or contaminating RNA species.

[0369] In some embodiments, enrichment is achieved by incubation with a kinase. In some embodiments, the kinase comprises polynucleotide kinase (PNK). In some embodiments, enrichment is achieved by incubation with a phosphatase.

[0370] In some embodiments, the kinase comprises polynucleotide kinase (PNK). In some embodiments, the PNK comprises T4 PNK, which is a bifunctional enzyme with 5'-kinase activity and 3'-phosphatase activity, and plays an important role in RNA repair and DNA repair. In some embodiments, the physiological substrate of PNK phosphatase is 2',3'-cyclic phosphate terminus of RNA (Das et al., Nucleic Acids Res, 2013, 41(1):355-365).

[0371] In some cases, treatment with PNK phosphorylates the 5' end of non-circularized RNA species (e.g., mono-terminally truncated RNA molecules that cannot be circularized because only the 5' ribozyme has been cleaved; see left side of Figure 11A). In some embodiments, such 5'-terminally phosphorylated RNA species can be degraded by a 5' phosphate-dependent exonuclease (see left side of Figure 11A). In some embodiments, the methods provided herein comprise incubating an RNA molecule preparation with PNK. In some embodiments, the methods provided herein comprise incubating an RNA molecule preparation with a 5' phosphate-dependent exonuclease.

[0372] In some cases, treatment with PNK removes the 2',3'-cyclic phosphate from the 5' end of non-circularized RNA species (e.g., a mono-terminally truncated RNA molecule that cannot be circularized because only the 3' ribozyme has been cleaved; see the right side of Figure 11A). In some embodiments, such RNA species with a 3' hydroxyl group (after removal of the 2',3'-cyclic phosphate) can be degraded by specific nucleases, such as RNase R (see the right side of Figure 11A). In some embodiments, the methods provided herein comprise incubating an RNA molecule preparation with PNK. In some embodiments, the methods provided herein comprise incubating an RNA molecule preparation with RNase R.

[0373] In some embodiments, the enrichment step is performed with one or more ribonucleases (RNases). Exemplary ribonucleases that can be used in some embodiments to degrade undesired or contaminating RNA species include RNase A, RNase B, RNase C, RNase E, RNase H, RNase HI, RNase HII, RNase II, RNase III, RNase Fl, RNase L, RNase M, RNase Ms, RNase N, RNase P, RNase PhyM, RNase R, RNase Sa, RNase St, RNase Tl, RNase T2, RNase U2, RNase IV, RNase V, RNase E, RNase E, polynucleotide phosphorylase (PNPase), RNase PH, RNase, RNase BN, RNase D, RNase T, RNase 1, exonuclease, oligoribonuclease, exoribonuclease I, and exoribonuclease II. In some embodiments, the one or more RNases comprises RNase R. In some embodiments, the one or more RNases comprises a 5' phosphate-dependent exonuclease.

[0374] In some embodiments, enrichment is performed with restriction enzymes, including, but not limited to, EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI, HinFI, Sau3AI, PvuII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SpeI, SphI, StuI, and XbaI.

[0375] In some embodiments, the enrichment methods provided herein enrich circRNA molecules by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to a circRNA preparation that has not been subjected to an enrichment step. In some embodiments, in a population of RNA molecules produced by the methods provided herein, at least 70%, 80%, 90%, or 95% of the RNA molecules in the population are circular RNA molecules.

[0376] III. Systems for the production of circular ribonucleic acid (RNA) molecules Provided herein are systems, kits, compositions, and products for producing the RNA molecules provided herein (e.g., precursor RNA molecules, truncated RNA molecules, or circRNA molecules). In some embodiments, the systems, kits, compositions, and products provided herein comprise one or more components required for producing the RNA molecules provided herein (e.g., precursor RNA molecules, truncated RNA molecules, or circRNA molecules, or compositions comprising the RNA molecules provided herein). In some embodiments, the systems, kits, compositions, and products provided herein further comprise instructions for carrying out the methods described herein and / or instructions for use.

[0377] In some embodiments, the systems, kits, compositions, and articles of manufacture provided herein may include one or more of the following: deoxyribonucleic acid (DNA) molecules or vectors, including DNA molecules for generating combinations of RNA molecules or precursor RNA molecules; a system for in vitro transcription and / or a system for RNA synthesis; an RNA ligase; one or more solutions and components for cleavage reactions; one or more ligases; one or more components for purification; and / or one or more enzymes for concentration. In some embodiments, the systems, kits, compositions, and articles of manufacture provided herein may include components required to practice the methods or uses described herein, for example, as described in Sections I, II, and IV. In some embodiments, the systems, kits, compositions, and articles of manufacture provided herein may include instructions for practicing the methods or uses described herein, for example, as described in Sections I, II, and IV.

[0378] The components included in the systems, kits, compositions and products provided herein can be selected depending on the particular RNA molecule and method chosen to generate the circRNA.

[0379] A. Deoxyribonucleic Acid (DNA) Molecules and Vectors Provided herein are deoxyribonucleic acid (DNA) molecules encoding the RNA molecules provided herein. In some embodiments, the DNA molecules provided herein comprise a sequence described herein that encodes one or more of the RNA molecules described herein, or a portion or combination thereof. Also provided herein are vectors comprising the DNA molecules described herein. In some embodiments, the DNA molecule can be inserted into a nucleic acid vector. As used herein, the term "nucleic acid vector" refers to a nucleic acid that functions to deliver, carry, or express a nucleic acid of interest. Nucleic acid vectors can have specialized functions, such as expression, packaging, pseudotyping, transduction, and sequencing. Nucleic acid vectors can also have genetic engineering functions, such as cloning vectors and shuttle vectors. Nucleic acid vectors can also be engineered and have structures that contain desired forms for specific uses. Such forms include, for example, circular forms such as plasmids and phagemids, and linear or branched forms. Nucleic acid vectors can be composed of, for example, DNA or RNA, and can contain nucleotide derivatives, analogs, and mimetics, in whole or in part. Such nucleic acid vectors can be obtained from natural sources, produced by recombinant techniques, or chemically synthesized.

[0380] In some embodiments, the nucleic acid vector comprises a vector for in vitro transcription of an encoded RNA molecule, such as a precursor RNA molecule, a first RNA molecule, and / or a second RNA molecule described herein. In some embodiments, the nucleic acid vector is suitable for in vitro transcription and includes factors (e.g., regulatory factors) required for in vitro transcription, e.g., to produce any of the precursor RNA molecules, first RNA molecules, and / or second RNA molecules described herein.

[0381] In some embodiments, the systems and kits provided herein include components needed to generate an RNA molecule, such as, for example, any of the precursor RNA molecules, first RNA molecules, and / or second RNA molecules described herein, including, for example, an RNA polymerase, solutions, buffers, and cofactors for in vitro transcription.

[0382] B. Components for the cleavage reaction In some embodiments, the systems and kits provided herein include one or more solutions and components for a cleavage reaction. In some embodiments, the solutions and components include those described herein, such as those described in Section II.B.1 or in the Examples. In some embodiments, the solution includes sodium acetate (NaOAc), magnesium acetate (MgOAc), or both. In some embodiments, the solution includes cyclic diguanosine monophosphate (c-di-GMP).

[0383] C. Ligation Reaction Components In some embodiments, the systems and kits provided herein include an RNA ligase and components for a ligation reaction. In some embodiments, the RNA ligase and components include those described herein, such as those described in Section II.C.1 and in the Examples. In some embodiments, the RNA ligase includes an RtcB ligase. In some embodiments, the RNA ligase includes a sequence having at least 90%, 95%, 86%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, 95%, 86%, 97%, 98%, 99%, or 99.5% sequence identity, to the sequence set forth in SEQ ID NO:43. In some embodiments, the RNA ligase includes the sequence set forth in SEQ ID NO:43. In some embodiments, the RNA ligase is encoded by a sequence having at least 90%, 95%, 86%, 97%, 98%, 99%, or 99.5% sequence identity, or about 90%, about 95%, about 86%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 42. In some embodiments, the RNA ligase is encoded by the sequence set forth in SEQ ID NO: 42.

[0384] D. Purification and Concentration Components In some embodiments, the systems and kits provided herein include components for purification and / or enrichment steps. In some embodiments, the components include those described herein, such as those described in Sections II.D and II.E, and in the Examples. In some embodiments, the systems and kits provided herein include kinases, phosphatases, and / or ribonucleases.

[0385] IV. TREATMENT AND PROPHYLAXIS METHODS AND USES Provided herein are vaccination and / or treatment methods, for example, comprising administering an RNA molecule described herein, e.g., a circRNA or pre-circRNA, or a composition comprising the circRNA or pre-circRNA, to a subject at risk of or with a disease or disorder. In some embodiments, methods are further provided for administering an RNA molecule described herein, e.g., a circRNA or pre-circRNA, or a composition comprising the circRNA or pre-circRNA, to a subject, such as a subject at risk of or with a disease or disorder. The RNA molecules described herein, e.g., circRNA or pre-circRNA, or compositions comprising the circRNA or pre-circRNA, are useful for treating various diseases and disorders in subjects or vaccinating subjects against potential diseases or disorders. Such methods and uses include treatment methods and therapeutic uses, for example, comprising administering an RNA molecule described herein or a composition comprising the RNA molecule to a subject with a disease or disorder. Such methods and uses further include prophylactic methods and uses, for example, methods and uses for preventing a disease or disorder in a subject, comprising administering to the subject an RNA molecule described herein or a composition comprising the RNA molecule. Exemplary prophylactic uses include vaccination, for example, vaccination against an infectious disease antigen or a cancer antigen. In some embodiments, the RNA molecule described herein or a composition comprising the RNA molecule is administered in an effective amount to treat or prevent a disease or disorder. The above uses include the use of the RNA molecule described herein or a composition comprising the RNA molecule in the above methods, treatments, and preventions, as well as in the preparation of medicaments for carrying out the above methods. In some embodiments, the above methods are carried out by administering an RNA molecule described herein or a composition comprising the RNA molecule to a subject having a disease or disorder, or a subject suspected of having a disease or disorder.In some embodiments, performing the method treats a disease or disorder in a subject.

[0386] A. Administration In some embodiments, the methods and uses provided herein comprise administering to a subject an RNA molecule described herein or a composition comprising the RNA molecule. In some embodiments, the administrable RNA molecules described herein include truncated RNA molecules and circRNA molecules.

[0387] In some embodiments, the circRNA molecules or truncated RNA molecules (pre-circRNA) provided herein are administered to a subject for the purpose of vaccinating the subject. In some embodiments, vaccination includes vaccination against infectious diseases, such as viral infections, using an insert sequence encoding a viral antigen. In some embodiments, vaccination includes vaccination against cancer, using an insert sequence encoding a cancer antigen. In some embodiments, the circRNA molecules or truncated RNA molecules (pre-circRNA) provided herein are administered to treat a disease or disorder.

[0388] In some embodiments, a circRNA molecule, or a composition comprising a circRNA molecule, is administered to a subject.

[0389] In some embodiments, a truncated RNA molecule (i.e., pre-circRNA) or a composition comprising the truncated RNA molecule is administered to a subject. In some embodiments, when the truncated RNA molecule (pre-circRNA) is administered to a subject, the truncated RNA molecule is circularized in vivo in the subject's body by endogenous tRNA splicing enzymes such as endogenous RtcB ligase.

[0390] B. Delivery Vectors In some embodiments, the RNA molecules described herein or compositions comprising the RNA molecules are compatible with delivery methods using viral vectors or non-viral vectors. Exemplary delivery methods using non-viral vectors include physical delivery methods or chemical delivery methods. For example, a non-viral physical delivery method includes electroporation. Exemplary non-viral chemical delivery vectors include nanoparticles, lipid molecules (e.g., lipids), or polymers. In some embodiments, the RNA molecules described herein or compositions comprising the RNA molecules are delivered via nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles (LNPs).

[0391] V. Definition Unless otherwise specified, all technical terms, notations, and other technical and scientific terms or terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the presence of such definitions herein should not necessarily be construed as meanings significantly different from those commonly understood in the art.

[0392] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more."

[0393] As used herein, the term "about" refers to a normal error range associated with each numerical value, which is readily understandable to those skilled in the art. With respect to the term "about," a numerical value or parameter described herein includes (states) an embodiment relating to the numerical value or parameter itself.

[0394] Throughout this disclosure, various aspects of the claimed subject matter are presented in range format. Descriptions in range format are for convenience and brevity only and should not be construed as indefinitely limiting the scope of the claimed subject matter. Therefore, descriptions in range format should be considered to include all possible subranges specifically disclosed herein, as well as individual numerical values ​​subsumed within that range. For example, when a range of values ​​is recited, all values ​​between the upper and lower limits of that range, as well as any other value recited or subsumed within that range, are also encompassed within the claimed subject matter. The upper and lower limits of these narrower ranges may independently be included within the narrower range and may also be encompassed within the claimed subject matter, depending on whether or not the upper or lower limit is specifically excluded from the range stated herein. If one or both of these limits are included in a range stated herein, then ranges excluding either or both of those limits within the ranges stated herein are also encompassed within the claimed subject matter. This applies regardless of the size of the range.

[0395] In the claims, the use of order terms such as "first," "second," and "third" to modify claim elements does not, by itself, imply that a claim element is preferred, precedes, or is in a particular order relative to another element, nor does it imply a chronological order of acts in a method. Rather, these terms are used merely as markers to distinguish a particular named element from another element having the same name (when no order term is used) for the purpose of distinguishing between claim elements. Similarly, the use of terms such as "a)" and "b)" or "i)" and "ii) does not, by itself, imply that a step recited in the claim is preferred, precedes, or is in a particular order relative to another step. Similarly, the use of such terms in this specification does not, by itself, imply that one step needs to be preferred, precedes, or be in a particular order relative to another.

[0396] The terms "nucleic acid" and "nucleotide" include naturally occurring nucleic acid species or functional analogs or variants thereof. Nucleic acids can include nucleotides with various analogs of sugar moieties that are established in the art. Nucleic acids can include natural or non-natural nucleosides. In this regard, natural ribonucleic acid (RNA) can have one or more nucleosides selected from the group consisting of uridine (U), adenosine (A), cytidine (C), and guanosine (G). Useful non-natural nucleosides are established in the art and include pseudouridine in place of uridine.

[0397] The term "hybridize" or "hybridize" refers to the pairing of complementary or substantially complementary nucleic acid sequences within two different molecules. Pairing can be achieved by the process of a nucleic acid sequence binding to a substantially complementary or fully complementary sequence through base pairing to form a hybridization complex. For purposes of hybridization, two nucleic acid sequences are "substantially complementary" if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the individual bases of the two nucleic acid sequences are complementary to each other.

[0398] In some embodiments, "precursor RNA" may refer to an RNA molecule in which the substrate sequence for a ribozyme has not been cleaved.

[0399] In some embodiments, "cleaved RNA" or "pre-circRNA" may refer to an RNA molecule in which the ribozyme substrate sequences present at the 5' and 3' ends of the RNA molecule have been cleaved.

[0400] In some embodiments, "circular RNA" or "circRNA" may refer to a circularized RNA molecule generated by ligation of the 5' and 3' ends of a truncated RNA molecule (i.e., a pre-circRNA molecule).

[0401] In some embodiments, the "overhang sequence" of a ribozyme (sometimes also referred to as the "cleavage sequence") may refer to a portion of a substrate sequence that remains attached to an RNA molecule containing an insert sequence (the "cleaved RNA" or "pre-circRNA" after cleavage by a ribozyme) after cleavage by the ribozyme. In some embodiments, as used herein, the overhang sequence is a portion of the substrate sequence, and the substrate sequence comprises the overhang sequence.

[0402] In some embodiments, the "substrate sequence" of a ribozyme may refer to the portion of the ribozyme sequence that is cleaved by the catalytic portion of the ribozyme. In some embodiments, the substrate sequence of the ribozyme before cleavage may be contained in the same RNA molecule as the RNA molecule containing the catalytic sequence of the ribozyme (unimolecular ribozyme, i.e., cis-acting ribozyme). In some embodiments, the substrate sequence of the ribozyme before cleavage may be contained in an RNA molecule different from the RNA molecule containing the catalytic sequence of the ribozyme (binamolecular ribozyme, i.e., trans-acting ribozyme). In some embodiments, the substrate sequence comprises an overhanging sequence that remains attached to the cleaved RNA molecule containing the insert sequence after cleavage by the ribozyme.

[0403] In some embodiments, a "catalytic sequence" of a ribozyme can refer to a portion of the ribozyme sequence that catalyzes the cleavage of a substrate sequence. In some embodiments, the catalytic sequence cleaves the substrate sequence at or after the overhang sequence, leaving the overhang sequence attached to the cleaved RNA molecule containing the insert sequence after cleavage by the ribozyme.

[0404] VI. Illustrative Embodiments The embodiments provided herein include the following: 1. A ribonucleic acid (RNA) molecule, which, in order from the 5' end to the 3' end, a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme An RNA molecule comprising: 2. The RNA molecule of embodiment 1, wherein the 5' substrate sequence comprises a 5' overhang sequence and the 3' substrate sequence comprises a 3' overhang sequence. 3. The RNA molecule of embodiment 1 or 2, wherein the 5' ribozyme and the 3' ribozyme can cooperate to cleave the 5' substrate sequence and the 3' substrate sequence to generate a cleaved RNA molecule comprising the 5' overhang sequence, the insert sequence, and the 3' overhang sequence. 4. The RNA molecule according to any one of embodiments 1 to 3, further comprising a 5' homology region and a 3' homology region. 5. The RNA molecule of embodiment 4, wherein the 5' homology region is located 3' to the 5' ribozyme. 6. The RNA molecule of embodiment 4 or 5, wherein the 3' homology region is located 3' to the insert sequence. 7. A cleaved ribonucleic acid (RNA) molecule, in order from the 5' end to the 3' end: 5' overhang sequence of the Twister-Sister ribozyme, a 5' homologous region; an insert sequence; a 3' homologous region; 3' overhang sequence of Twister ribozyme An RNA molecule comprising: 8. An RNA molecule according to any one of embodiments 4 to 7, wherein at least a portion of the 5' homologous region and at least a portion of the 3' homologous region are complementary to each other and can form a stem structure. 9. The RNA molecule of any one of embodiments 3 to 8, wherein the cleaved RNA molecule comprises a hydroxyl group at the 5' end and a 2',3'-cyclic phosphate at the 3' end. 10. The RNA molecule of embodiment 9, wherein a circular RNA molecule can be generated by ligating the hydroxyl group at the 5' end of the cleaved RNA molecule to the 2',3'-cyclic phosphate at the 3' end in the presence of an RNA ligase. 11. The RNA molecule of embodiment 10, wherein the RNA ligase is a tRNA splicing ligase. 12. The RNA molecule of embodiment 10 or 11, wherein the RNA ligase is RtcB ligase. 13. The RNA molecule of any one of embodiments 10 to 12, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO: 43 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 43. 14. A circular ribonucleic acid (RNA) molecule comprising: 5' overhang sequence of the Twister-Sister ribozyme, a 5' homologous region; an insert sequence; a 3' homologous region; 3' overhang sequence of Twister ribozyme An RNA molecule comprising: 15. The RNA molecule of any one of embodiments 1 to 14, wherein the Twister-Sister ribozyme comprises a Twister-Sister-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme. 16. The RNA molecule of any one of embodiments 1 to 15, wherein the Twister-Sister ribozyme comprises a TS-1 ribozyme. 17. The RNA molecule of any one of embodiments 1 to 16, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO: 6 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 6. 18. The RNA molecule of any one of embodiments 1 to 17, wherein the 5' overhang sequence comprises the sequence shown in SEQ ID NO:6. 19. The RNA molecule of any one of embodiments 1 to 18, wherein the catalytic sequence of the Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO: 4 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 4. 20. The RNA molecule of any one of embodiments 1 to 19, wherein the catalytic sequence of the Twister-Sister ribozyme comprises the sequence shown in SEQ ID NO:4. 21. The RNA molecule of any one of embodiments 1 to 20, wherein the Twister ribozyme comprises a P1-type Twister ribozyme. 22. The RNA molecule of any one of embodiments 1 to 21, wherein the Twister ribozyme comprises a P1-type Twister ribozyme from Parasitoid wasp. 23. The RNA molecule of any one of embodiments 1 to 22, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO: 21 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 21. 24. The RNA molecule of any one of embodiments 1 to 23, wherein the 3' overhang sequence comprises the sequence shown in SEQ ID NO: 21. 25. The RNA molecule of any one of embodiments 1 to 24, wherein the catalytic sequence of the Twister ribozyme comprises the sequence set forth in SEQ ID NO: 23 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 23. 26. The RNA molecule of any one of embodiments 1 to 25, wherein the catalytic sequence of the Twister ribozyme comprises the sequence shown in SEQ ID NO: 23. 27. The RNA molecule of any one of embodiments 1 to 26, wherein the 5' homologous region comprises the sequence set forth in SEQ ID NO: 10 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 10. 28. The RNA molecule of any one of embodiments 1 to 27, wherein the 5' homologous region comprises the sequence shown in SEQ ID NO: 10. 29. The RNA molecule of any one of embodiments 1 to 28, wherein the 3' homologous region comprises the sequence set forth in SEQ ID NO: 19 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 19. 30. The RNA molecule of any one of embodiments 1 to 29, wherein the 3' homologous region comprises the sequence set forth in SEQ ID NO: 19. 31. The RNA molecule of any one of embodiments 1 to 30, wherein the insert sequence comprises a translation initiation element. 32. The RNA molecule of embodiment 31, wherein the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element. 33. The RNA molecule of embodiment 31 or 32, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 14. 34. The RNA molecule of any one of embodiments 31 to 33, wherein the translation initiation element comprises the sequence shown in SEQ ID NO: 14. 35. The RNA molecule of any one of embodiments 1 to 34, wherein the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules. 36. The RNA molecule of embodiment 35, wherein the one or more exogenous molecules are selected from a vaccine antigen, a cancer antigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, and a reporter molecule. 37. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecules comprise a vaccine antigen. 38. The RNA molecule of embodiment 37, wherein the vaccine antigen comprises a viral vaccine antigen. 39. The RNA molecule of embodiment 38, wherein the vaccine antigen comprises a cancer antigen and optionally a cancer neoantigen. 40. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecules comprises a sequence-specific nuclease. 41. The RNA molecule of embodiment 40, wherein the sequence-specific nuclease is a Cas nuclease. 42. The RNA molecule of embodiment 41, wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas12 nuclease, or a Cas13 nuclease. 43. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecules comprise an antibody or an antigen-binding fragment thereof. 44. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecules comprise an immunomodulatory polypeptide. 45. The RNA molecule of embodiment 44, wherein the immunomodulatory polypeptide comprises a cytokine. 46. ​​The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecules comprise a transcription factor. 47. The RNA molecule of embodiment 35 or 36, wherein the one or more exogenous molecules comprises a reporter molecule. 48. The RNA molecule of embodiment 47, wherein the reporter molecule comprises firefly luciferase, enhanced green fluorescent protein (eGFP), or red fluorescent protein (RFP). 49. The RNA molecule of embodiment 48, wherein the reporter molecule comprises firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16 or in a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 16. 50. The RNA molecule of any one of embodiments 1 to 49, wherein the length of the insert sequence is at least about 500 nucleotides (nt), about 750 nt, about 1000 nt, about 1250 nt, about 1500 nt, about 2000 nt, about 2500 nt, about 3000 nt, about 3500 nt, about 4000 nt, about 4500 nt, about 5000 nt, about 6000 nt, about 7000 nt, about 8000 nt, about 9000 nt, or about 10000 nt. 51. An RNA molecule described in any one of embodiments 1 to 50, wherein in the population of RNA molecules, cleavage of the 5' substrate sequence and / or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. 52. The RNA molecule of any one of embodiments 3 to 13 and 15 to 51, wherein in the population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules. 53. An RNA molecule described in any one of embodiments 1 to 52, wherein in the population of RNA molecules, ligation of the 5' end and the 3' end occurs in the presence of an RNA ligase in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. 54. The RNA molecule of any one of embodiments 10 to 53, wherein in the population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules. 55. The RNA molecule of any one of embodiments 1 to 54, comprising modified nucleosides. 56. The modified nucleoside is pseudouridine or N 1 56. The RNA molecule of embodiment 55, comprising methyl methylpseudouridine. 57. An RNA molecule according to any one of embodiments 1 to 56, which, when incubated with cells containing Toll-like receptors (TLRs), reduces activation of one or more TLRs or avoids detection by one or more TLRs. 58. A combination of ribonucleic acid (RNA) molecules, a first RNA molecule and a second RNA molecule; The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; combination. 59. The combination of embodiment 58, wherein the 5' substrate sequence comprises a 5' overhang sequence. 60. The combination of embodiment 58 or 59, wherein the 5' ribozyme of a first RNA molecule and the trans-acting ribozyme of a second RNA molecule can cooperate to cleave the 5' substrate sequence and the 3' substrate sequence, thereby generating a cleaved RNA molecule comprising the 5' overhang sequence, the insert sequence, and the 3' overhang sequence. 61. The combination of embodiment 60, wherein the cleaved RNA molecule further comprises a 5' homology region and a 3' homology region. 62. The combination of embodiment 61, wherein the 5' homology region is located 3' to the 5' ribozyme. 63. The combination according to embodiment 61 or 62, wherein the 3' homology region is located 3' to the insert sequence. 64. A combination according to any one of embodiments 61 to 63, wherein at least a portion of the 5' homologous region and at least a portion of the 3' homologous region are complementary and can form a stem structure. 65. The combination according to any one of embodiments 60 to 64, wherein the cleaved RNA molecule comprises a hydroxyl group at the 5' end and a 2',3'-cyclic phosphate at the 3' end. 66. The combination described in embodiment 65, wherein a circular RNA molecule can be generated by ligating the hydroxyl group at the 5' end of the cleaved RNA molecule with the 2',3'-cyclic phosphate at the 3' end in the presence of an RNA ligase. 67. The combination of embodiment 66, wherein the RNA ligase is a tRNA splicing ligase. 68. The combination of embodiment 66 or 67, wherein the RNA ligase is RtcB ligase. 69. The combination of any one of embodiments 66 to 68, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO: 43 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 43. 70. The combination according to any one of embodiments 58 to 69, wherein the Twister-Sister ribozyme comprises a Twister-Sister-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme or a TS-4 ribozyme. 71. A combination according to any one of embodiments 58 to 70, wherein the Twister-Sister ribozyme comprises a TS-1 ribozyme. 72. The combination of any one of embodiments 58 to 71, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO: 6 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 6. 73. The combination according to any one of embodiments 58 to 72, wherein the 5' overhang sequence comprises the sequence shown in SEQ ID NO: 6. 74. The combination of any one of embodiments 58 to 73, wherein the catalytic sequence of the Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO: 4 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 4. 75. The combination according to any one of embodiments 58 to 74, wherein the catalytic sequence of the Twister-Sister ribozyme comprises the sequence shown in SEQ ID NO:4. 76. A combination according to any one of embodiments 58 to 75, wherein said Twister ribozyme comprises a P1-type Twister ribozyme. 77. A combination according to any one of embodiments 58 to 76, wherein said Twister ribozyme comprises a P1-type Twister ribozyme from Parasitoid wasp. 78. The combination of any one of embodiments 58 to 77, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO: 21 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 21. 79. The combination according to any one of embodiments 58 to 78, wherein the 3' overhang sequence comprises the sequence shown in SEQ ID NO: 21. 80. The combination of any one of embodiments 58 to 79, wherein the catalytic sequence of the Twister ribozyme comprises the sequence set forth in SEQ ID NO: 23 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 23. 81. The combination according to any one of embodiments 58 to 80, wherein the catalytic sequence of the Twister ribozyme comprises the sequence shown in SEQ ID NO: 23. 82. The combination according to any one of embodiments 58 to 81, wherein the 5' homologous region comprises the sequence set forth in SEQ ID NO: 10 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 10. 83. The combination according to any one of embodiments 58 to 82, wherein the 5' homology region comprises the sequence shown in SEQ ID NO: 10. 84. The combination according to any one of embodiments 58 to 83, wherein the 3' homologous region comprises the sequence set forth in SEQ ID NO: 19 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 19. 85. The combination according to any one of embodiments 58 to 84, wherein the 3' homology region comprises the sequence shown in SEQ ID NO: 19. 86. The combination according to any one of embodiments 58 to 85, wherein the insert sequence comprises a translation initiation element. 87. The combination according to embodiment 86, wherein the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element. 88. The combination of embodiment 86 or 87, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO: 14. 89. The combination according to any one of embodiments 86 to 88, wherein the translation initiation element comprises the sequence shown in SEQ ID NO: 14. 90. The combination according to any one of embodiments 58 to 89, wherein the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules. 91. The combination of embodiment 90, wherein the one or more exogenous molecules are selected from a vaccine antigen, a cancer antigen which may be a cancer neoantigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, and a reporter molecule. 92. The combination according to embodiment 90 or 91, wherein the one or more exogenous molecules comprise a vaccine antigen. 93. The combination according to embodiment 92, wherein the vaccine antigen comprises a viral vaccine antigen. 94. The combination according to embodiment 92, wherein the vaccine antigen comprises a cancer antigen and optionally a cancer neoantigen. 95. The combination of embodiment 90 or 91, wherein the one or more exogenous molecules comprises a sequence-specific nuclease. 96. The combination of embodiment 95, wherein said sequence-specific nuclease is a Cas nuclease. 97. The combination of embodiment 96, wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas12 nuclease, or a Cas13 nuclease. 98. The combination of embodiment 90 or 91, wherein the one or more exogenous molecules comprise an antibody or an antigen-binding fragment thereof. 99. The combination of embodiment 90 or 91, wherein the one or more exogenous molecules comprise an immunomodulatory polypeptide. 100. The combination according to embodiment 99, wherein the immunomodulatory polypeptide comprises a cytokine. 101. The combination of embodiment 90 or 91, wherein the one or more exogenous molecules comprise a transcription factor. 102. The combination according to embodiment 90 or 91, wherein the one or more exogenous molecules comprise a reporter molecule. 103. The combination of embodiment 102, wherein the reporter molecule comprises firefly luciferase, enhanced green fluorescent protein (eGFP) or red fluorescent protein (RFP). 104. The combination of embodiment 103, wherein the reporter molecule comprises firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16 or in a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO: 16. 105. The combination according to any one of embodiments 58 to 104, wherein the length of the insert sequence is at least about 500 nucleotides (nt), about 750 nt, about 1000 nt, about 1250 nt, about 1500 nt, about 2000 nt, about 2500 nt, about 3000 nt, about 3500 nt, about 4000 nt, about 4500 nt, about 5000 nt, about 6000 nt, about 7000 nt, about 8000 nt, about 9000 nt or about 10000 nt. 106. The combination of any one of embodiments 58 to 105, wherein in the population of RNA molecules, cleavage of the 5' substrate sequence and / or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. 107. The combination according to any one of embodiments 60 to 106, wherein in the population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules. 108. A combination described in any one of embodiments 58 to 107, wherein in the population of RNA molecules, ligation of the 5' end and the 3' end occurs in the presence of an RNA ligase in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. 109. The combination according to any one of embodiments 66 to 108, wherein in the population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules. 110. The combination according to any one of embodiments 58 to 109, wherein the RNA molecule comprises modified nucleosides. 111. The modified nucleoside is pseudouridine or N 1 The combination according to embodiment 110, comprising methylpseudouridine. 112. A combination according to any one of embodiments 58 to 111, wherein the RNA molecule reduces activation of or avoids detection by one or more Toll-like receptors (TLRs) when incubated with cells containing TLRs. 113. A deoxyribonucleic acid (DNA) molecule encoding an RNA molecule according to any one of embodiments 1 to 57, a first RNA molecule of a combination according to any one of embodiments 58 to 112, a second RNA molecule of a combination according to any one of embodiments 58 to 112, or the first and second RNA molecules of a combination according to any one of embodiments 58 to 112. 114. A system for producing a circular RNA molecule, comprising an RNA molecule according to any one of embodiments 1 to 57, or a combination according to any one of embodiments 58 to 112. 115. A system for producing circular RNA molecules, comprising a DNA molecule according to embodiment 113 and reagents for in vitro transcription. 116. The system of embodiment 114 or 115, further comprising an RNA ligase. 117. The system described in embodiment 116, wherein the RNA ligase is a tRNA splicing ligase. 118. The system described in embodiment 116 or 117, wherein the RNA ligase is RtcB ligase. 119. The system of any one of embodiments 116 to 118, wherein the RNA ligase comprises a sequence set forth in SEQ ID NO: 43 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence set forth in SEQ ID NO: 43, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity. 120. A method for producing a ribonucleic acid (RNA) molecule, comprising producing an RNA molecule according to any one of embodiments 1-57. 121. A method for producing a ribonucleic acid (RNA) molecule, comprising: From the 5' end to the 3' end, a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme A method comprising the step of producing an RNA molecule comprising: 122. A method for producing a combination of ribonucleic acid (RNA) molecules, comprising producing a combination according to any one of embodiments 58 to 112. 123. A method for producing a combination of ribonucleic acid (RNA) molecules, comprising: generating a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; method. 124. The method of any one of embodiments 120-123, further comprising the step of producing cleaved RNA molecules by incubating said RNA molecules in solution. 125. A method for producing a truncated RNA molecule, comprising: (1) preparing an RNA molecule according to any one of embodiments 1 to 57; and (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; A method comprising: 126. A method for producing a truncated RNA molecule, comprising: (1) In the order from the 5' end to the 3' end, a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme producing an RNA molecule comprising: (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; A method comprising: 127. A method for producing a truncated RNA molecule, comprising: (1) preparing a combination according to any one of embodiments 58 to 112; and (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; A method comprising: 128. A method for producing a truncated RNA molecule, comprising: (1) generating a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; and (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; Including, The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; method. 129. The method of any one of embodiments 124-128, further comprising the step of generating a circular RNA molecule by incubating the RNA molecule with an RNA ligase. 130. A method for producing a circular RNA molecule, comprising: (1) preparing an RNA molecule according to any one of embodiments 1 to 57; (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; A method comprising: 131. A method for producing a circular RNA molecule, comprising: (1) In the order from the 5' end to the 3' end, a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme producing an RNA molecule comprising: (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; A method comprising: 132. A method for producing a circular RNA molecule, comprising: (1) preparing a combination according to any one of embodiments 58 to 112; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; A method comprising: 133. A method for producing a circular RNA molecule, comprising: (1) preparing a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; Including, The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; method. 134. A method for producing a circular RNA molecule, comprising: (1) preparing an RNA molecule according to any one of embodiments 1 to 57; (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. A method comprising: 135. A method for producing a circular RNA molecule, comprising: (1) In the order from the 5' end to the 3' end, a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' ribozymes containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme producing an RNA molecule comprising: (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. A method comprising: 136. A method for producing a circular RNA molecule, comprising: (1) preparing a combination according to any one of embodiments 58 to 112; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. A method comprising: 137. A method for producing a circular RNA molecule, comprising: (1) preparing a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. Including, The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme containing a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; 3' substrate sequence of Twister ribozyme containing 3' overhang sequence Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; method. 138. The method of any one of embodiments 134 to 137, wherein the circular RNA is generated by an RNA ligase endogenously present in the subject. 139. The method of any one of embodiments 120 to 138, wherein the RNA molecule, or the first RNA molecule or the second RNA molecule of the combination, is produced by in vitro transcription. 140. The method of any one of embodiments 120 to 139, wherein the RNA molecule, or the first RNA molecule or the second RNA molecule of the combination, is produced by RNA synthesis. 141. The method of any one of embodiments 120 to 140, wherein in the incubation step of step (2), a cleaved RNA molecule is produced after the 5' substrate sequence and the 3' substrate sequence are cleaved by the catalytic sequence. 142. The method according to any one of embodiments 120 to 141, wherein in the incubation step of step (2), a hydroxyl group at the 5' end and a 2',3'-cyclic phosphate at the 3' end of the cleaved RNA molecule are generated. 143. The method of any one of embodiments 124-142, wherein the solution comprises sodium acetate (Na2OAc), magnesium acetate (MgOAc2), or both. 144. The method of any one of embodiments 124-143, wherein the solution does not contain potassium chloride (KCl) or magnesium chloride (MgCl2). 145. The method of any one of embodiments 124 to 144, wherein the solution comprises cyclic diguanosine monophosphate (c-di-GMP). 146. The method of embodiment 145, wherein the solution comprises c-di-GMP at a concentration of about 0.5 mM to about 10 mM. 147. The method of embodiment 145 or 146, wherein the solution comprises c-di-GMP at a concentration of about 5 mM. 148. The method of any one of claims 124-142, wherein the solution comprises distilled water (DW). 149. The method of any one of claims 124-142 and 148, wherein the solution consists of distilled water (DW). 150. The method of any one of claims 124-142, wherein the solution comprises a Tris-EDTA (TE) buffer, and optionally a TE buffer of pH 7.0 or a TE buffer of pH 8.0. 151. The method of any one of claims 124-150, further comprising a denaturation step and a renaturation step. 152. The method of embodiment 151, wherein the denaturing step is carried out at about 60°C to about 85°C, about 65°C to about 80°C, about 65°C, or about 80°C. 153. The method according to embodiment 151 or 152, wherein the renaturation step comprises incubating at ambient temperature or at 4°C after the denaturation step. 154. The method of any one of claims 151 to 153, wherein the denaturing step and the renaturing step are carried out in the solution for producing the cleaved RNA molecule. 155. The method of embodiment 154, wherein the solution comprises a Tris-EDTA (TE) buffer, and may comprise a TE buffer of pH 7.0 or a TE buffer of pH 8.0. 156. The method of any one of claims 120 to 155, wherein in a population of RNA molecules produced by said method, cleavage of the 5' substrate sequence and / or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. 157. The method of any one of claims 124-156, wherein in the population of RNA molecules produced by said method, at least 70%, 80%, 90%, or 95% of the RNA molecules in said population are cleaved RNA molecules. 158. A method according to any one of claims 144 to 157, wherein a circular RNA molecule can be produced by ligating the hydroxyl group at the 5' end of the cleaved RNA molecule with the 2',3'-cyclic phosphate at the 3' end in the presence of an RNA ligase. 159. The method of any one of claims 129 to 133 and 139 to 158, wherein the incubation step with the RNA ligase in step (3) is carried out for about 5 to about 60 minutes, about 10 to about 30 minutes, about 15 to about 25 minutes, or about 10 to about 20 minutes. 160. The method of any one of claims 129-133 and 139-159, wherein the incubation step with RNA ligase in step (3) is carried out for about 20 minutes. 161. A method according to any one of claims 129 to 133 and 139 to 160, wherein the incubation step with the RNA ligase in step (3) is carried out at about 30°C to about 40°C, about 35°C to about 39°C, or about 36°C to about 38°C. 162. The method of any one of claims 129-133 and 139-161, wherein the incubation step with RNA ligase in step (3) is carried out at about 37°C. 163. The incubation step with the RNA ligase in step (3) is carried out using Mg 2+ The method according to any one of claims 129 to 133 and 139 to 162, which is carried out in a buffer comprising: 164. The method of embodiment 163, wherein the incubation step with RNA ligase in step (3) is carried out in a buffer containing Tris-HCl, KCl, MgCl2 and DTT. 165. The method of embodiment 163 or 164, wherein the incubation step with RNA ligase in step (3) is carried out in a buffer comprising 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2 and 10 mM DTT. 166. The incubation step with the RNA ligase in step (3) is carried out using Mg 2+ The method according to any one of claims 129 to 133 and 139 to 162, which is carried out in a buffer that does not contain 167. The method of any one of claims 129-166, wherein the RNA ligase is a tRNA splicing ligase. 168. The method of any one of claims 129 to 167, wherein the RNA ligase is RtcB ligase. 169. The method of any one of claims 129-168, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO:43, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:43. 170. The method of any one of claims 120-169, further comprising purifying the cleaved linear RNA molecule or the circular RNA molecule. 171. The method of embodiment 170, wherein said purification is carried out by chromatography. 172. The method of embodiment 171, wherein said chromatography is high-performance liquid chromatography (HPLC), size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), or size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC). 173. The method of any one of claims 120 to 172, further comprising the step of concentrating the circular RNA molecules. 174. The method of embodiment 173, wherein the enrichment step is carried out by incubation with a kinase. 175. The method of embodiment 174, wherein the kinase comprises polynucleotide kinase (PNK). 176. The method of embodiment 173, wherein the concentrating step is carried out by incubation with a phosphatase. 177. The method of any one of claims 173-176, wherein the enrichment step is carried out by incubation with one or more ribonucleases. 178. The method of embodiment 177, wherein the one or more ribonucleases comprise RNase R and / or a 5' phosphate-dependent exonuclease. 179. The method of any one of claims 120 to 178, wherein in a population of RNA molecules produced by said method, ligation of the 5' end to the 3' end occurs in the presence of an RNA ligase in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population. 180. The method of any one of claims 120 to 179, wherein in the population of RNA molecules produced by said method, at least 70%, 80%, 90%, or 95% of the RNA molecules in said population are circular RNA molecules. 181. The method of any one of claims 120-180, wherein the RNA molecule comprises modified nucleosides. 182. The modified nucleoside is pseudouridine or N 1 The method of embodiment 181, comprising methyl methylpseudouridine. 183. An RNA molecule produced by the method of any one of claims 120 to 182. 184. A truncated RNA molecule produced by the method of any one of claims 124-182. 185. The RNA molecule of embodiment 183 or 184, wherein the RNA molecule is a linear RNA molecule. 186. A circular RNA molecule produced by the method of any one of claims 129 to 182. 187. A composition comprising an RNA molecule according to any one of claims 1-57, 183 and 185. 188. A composition comprising the truncated RNA molecule of any one of claims 7-57, 184 and 185. 189. A composition comprising the circular RNA molecule of any one of claims 14-57 and 186. 190. A composition comprising a combination according to any one of claims 58-112. 191. The composition of any one of claims 187 to 190, which is a pharmaceutical composition. 192. The composition according to embodiment 191, comprising a pharmaceutically acceptable excipient. 193. The composition of any one of claims 187-192, comprising a lipid nanoparticle (LNP). 194. A method of vaccinating a subject, the method comprising administering an RNA molecule according to any one of claims 1 to 57 and 183 to 186, a combination according to any one of claims 58 to 112, an RNA molecule produced by the method according to any one of claims 120 to 182, or a composition according to any one of claims 187 to 193. 195. A method of treating a disease or disorder in a subject, comprising administering an RNA molecule of any one of claims 1-57 and 183-186, a combination of any one of claims 58-112, an RNA molecule produced by the method of any one of claims 120-182, or a composition of any one of claims 187-193. 196. An RNA molecule according to any one of claims 1 to 57 and 183 to 186, a combination according to any one of claims 58 to 112, an RNA molecule produced by the method of any one of claims 120 to 182, or a composition according to any one of claims 187 to 193 for use in vaccinating a subject, wherein the RNA molecule or composition is administered to the subject. 197. An RNA molecule according to any one of claims 1 to 57 and 183 to 186, a combination according to any one of claims 58 to 112, an RNA molecule produced by a method according to any one of claims 120 to 182, or a composition according to any one of claims 187 to 193 for use in treating a disease or disorder in a subject, wherein the RNA molecule or composition is administered to the subject. 198. Use of an RNA molecule according to any one of claims 1 to 57 and 183 to 186, a combination according to any one of claims 58 to 112, an RNA molecule produced by a method according to any one of claims 120 to 182, or a composition according to any one of claims 187 to 193 in the manufacture of a medicament for vaccination of a subject, wherein the medicament is administered to the subject. 199. Use of an RNA molecule according to any one of claims 1-57 and 183-186, a combination according to any one of claims 58-112, an RNA molecule produced by a method according to any one of claims 120-182, or a composition according to any one of claims 187-193 in the manufacture of a medicament for the treatment of a disease or disorder in a subject, wherein the medicament is administered to the subject. [Example]

[0405] VII. Working Examples The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0406] Example 1: Preparation of constructs encoding Twister-Sister ribozymes and RNA molecules containing Twister ribozymes to generate circular RNAs (circRNAs) Various DNA constructs encoding RNA molecules containing various ribozymes were designed and tested for their efficiency in generating circular RNA (circRNA) molecules.

[0407] Two general approaches for designing the constructs were used: (1) a unimolecular approach using an RNA molecule containing a 5′ Twister-Sister ribozyme, an insert, and a 3′ Twister ribozyme, and (2) a bimolecular approach using an RNA molecule containing a 5′ Twister-Sister ribozyme, an insert, and a 3′ Twister substrate sequence, and another RNA molecule containing a trans-acting Twister ribozyme.

[0408] An exemplary construct for the single-molecule approach contained, from 5' to 3', a sequence encoding an RNA molecule (also referred to as "precursor RNA") containing a 5' Twister-Sister-1 (TS-1) ribozyme derived from a microbial metagenomic DNA source, an internal ribosome entry site from Coxsackievirus type B3 (CVB3 IRES), a coding sequence for firefly luciferase (FLuc), and a 3' Twister ribozyme from N. vitripennis (Figure 1A, construct U1; the DNA sequence is shown in SEQ ID NO:28, and the generated RNA sequence is shown in SEQ ID NO:29). An exemplary construct for the bimolecular approach included, from 5' to 3', a sequence encoding an RNA molecule containing a 5' Twister-Sister-1 (TS-1) ribozyme, a Coxsackievirus type B3 internal ribosome entry site (CVB3 IRES), a coding sequence for firefly luciferase (FLuc), and a 3' substrate sequence for the P1-type Twister ribozyme from Parasitoid wasp (Figure 1B, Construct B1; DNA sequence shown in SEQ ID NO: 36, and the resulting RNA sequence shown in SEQ ID NO: 37). Additionally, a separate molecule encoding another RNA molecule containing the trans-acting P1-type Twister ribozyme from Parasitoid wasp was included (Figure 1B, Construct B2; DNA sequence shown in SEQ ID NO: 34, and the resulting RNA sequence shown in SEQ ID NO: 35). The substrate of construct B1 was designed to be cleaved only in the presence of the trans-acting Twister ribozyme contained in construct B2.

[0409] Using existing circRNA production methods, it has been difficult and inefficient to produce circRNAs containing large inserts. Therefore, we tested the cleavage and circularization efficiencies using a large insert sequence containing a total of 2394 nt, consisting of an internal ribosome entry site derived from Coxsackievirus type B3 (CVB3 IRES; DNA shown in SEQ ID NO: 13, encoded RNA shown in SEQ ID NO: 14; 741 nt) and a coding sequence for firefly luciferase (FLuc; DNA shown in SEQ ID NO: 15, encoded RNA shown in SEQ ID NO: 16; 1653 nt), between the 5' and 3' ribozyme sequences.

[0410] In the unimolecular approach, each construct was designed so that the 5' ribozyme and 3' ribozyme each self-cleave at a specific cleavage site. In the bimolecular approach, each construct was designed so that the 5' ribozyme self-cleaves at its cleavage site, and the 3' substrate sequence of the first RNA molecule is cleaved by a trans-acting ribozyme contained in a separate RNA molecule (i.e., the second RNA molecule). In both cases, the cleavage reaction generates a cleaved RNA molecule (also referred to as "pre-circRNA") containing a 5' cleavage product with a 5'-terminal hydroxyl group and a 3' cleavage product with a 2',3'-cyclic phosphate group, which serves as a substrate for ligase-mediated ligation. In some embodiments, the portions of the substrate sequence remaining on the RNA molecule after cleavage are referred to as overhang sequences (i.e., the 5' overhang sequence and the 3' overhang sequence). Each construct was designed so that the cleaved RNA molecule would form a stem-and-loop structure similar to that of tRNA due to the homologous regions near the 5' and 3' cleavage ends (Figures 1A-1B). Each construct was designed to form a 21-nucleotide (nt) stem with a 6-nucleotide overhang at the 5' end and a 4-nucleotide overhang at the 3' end (Figures 1C and 1D). In some embodiments, circular RNA molecules produced by the methods described herein are referred to as "circular RNAs (circRNAs)." Regarding the intermediates required for circRNA production, the linear RNA containing the ribozyme before cleavage is referred to as "precursor RNA," and the linear RNA that has been cleaved but not yet circularized is referred to as "pre-circRNA."

[0411] Example 2: Evaluation of the cleavage efficiency of unimolecular constructs encoding Twister-Sister ribozymes and RNA molecules containing Twister ribozymes to generate circular RNAs (circRNAs) The cleavage efficiency of various single-molecule constructs for circRNA generation was evaluated.

[0412] The exemplary construct U1 described above in Example 1 was used to test the efficiency of the single-molecule method for forming circular RNA using RNA containing the 5'TS-1 and 3'Twister ribozymes, and compared with various other constructs, including a construct encoding RNA containing the 5'P3 Twister U2A ribozyme (construct 3).

[0413] DNA constructs encoding the RNA molecules for circRNA production were synthesized and cloned into the pF1A T7 Flexi vector (Promega) using Gibson assembly. After colony screening and sequence verification, each plasmid was linearized and in vitro transcribed at 37°C for 3 hours using the MEGAScript T7 Transcription Kit (Thermo Fisher Scientific). Following transcription, the plasmids were digested with DNase A. The RNA was then purified using the Monarch RNA Cleanup Kit (NEB) to obtain purified precursor RNA.

[0414] After in vitro transcription, linear RNA (precursor RNA) was incubated in cleavage buffer 1 (30 mM HEPES pH 7.5, 100 mM KCl, 20 mM MgCl) at room temperature for 1 h, and the RNA was purified to obtain purified pre-circRNA.

[0415] Next, the pre-circRNA was ligated in ligase buffer 1 (0.1 mM GTP, 1 mM MnCl2) containing mouse RNase inhibitor (New England BioLabs) to prevent RNase degradation in the presence of RtcB RNA ligase (New England BioLabs) at a concentration of approximately 10 pmol pre-circRNA and 15 pmol RtcB RNA ligase for 1.5 h at 37°C, and the RNA was purified to obtain purified circRNA.

[0416] After cleanup, circRNAs were enriched by RNase R digestion to remove all linear RNAs by incubating 10 μg of total RNA sample with 2 μl of RNase R (Abcam, ab286929) at 37°C for 1 hour (in the presence of an RNase inhibitor that does not inhibit RNase R). The RNA was purified to obtain enriched circRNAs. At various stages, samples were taken and subjected to electrophoresis, and the concentrations of various RNA species in the samples were measured by densitometry.

[0417] Figure 2A shows the electrophoretic profiles of various RNA intermediates and products from construct 3 (RNA containing a 5' P3 Twister U2A ribozyme and a 3' P1-type Twister ribozyme) after generation, cleavage, ligation (with RtcB), and enrichment (with RNase R). Without RNase R treatment, the circularization efficiency of construct 3 (circRNA / (circRNA + linear RNA) × 100) was approximately 10%. This low circularization efficiency is likely due to the relatively low cleavage efficiency of the 5' P3 Twister U2A ribozyme and / or the 3' P1-type Twister ribozyme, resulting in a low number of molecules with a 5' hydroxyl terminus and a 2',3'-cyclic phosphate group, which are the 3' cleavage products, that can be ligated by RtcB.

[0418] We evaluated multiple DNA constructs encoding various ribozyme pairs, consisting of a 5' ribozyme and a 3' ribozyme, to identify a ribozyme pair that could efficiently cleave both the 5' and 3' ends of precursor RNAs to generate pre-circRNAs that could be ligated by RtcB. Twister-Sister 1 (TS-1) was selected as the 5' end candidate because its cleavage site is located near the 3' end of the ribozyme, minimizing the length of the residual ribozyme sequence and leaving a 5' hydroxyl group at the 5' end of the cleaved pre-circRNA. For the 3' ribozyme, we selected and tested several ribozymes that cleave near the 5' end, minimizing the length of the residual ribozyme sequence and leaving a 2',3'-cyclic phosphate group at the 3' end of the cleaved pre-circRNA. Specifically, we used the Twister ribozyme from Parasitoid wasp, the hatchet ribozyme, and the P1-type Twister ribozymes Osa-1 to Osa-4 from rice (Oryza sativa). As shown in Figures 2B and 2C, overall, the cleavage efficiency of the 5′TS-1 ribozyme was at least 3-5 times higher than that of the 5′Twister U2A ribozyme. The highest cleavage efficiency was observed in construct U1, which contains the 5′TS-1 ribozyme and the 3′P1-type Twister ribozyme. These results support the practicality and advantages of using 5′Twister-Sister ribozymes and 3′Twister ribozymes to generate circRNAs, particularly for the generation of circRNAs with large inserts.

[0419] Example 3: Evaluation of reaction conditions for the formation of circular RNA (circRNA) Various reaction conditions, including buffers and buffer components, were evaluated for their effect on the cleavage and ligation / circularization of RNA molecules to generate circular RNAs (circRNAs).

[0420] A. Cleavage buffer The low efficiency of direct circularization indicated that the majority of RNA species in the in vitro transcription samples were either uncleaved precursor RNA or RNA cleaved at only one end (5' or 3'). This was likely due to the low cleavage efficiency, the large insert size, and the large distance between the 5' and 3' ribozymes. In contrast, treatment with a ligase such as RtcB resulted in relatively high circularization efficiency, suggesting that the majority of the linear RNA generated after the separate cleavage reaction was pre-circRNA. Furthermore, chelating agents such as EDTA in the T7 RNA polymerase buffer and pyrophosphate generated during transcription may contribute to the reduction of Mg, which promotes ribozyme activity. 2+ Based on these findings, we performed tests using various buffers to investigate whether the buffer components used in the cleavage reaction had any effect on the cleavage efficiency or circularization efficiency.

[0421] The buffers tested for the cleavage reaction included T7 RNA transcription buffers obtained from various manufacturers (ThermoFisher, Promega, Roche, and MEGAScript) and cleavage buffer 1 (30 mM HEPES pH 7.5, 100 mM KCl, 20 mM MgCl). RNA in vitro transcribed from construct U1 was incubated in these various buffers. Each sample was then circularized by incubation with RtcB ligase, as described in Example 2 above, followed by electrophoresis and densitometry.

[0422] Figure 3A shows the electrophoretic profiles and densitometric quantification of circularization efficiencies of RNA molecules subjected to cleavage reactions using T7 RNA polymerase buffers from Thermo Fisher Scientific (lane 2), Promega Biosciences (lane 3), Roche Biosciences (lane 4), or MEGAScript Biosciences (lane 6), or Cleavage Buffer 1 (lane 5), or in vitro transcribed RNA without a separate cleavage reaction as a negative control (lane 1). Of the buffers tested, Cleavage Buffer 1 and MEGAScript Biosciences buffers yielded the highest circularization efficiencies (see Figure 3A).

[0423] Taking this result into consideration, various buffer components were further investigated to evaluate their effect on cleavage activity and circularization. Cleavage buffer 2 (40 mM HEPES pH 7.5, 40 mM DTT, 10 mM NaOAc, 75 mM MgOAc and 0.2 mM spermidine) was tested as a buffer containing sodium acetate (NaOAc) and magnesium acetate (MgOAc) instead of potassium chloride (KCl) and magnesium chloride (MgCl). Acetic acid (CHCOOH) is a weak acid, but acetate ions (CHCOOH) - Since HCl is a strong base and can only deprotonate acids with a pKa less than 5.0, we hypothesized that acetate ions could deprotonate the hydroxyl groups of ribose in the catalytic site of the ribozyme, improving cleavage and cyclization efficiencies. Furthermore, we also tested another buffer containing MgCl, cleavage buffer 3 (100 mM HEPES pH 7.5, 10 mM MgCl, 2 mM spermidine, 40 mM DTT, and 0.1 mg / ml BSA).

[0424] As shown in Figure 3B, cleavage buffer 2 containing sodium acetate and magnesium acetate (lane 4; 63.44%) increased the circularization efficiency by 1.47-fold compared to cleavage buffer 1 (lane 1; 54.1%).

[0425] Additionally, we tested the addition of cyclic diguanosine monophosphate (c-di-GMP) as an additive in the cleavage buffer. c-di-GMP exhibits picomolar and nanomolar dissociation constants (K) for the riboswitch. D ), which is more than three orders of magnitude higher than that of guanine analogs (Lee et al. Science, 329(5993), 845-848, Sudarsan et al., Science, 321(5887), 411-413). Such strong binding affinity may allow c-di-GMP to access the catalytic site of the ribozyme, potentially enhancing its cleavage activity by creating a highly nucleophilic environment at the catalytic site.

[0426] Furthermore, as shown in Figure 3B, adding a high concentration (5 mM) of c-di-GMP to cleavage buffer 1 increased the circularization efficiency by 1.61-fold (lane 3; 65.5%) compared to cleavage buffer 1 (lane 1; 54.1%). Also shown in Figure 3B, adding cleavage buffer 2 containing sodium acetate and magnesium acetate (lane 4; 63.44%) increased the circularization efficiency by 1.47-fold compared to cleavage buffer 1 (lane 1; 54.1%).

[0427] We also measured the effect of c-di-GMP on circularization efficiency in cleavage buffer 1 and cleavage buffer 2. Unexpectedly, the enhancing effect of c-di-GMP on circularization efficiency was more pronounced in cleavage buffer 1 than in cleavage buffer 2 (Figure 3C). This result supports the benefit of using c-di-GMP in the cleavage reaction to promote circRNA production.

[0428] Furthermore, magnesium ions (Mg 2+ The effect of the presence or absence of Mg was also evaluated. 2+It has been reported that MgCl2 induces nicks in generated circRNAs. The generated circRNAs were gel-purified and then incubated for 16 hours at various temperatures (room temperature, 4°C, -20°C, or -80°C) in the presence or absence of 10 mM MgCl2. The bands of nicked circRNAs appear as single nicks at random positions in non-nicked circRNAs (Figure 3D). As shown in Figure 3D, MgCl2 induced nicks at all temperatures tested.

[0429] B. Ligase Conditions We also evaluated the effect of the amount of ligase and reaction time on the efficiency of RNA circularization. Ligation reactions were performed using 500 ng of cleaved RNA prepared from construct U1. The amount of RtcB ligase (15 pmol / μL) was varied by adding 0.5 μL, 1 μL, 2 μL, 3 μL, 4 μL, or 5 μL to cleavage buffer 1. The reaction was carried out at 37°C for 1 hour and 30 minutes.

[0430] As shown in Figure 3E, the circularization efficiency gradually increased in a dose-dependent manner and reached a plateau at 2 μL of RtcB ligase (30 pmol), indicating that RtcB-mediated circularization was generally dose-dependent until it reached a plateau.

[0431] We also evaluated the effect of ligase reaction time on ligation efficiency. 1.5 mg of cleaved pre-circRNA, generated from construct U1 by in vitro transcription and cleavage in the presence of c-di-GMP, was column-purified and incubated with RtcB for 5, 10, 20, 30, 60, or 90 minutes at 37°C, or at room temperature for 90 minutes. After purification, each RNA sample was subjected to electrophoresis.

[0432] Unexpectedly, the highest circularization efficiency (75.5%) was observed when incubated with RtcB at 37°C for 20 minutes (see Figure 3F). After 20 minutes, the circularization efficiency gradually decreased, and linear RNA gradually increased (see Figure 3F). This result suggests that extending the incubation time beyond the maximum circularization efficiency may result in linearization of circular RNA. One possible reason for the apparent linearization of the generated circRNA is the Mg concentration shown in Example 3A and Figure 3D. 2+ Similarly, Mn in the ligase buffer 2+ The presence of ions likely induced nicks in the circRNA, ultimately linearizing the resulting circRNA. This result supports the benefit of limiting the ligation reaction time when generating circRNA.

[0433] Example 4: Stable expression of gene products in cells using circRNAs and intermediates Circular RNAs (circRNAs) or various intermediate preparations generated using the single-molecule methods described above in Examples 1-4 were introduced into cell lines, and expression of the encoded gene products was assessed.

[0434] HEK 293T cells were cultured and seeded into 12-well cell culture surface-treated dishes the day before transfection and grown to 60–80% confluence before transfection. Three RNA intermediate samples generated using the exemplary construct U1 (described in Figure 1A and Example 2: RNA preparation obtained after in vitro transcription ("IVT"); RNA preparation obtained after the cleavage reaction ("CLV"); or concentrated RNA obtained after the ligation reaction and RNase R treatment ("RtcB+RR")) were transfected into cells using lipofectamine as follows: First, 3 μL of Messenger Max lipid was added to 50 μL of prewarmed OPTI-MEM (Gibco). Next, 1 μg of RNA was added to the OPTI-MEM and incubated at room temperature for 5 minutes. The resulting lipid / RNA mixture was then added to HEK 293T cells.

[0435] As shown in Figure 4A , generating and enriching circRNAs by a cleavage reaction followed by treatment with ligase and RNase R (enrichment) before transfecting cells significantly increased the content of circRNAs in the RNA sample (73%, lane 3, “RtcB+RR”).

[0436] Twenty-four hours after transfection, the conversion of linear RNA to circRNA was analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using primers specific for the circRNA junction site. The primers for the circRNA junction site were 5′-GCAAGACCATGACCGAGAAG-3′ (forward primer; SEQ ID NO: 38) and 5′-GATCAAAACGTGGCTGGTGT-3′ (reverse primer; SEQ ID NO: 39). Total RNA was extracted from transfected HEK293 cells and reverse-transcribed using a cDNA synthesis kit with random hexamers or gene-specific primers.

[0437] In some embodiments, because RtcB is ubiquitously endogenously expressed in mammalian cells, transfection of linear RNA intermediates (IVT or CLV samples) into mammalian cells can also produce circRNAs in those cells. As shown in Figure 4B, transfection of IVT or CLV samples also produced circRNAs detectable by qPCR of junction sites. However, the amount of circRNA produced by transfection of IVT or CLV preparations was approximately 1.3-fold lower than the amount of circRNA produced in cells transfected with enriched circRNA preparations (Figure 4B). These data demonstrate that in vitro transcribed and cleaved linear RNA from construct U1 (lane 2; "CLV") can be circularized in mammalian cells.

[0438] Protein expression levels in cells transfected with various RNA intermediates were measured by measuring luciferase activity after transfection. At 24, 48, 72, 96, and 120 hours posttransfection, cells were washed with PBS and lysed in 200 μL of passive lysis buffer (Promega) for 15 minutes at 4°C. 10 μL of cell lysate was transferred to a 96-well solid white plate, and 90 μL of Bio-Glo substrate (Promega) was added. The mixture was incubated at room temperature for an additional 5 minutes. Relative luminescence was measured using a plate reader. As shown in Figure 4C, luciferase expression was significantly increased by ligase treatment and enrichment with RNase R (enriched circRNA samples) after the cleavage reaction.

[0439] Furthermore, we measured the kinetics of protein production and confirmed that the generated circRNA molecules or intermediates were not derived from linear mRNA molecules (unmodified uridine or modified N-terminal uridine, as the case may be) that were not designed to generate circular RNAs. 1Based on the enhanced stability or expression compared to the same protein encoded by a circRNA containing -methylpseudouridine (pU), we assessed whether the protein expressed during its lifespan was substantially greater. As shown in Figure 4D, the half-lives of protein production by transfected linear IVT and CLV preparations were 109.2 and 121.1 h, respectively, which was comparable to the half-life of protein production by enriched circRNA (119.6 h). In contrast, the half-lives of luciferase production by linear mRNA containing unmodified or modified uridines were approximately 56.9 and 58.9 h, respectively (Figure 4D). These results indicate that transfection of IVT or CLV preparations, which are intermediates of constructs designed to generate circRNAs, or enriched circRNA preparations extended the half-life of protein expression compared to expression by linear RNA expressing the same protein. These results support the utility of the exemplary constructs and RNA intermediates (e.g., IVT and CLV preparations) for generating circRNAs when introduced into mammalian cells, and the utility of the generated circRNAs for stable expression of the delivered encoded proteins for significantly longer periods of time compared to the same proteins expressed using linear mRNA.

[0440] Example 5: Evaluation of the cleavage efficiency of bimolecular constructs encoding Twister-Sister ribozymes and RNA molecules containing Twister ribozymes to generate circular RNAs (circRNAs) We evaluated the cleavage and ligation efficiencies of various bimolecular constructs generated using trans-acting ribozymes for circRNA generation.

[0441] In the bimolecular method, the cleavage efficiency and production of precursor RNA were tested using a first RNA molecule (exemplary construct B1) containing a 5' TS-1, an insert, and a 3' substrate, and a second RNA molecule containing trans-acting ribozymes from various species. The trans-acting ribozymes used were the trans-acting P1-type Twister ribozyme from Parasitoid wasp (exemplary construct B2), the pistol ribozyme, the hatchet ribozyme, and the VS ribozyme from Neurospora crassa. The 3' substrate sequence of the first RNA molecule contained the substrate sequence corresponding to each trans-acting ribozyme. The trans-acting ribozymes with cleavage activity were synthesized separately from the first RNA molecule containing the substrate. The first RNA molecule containing the substrate and the second RNA molecule containing the trans-acting ribozyme were each heated at 80°C for 1 minute and cooled to room temperature. The cleavage reaction was carried out by mixing 100 nM of the first RNA molecule containing the substrate and 5 μM of the trans-acting ribozyme in cleavage buffer 1 containing MgCl 2 or MEGAScript T7 RNA transcription buffer and incubating the mixture.

[0442] A trans-acting ribozyme cleaved the 5′ TS-1 ribozyme sequence and 3′ substrate sequence of the construct, followed by ligation with RtcB. Of the trans-acting ribozymes tested, only the construct B1-B2 pair, containing the trans-acting P1-type Twister ribozyme from Parasitoid wasp, successfully generated circRNA from an RNA molecule containing its cognate substrate (the 3′ P1-type Twister ribozyme substrate sequence from Parasitoid wasp). Densitometric analysis of each band revealed that the circularization efficiency without enrichment using RNase R was 35% when cleaved in Cleavage Buffer 1 and 27% when cleaved in MEGAScript T7 RNA Transcription Buffer (Figure 5), indicating a relatively lower circularization efficiency than the single-molecule method using construct U1. Although linear RNAs generated by trans-acting ribozymes showed relatively low cleavage efficiency, the generated circular RNAs were completely resistant to RNase R, enabling enrichment of circRNAs using RNase R (Figure 5). These results support the practicality of the exemplary bimolecular method described herein, using 5′ Twister-Sister ribozymes and trans-acting Twister ribozymes, to generate cleaved RNA intermediates (pre-circRNAs) that can be further ligated to generate circRNAs. Furthermore, these results demonstrate that the generated circRNAs can be further enriched using RNase R, demonstrating that the generated circRNAs have similar characteristics and behavior to those generated using the exemplary unimolecular method.

[0443] Example 6: DNA sequencing of junction sites for circularization in the production of circular RNA (circRNA) using unimolecular or bimolecular methods To confirm that the pre-circRNAs were circularized by using the exemplary unimolecular or bimolecular methods described above in Examples 1–5, reverse transcriptase polymerase chain reaction (RT-PCR) was performed using primers targeting the junction site of the circularized circRNA, and the resulting products were sequenced.

[0444] Pre-circRNAs were prepared by in vitro transcription of construct U1 or construct B1, followed by cleavage (in the case of construct B1, incubation with construct B2). The resulting circRNAs were then ligated in the prese...

Claims

1. A ribonucleic acid (RNA) molecule, consisting of, in order from the 5' end to the 3' end: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; A 3' ribozyme containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme. An RNA molecule comprising:

2. The RNA molecule of claim 1 , wherein the 5′ substrate sequence comprises a 5′ overhang sequence and the 3′ substrate sequence comprises a 3′ overhang sequence.

3. The RNA molecule of claim 1 or 2, wherein the 5' ribozyme and the 3' ribozyme work together to cleave the 5' substrate sequence and the 3' substrate sequence, thereby generating a cleaved RNA molecule comprising the 5' overhang sequence, the insert sequence, and the 3' overhang sequence.

4. The RNA molecule according to any one of claims 1 to 3, further comprising a 5' homologous region and a 3' homologous region.

5. The RNA molecule of claim 4, wherein the 5' homologous region is located 3' to the 5' ribozyme.

6. The RNA molecule of claim 4 or 5, wherein the 3' homologous region is located 3' to the insert sequence.

7. A truncated ribonucleic acid (RNA) molecule, which, in order from the 5' end to the 3' end, a 5' overhang sequence of the Twister-Sister ribozyme; a 5' homologous region; an insert sequence; a 3' homologous region; 3' overhang sequence of Twister ribozyme An RNA molecule comprising:

8. The RNA molecule according to any one of claims 4 to 7, wherein at least a portion of the 5' homologous region and at least a portion of the 3' homologous region are complementary to each other and can form a stem structure.

9. The RNA molecule according to any one of claims 3 to 8, wherein the cleaved RNA molecule comprises a hydroxyl group at the 5'-end and a 2',3'-cyclic phosphate at the 3'-end.

10. The RNA molecule of claim 9, which can generate a circular RNA molecule by ligating the hydroxyl group at the 5' end of the cleaved RNA molecule to the 2',3'-cyclic phosphate at the 3' end in the presence of an RNA ligase.

11. The RNA molecule of claim 10, wherein the RNA ligase is a tRNA splicing ligase.

12. The RNA molecule of claim 10 or 11, wherein the RNA ligase is RtcB ligase.

13. 13. The RNA molecule of any one of claims 10 to 12, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO: 43 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

43.

14. A circular ribonucleic acid (RNA) molecule comprising: a 5' overhang sequence of the Twister-Sister ribozyme; a 5' homologous region; an insert sequence; a 3' homologous region; 3' overhang sequence of Twister ribozyme An RNA molecule comprising:

15. The RNA molecule of any one of claims 1 to 14, wherein the Twister-Sister ribozyme comprises a Twister-Sister-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme.

16. The RNA molecule of any one of claims 1 to 15, wherein the Twister-Sister ribozyme comprises a TS-1 ribozyme.

17. 17. The RNA molecule of any one of claims 1 to 16, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO: 6 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

6.

18. The RNA molecule of any one of claims 1 to 17, wherein the 5' overhang sequence comprises the sequence shown in SEQ ID NO:

6.

19. 19. The RNA molecule of any one of claims 1 to 18, wherein the catalytic sequence of the Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO: 4 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

4.

20. 20. The RNA molecule of claim 1, wherein the catalytic sequence of the Twister-Sister ribozyme comprises the sequence shown in SEQ ID NO:

4.

21. The RNA molecule according to any one of claims 1 to 20, wherein the twister ribozyme comprises a P1-type twister ribozyme.

22. The RNA molecule according to any one of claims 1 to 21, wherein the Twister ribozyme comprises a P1-type Twister ribozyme derived from Parasitic wasp.

23. 23. The RNA molecule of any one of claims 1 to 22, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

21.

24. The RNA molecule of any one of claims 1 to 23, wherein the 3' overhang sequence comprises the sequence shown in SEQ ID NO:

21.

25. 25. The RNA molecule of any one of claims 1 to 24, wherein the catalytic sequence of the twister ribozyme comprises the sequence set forth in SEQ ID NO: 23 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

23.

26. 26. The RNA molecule of any one of claims 1 to 25, wherein the catalytic sequence of the Twister ribozyme comprises the sequence shown in SEQ ID NO:

23.

27. 27. The RNA molecule of any one of claims 1 to 26, wherein the 5' homologous region comprises the sequence set forth in SEQ ID NO: 10 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

10.

28. The RNA molecule according to any one of claims 1 to 27, wherein the 5' homologous region comprises the sequence shown in SEQ ID NO:

10.

29. 29. The RNA molecule of any one of claims 1 to 28, wherein the 3' homologous region comprises the sequence set forth in SEQ ID NO: 19 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

19.

30. The RNA molecule according to any one of claims 1 to 29, wherein the 3' homologous region comprises the sequence shown in SEQ ID NO:

19.

31. The RNA molecule of any one of claims 1 to 30, wherein the insert sequence comprises a translation initiation element.

32. 32. The RNA molecule of claim 31, wherein the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element.

33. 33. The RNA molecule of claim 31 or 32, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

14.

34. The RNA molecule of any one of claims 31 to 33, wherein the translation initiation element comprises the sequence shown in SEQ ID NO:

14.

35. The RNA molecule of any one of claims 1 to 34, wherein the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules.

36. 36. The RNA molecule of claim 35, wherein the one or more exogenous molecules are selected from a vaccine antigen, a cancer antigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, and a reporter molecule.

37. 37. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecules comprise a vaccine antigen.

38. 38. The RNA molecule of claim 37, wherein the vaccine antigen comprises a viral vaccine antigen.

39. 39. The RNA molecule of claim 38, wherein the vaccine antigen comprises a cancer antigen and optionally a cancer neoantigen.

40. 37. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecules comprises a sequence-specific nuclease.

41. The RNA molecule of claim 40, wherein the sequence-specific nuclease is a Cas nuclease.

42. 42. The RNA molecule of claim 41, wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas12 nuclease, or a Cas13 nuclease.

43. 37. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecules comprise an antibody or an antigen-binding fragment thereof.

44. 37. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecules comprises an immunomodulatory polypeptide.

45. The RNA molecule of claim 44, wherein the immunomodulatory polypeptide comprises a cytokine.

46. 37. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecules comprises a transcription factor.

47. 37. The RNA molecule of claim 35 or 36, wherein the one or more exogenous molecules comprises a reporter molecule.

48. 48. The RNA molecule of claim 47, wherein the reporter molecule comprises firefly luciferase, enhanced green fluorescent protein (eGFP), or red fluorescent protein (RFP).

49. 49. The RNA molecule of claim 48, wherein the reporter molecule comprises firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity, to the sequence set forth in SEQ ID NO:

16.

50. 50. The RNA molecule of any one of claims 1 to 49, wherein the length of the insert sequence is at least about 500 nucleotides (nt), about 750 nt, about 1000 nt, about 1250 nt, about 1500 nt, about 2000 nt, about 2500 nt, about 3000 nt, about 3500 nt, about 4000 nt, about 4500 nt, about 5000 nt, about 6000 nt, about 7000 nt, about 8000 nt, about 9000 nt, or about 10000 nt.

51. 51. The RNA molecule of any one of claims 1 to 50, wherein in the population of RNA molecules, cleavage of the 5' substrate sequence and / or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

52. 52. The RNA molecule of any one of claims 3 to 13 and 15 to 51, wherein in the population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules.

53. 53. The RNA molecule of any one of claims 1 to 52, wherein in the population of RNA molecules, ligation of the 5' end to the 3' end occurs in the presence of an RNA ligase in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

54. 54. The RNA molecule of any one of claims 10 to 53, wherein in the population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

55. 55. The RNA molecule of any one of claims 1 to 54, comprising modified nucleosides.

56. The modified nucleoside is pseudouridine or N 1 56. The RNA molecule of claim 55, comprising -methylmethylpseudouridine.

57. 57. The RNA molecule of any one of claims 1 to 56, which, when incubated with a cell containing a Toll-like receptor (TLR), reduces activation of or avoids detection by one or more TLRs.

58. A combination of ribonucleic acid (RNA) molecules, a first RNA molecule and a second RNA molecule; The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; a 3' substrate sequence for the Twister ribozyme containing a 3' overhang sequence; Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; combination.

59. 59. The combination of claim 58, wherein the 5' substrate sequence comprises a 5' overhang sequence.

60. The combination of claim 58 or 59, wherein the 5' ribozyme of the first RNA molecule and the trans-acting ribozyme of the second RNA molecule cooperate to cleave the 5' substrate sequence and the 3' substrate sequence, thereby generating a cleaved RNA molecule comprising the 5' overhang sequence, the insert sequence, and the 3' overhang sequence.

61. 61. The combination of claim 60, wherein the cleaved RNA molecule further comprises a 5' homology region and a 3' homology region.

62. 62. The combination of claim 61, wherein the 5' homology region is located 3' to the 5' ribozyme.

63. 63. The combination of claim 61 or 62, wherein the 3' homology region is located 3' to the insert sequence.

64. 64. The combination according to any one of claims 61 to 63, wherein at least a portion of the 5' homologous region and at least a portion of the 3' homologous region are complementary and can form a stem structure.

65. 65. The combination of any one of claims 60 to 64, wherein the cleaved RNA molecule comprises a hydroxyl group at the 5' end and a 2',3'-cyclic phosphate at the 3' end.

66. The combination described in claim 65, wherein a circular RNA molecule can be generated by ligating the hydroxyl group at the 5' end of the cleaved RNA molecule to the 2',3'-cyclic phosphate at the 3' end in the presence of an RNA ligase.

67. 67. The combination of claim 66, wherein the RNA ligase is a tRNA splicing ligase.

68. 68. The combination of claim 66 or 67, wherein the RNA ligase is RtcB ligase.

69. 69. The combination of any one of claims 66 to 68, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO: 43 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

43.

70. 70. The combination of any one of claims 58 to 69, wherein the Twister-Sister ribozyme comprises a Twister-Sister-1 (TS-1) ribozyme, a TS-2 ribozyme, a TS-3 ribozyme, or a TS-4 ribozyme.

71. 71. The combination of any one of claims 58 to 70, wherein the Twister-Sister ribozyme comprises a TS-1 ribozyme.

72. 72. The combination of any one of claims 58 to 71, wherein the 5' overhang sequence comprises the sequence set forth in SEQ ID NO:6 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

6.

73. 73. The combination of any one of claims 58 to 72, wherein the 5' overhang sequence comprises the sequence shown in SEQ ID NO:

6.

74. 74. The combination of any one of claims 58 to 73, wherein the catalytic sequence of the Twister-Sister ribozyme comprises the sequence set forth in SEQ ID NO:4 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

4.

75. 75. The combination of any one of claims 58 to 74, wherein the catalytic sequence of the Twister-Sister ribozyme comprises the sequence shown in SEQ ID NO:

4.

76. The combination according to any one of claims 58 to 75, wherein the twister ribozyme comprises a P1-type twister ribozyme.

77. The combination according to any one of claims 58 to 76, wherein the Twister ribozyme comprises a P1-type Twister ribozyme derived from Parasitic wasp.

78. 78. The combination of any one of claims 58 to 77, wherein the 3' overhang sequence comprises the sequence set forth in SEQ ID NO:21 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

21.

79. 79. The combination of any one of claims 58 to 78, wherein the 3' overhang sequence comprises the sequence shown in SEQ ID NO:

21.

80. 80. The combination of any one of claims 58 to 79, wherein the catalytic sequence of the twister ribozyme comprises the sequence set forth in SEQ ID NO:23 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

23.

81. 81. The combination of any one of claims 58 to 80, wherein the catalytic sequence of the Twister ribozyme comprises the sequence shown in SEQ ID NO:

23.

82. 82. The combination of any one of claims 58 to 81, wherein the 5' homologous region comprises the sequence set forth in SEQ ID NO: 10 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

10.

83. 83. The combination according to any one of claims 58 to 82, wherein the 5' homologous region comprises the sequence shown in SEQ ID NO:

10.

84. 84. The combination of any one of claims 58 to 83, wherein the 3' homologous region comprises the sequence set forth in SEQ ID NO: 19 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

19.

85. The combination according to any one of claims 58 to 84, wherein the 3' homologous region comprises the sequence shown in SEQ ID NO:

19.

86. 86. The combination of any one of claims 58 to 85, wherein the insert sequence comprises a translation initiation element.

87. 87. The combination of claim 86, wherein the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element.

88. 88. The combination of claim 86 or 87, wherein the translation initiation element comprises the sequence set forth in SEQ ID NO: 14 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

14.

89. 89. The combination of any one of claims 86 to 88, wherein the translation initiation element comprises the sequence shown in SEQ ID NO:

14.

90. 90. The combination of any one of claims 58 to 89, wherein the insert sequence comprises a nucleic acid sequence encoding one or more exogenous molecules.

91. 91. The combination of claim 90, wherein the one or more exogenous molecules are selected from a vaccine antigen, a cancer antigen which may be a cancer neo-antigen, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, and a reporter molecule.

92. 92. The combination of claim 90 or 91, wherein the one or more exogenous molecules comprise a vaccine antigen.

93. 93. The combination of claim 92, wherein the vaccine antigen comprises a viral vaccine antigen.

94. 93. The combination of claim 92, wherein the vaccine antigen comprises a cancer antigen and optionally a cancer neo-antigen.

95. 92. The combination of claim 90 or 91, wherein the one or more exogenous molecules comprises a sequence-specific nuclease.

96. 96. The combination of claim 95, wherein the sequence-specific nuclease is a Cas nuclease.

97. 97. The combination of claim 96, wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas12 nuclease, or a Cas13 nuclease.

98. 92. The combination of claim 90 or 91, wherein the one or more exogenous molecules comprise an antibody or an antigen-binding fragment thereof.

99. 92. The combination of claim 90 or 91, wherein the one or more exogenous molecules comprises an immunomodulatory polypeptide.

100. 100. The combination of claim 99, wherein the immunomodulatory polypeptide comprises a cytokine.

101. 92. The combination of claim 90 or 91, wherein the one or more exogenous molecules comprises a transcription factor.

102. 92. The combination of claim 90 or 91, wherein the one or more exogenous molecules comprises a reporter molecule.

103. 103. The combination of claim 102, wherein the reporter molecule comprises firefly luciferase, enhanced green fluorescent protein (eGFP), or red fluorescent protein (RFP).

104. 104. The combination of claim 103, wherein the reporter molecule comprises firefly luciferase, and the nucleic acid sequence encoding the firefly luciferase is set forth in SEQ ID NO: 16 or in a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

16.

105. 105. The combination of any one of claims 58 to 104, wherein the insert sequence is at least about 500 nucleotides (nt), about 750 nt, about 1000 nt, about 1250 nt, about 1500 nt, about 2000 nt, about 2500 nt, about 3000 nt, about 3500 nt, about 4000 nt, about 4500 nt, about 5000 nt, about 6000 nt, about 7000 nt, about 8000 nt, about 9000 nt, or about 10000 nt in length.

106. 106. The combination of any one of claims 58 to 105, wherein in the population of RNA molecules, cleavage of the 5' substrate sequence and / or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

107. 107. The combination of any one of claims 60 to 106, wherein in the population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are cleaved RNA molecules.

108. 108. The combination of any one of claims 58 to 107, wherein in the population of RNA molecules, ligation of the 5' end to the 3' end occurs in the presence of an RNA ligase in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

109. 109. The combination of any one of claims 66 to 108, wherein in the population of RNA molecules, at least 70%, 80%, 90% or 95% of the RNA molecules in the population are circular RNA molecules.

110. 110. The combination of any one of claims 58 to 109, wherein the RNA molecule comprises modified nucleosides.

111. The modified nucleoside is pseudouridine or N 1 111. The combination of claim 110, comprising methylpseudouridine.

112. 112. The combination of any one of claims 58 to 111, wherein the RNA molecule reduces activation of or avoids detection by one or more Toll-like receptors (TLRs) when incubated with cells containing TLRs.

113. A deoxyribonucleic acid (DNA) molecule encoding an RNA molecule according to any one of claims 1 to 57, a first RNA molecule of a combination according to any one of claims 58 to 112, a second RNA molecule of a combination according to any one of claims 58 to 112, or the first and second RNA molecules of a combination according to any one of claims 58 to 112.

114. A system for producing a circular RNA molecule, comprising the RNA molecule according to any one of claims 1 to 57, or the combination according to any one of claims 58 to 112.

115. A system for producing a circular RNA molecule, comprising the DNA molecule of claim 113 and an in vitro transcription reagent.

116. 116. The system of claim 114 or 115, further comprising an RNA ligase.

117. The system of claim 116, wherein the RNA ligase is a tRNA splicing ligase.

118. The system of claim 116 or 117, wherein the RNA ligase is RtcB ligase.

119. 119. The system of any one of claims 116 to 118, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO: 43 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

43.

120. 58. A method of making a ribonucleic acid (RNA) molecule, the method comprising the step of making an RNA molecule according to any one of claims 1 to 57.

121. 1. A method for making a ribonucleic acid (RNA) molecule, comprising: In the order from the 5' end to the 3' end, a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; A 3' ribozyme containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme. A method comprising the step of producing an RNA molecule comprising:

122. 113. A method of making a combination of ribonucleic acid (RNA) molecules, the method comprising the step of making a combination according to any one of claims 58 to 112.

123. 1. A method for making a combination of ribonucleic acid (RNA) molecules, comprising: generating a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; a 3' substrate sequence for the Twister ribozyme containing a 3' overhang sequence; Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; method.

124. 124. The method of any one of claims 120 to 123, further comprising the step of producing cleaved RNA molecules by incubating the RNA molecules in solution.

125. 1. A method of making a truncated RNA molecule, comprising: (1) producing an RNA molecule according to any one of claims 1 to 57; and (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; A method comprising:

126. 1. A method of making a truncated RNA molecule, comprising: (1) In the order from the 5' end to the 3' end, a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; A 3' ribozyme containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme. producing an RNA molecule comprising: (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; A method comprising:

127. 1. A method of making a truncated RNA molecule, comprising: (1) preparing a combination according to any one of claims 58 to 112; and (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; A method comprising:

128. 1. A method of making a truncated RNA molecule, comprising: (1) generating a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; and (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; Including, The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; a 3' substrate sequence for the Twister ribozyme containing a 3' overhang sequence; Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; method.

129. 129. The method of any one of claims 124 to 128, further comprising the step of generating a circular RNA molecule by incubating the RNA molecule with an RNA ligase.

130. 1. A method for producing a circular RNA molecule, comprising: (1) producing an RNA molecule according to any one of claims 1 to 57; (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; A method comprising:

131. 1. A method for producing a circular RNA molecule, comprising: (1) In the order from the 5' end to the 3' end, a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; A 3' ribozyme containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme. producing an RNA molecule comprising: (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; A method comprising:

132. 1. A method for producing a circular RNA molecule, comprising: (1) preparing a combination according to any one of claims 58 to 112; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; A method comprising:

133. 1. A method for producing a circular RNA molecule, comprising: (1) preparing a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) generating circular RNA molecules by incubating the cleaved RNA molecules with RNA ligase; Including, The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; a 3' substrate sequence for the Twister ribozyme containing a 3' overhang sequence; Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; method.

134. 1. A method for producing a circular RNA molecule, comprising: (1) producing an RNA molecule according to any one of claims 1 to 57; (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. A method comprising:

135. 1. A method for producing a circular RNA molecule, comprising: (1) In the order from the 5' end to the 3' end, a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; A 3' ribozyme containing the catalytic sequence and 3' substrate sequence of the Twister ribozyme. producing an RNA molecule comprising: (2) generating cleaved RNA molecules by incubating the RNA molecules in a solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. A method comprising:

136. 1. A method for producing a circular RNA molecule, comprising: (1) preparing a combination according to any one of claims 58 to 112; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. A method comprising:

137. 1. A method for producing a circular RNA molecule, comprising: (1) preparing a combination of RNA molecules comprising a first RNA molecule and a second RNA molecule; (2) generating cleaved RNA molecules by incubating the first RNA molecule and the second RNA molecule in solution; and (3) administering the cleaved RNA molecule to a subject to generate a circular RNA molecule. Including, The first RNA molecule is composed of, in order from the 5' end to the 3' end: a 5' ribozyme comprising a catalytic sequence and a 5' substrate sequence of a Twister-Sister ribozyme; an insert sequence; a 3' substrate sequence for the Twister ribozyme containing a 3' overhang sequence; Including, the second RNA molecule comprises a trans-acting ribozyme comprising the catalytic sequence of a Twister ribozyme; method.

138. The method of any one of claims 134 to 137, wherein the circular RNA is generated by an RNA ligase endogenously present in the subject.

139. 139. The method of any one of claims 120 to 138, wherein the RNA molecule, or the first or second RNA molecule of the combination, is produced by in vitro transcription.

140. 140. The method of any one of claims 120 to 139, wherein the RNA molecule, or the first or second RNA molecule of the combination, is produced by RNA synthesis.

141. 141. The method of any one of claims 120 to 140, wherein in the incubation step of step (2), a cleaved RNA molecule is produced after the 5' substrate sequence and the 3' substrate sequence are cleaved by the catalytic sequence.

142. The method of any one of claims 120 to 141, wherein in the incubation step of step (2), a hydroxyl group at the 5' end and a 2',3'-cyclic phosphate at the 3' end of the cleaved RNA molecule are generated.

143. The solution contains sodium acetate (Na 2 OAc), magnesium acetate (MgOAc 2 143. The method of any one of claims 124 to 142, comprising:

144. The solution contains neither potassium chloride (KCl) nor magnesium chloride (MgCl 2 144. The method of any one of claims 124 to 143, wherein the method does not include any of the following:

145. 145. The method of any one of claims 124 to 144, wherein the solution comprises cyclic diguanosine monophosphate (c-di-GMP).

146. 146. The method of claim 145, wherein the solution comprises c-di-GMP at a concentration of about 0.5 mM to about 10 mM.

147. 147. The method of claim 145 or 146, wherein the solution comprises c-di-GMP at a concentration of about 5 mM.

148. 143. The method of any one of claims 124 to 142, wherein the solution comprises distilled water (DW).

149. 149. The method of any one of claims 124-142 and 148, wherein the solution consists of distilled water (DW).

150. 143. The method of any one of claims 124 to 142, wherein the solution comprises a Tris-EDTA (TE) buffer, and optionally a TE buffer of pH 7.0 or a TE buffer of pH 8.

0.

151. 151. The method of any one of claims 124 to 150, further comprising a denaturation step and a renaturation step.

152. 152. The method of claim 151, wherein the denaturing step is carried out at about 60°C to about 85°C, about 65°C to about 80°C, about 65°C, or about 80°C.

153. 153. The method of claim 151 or 152, wherein the renaturation step comprises incubating at ambient temperature or 4°C after the denaturation step.

154. 154. The method of any one of claims 151 to 153, wherein the denaturing and renaturing steps are carried out in the solution for producing the cleaved RNA molecules.

155. 155. The method of claim 154, wherein the solution comprises Tris-EDTA (TE) buffer, and optionally comprises a TE buffer of pH 7.0 or a TE buffer of pH 8.

0.

156. 156. The method of any one of claims 120 to 155, wherein in a population of RNA molecules produced by said method, cleavage of the 5' substrate sequence and / or the 3' substrate sequence occurs in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

157. 157. The method of any one of claims 124 to 156, wherein in the population of RNA molecules produced by said method, at least 70%, 80%, 90% or 95% of the RNA molecules in said population are cleaved RNA molecules.

158. The method according to any one of claims 144 to 157, wherein a circular RNA molecule can be generated by ligating the hydroxyl group at the 5' end of the cleaved RNA molecule to the 2',3'-cyclic phosphate at the 3' end in the presence of an RNA ligase.

159. 159. The method of any one of claims 129 to 133 and 139 to 158, wherein the incubation with RNA ligase in step (3) is carried out for about 5 to about 60 minutes, about 10 to about 30 minutes, about 15 to about 25 minutes, or about 10 to about 20 minutes.

160. 160. The method of any one of claims 129 to 133 and 139 to 159, wherein the incubation with RNA ligase in step (3) is carried out for about 20 minutes.

161. 161. The method of any one of claims 129 to 133 and 139 to 160, wherein the incubation step with the RNA ligase in step (3) is carried out at about 30°C to about 40°C, about 35°C to about 39°C, or about 36°C to about 38°C.

162. 162. The method of any one of claims 129 to 133 and 139 to 161, wherein the incubation step with the RNA ligase in step (3) is carried out at about 37°C.

163. The incubation step with RNA ligase in step (3) is carried out in the presence of Mg 2+ The method of any one of claims 129 to 133 and 139 to 162, which is carried out in a buffer comprising:

164. The incubation step with the RNA ligase in step (3) is carried out in a solution of Tris-HCl, KCl, MgCl 2 and DTT.

165. The incubation step with RNA ligase in step (3) is carried out in 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl 2 and 10 mM DTT.

166. The incubation step with RNA ligase in step (3) is carried out in the presence of Mg 2+ The method of any one of claims 129 to 133 and 139 to 162, wherein the method is carried out in a buffer that does not contain

167. The method of any one of claims 129 to 166, wherein the RNA ligase is a tRNA splicing ligase.

168. The method of any one of claims 129 to 167, wherein the RNA ligase is RtcB ligase.

169. 169. The method of any one of claims 129 to 168, wherein the RNA ligase comprises the sequence set forth in SEQ ID NO: 43, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity to the sequence set forth in SEQ ID NO:

43.

170. 170. The method of any one of claims 120 to 169, further comprising purifying the cleaved linear or circular RNA molecules.

171. 171. The method of claim 170, wherein the purification is performed by chromatography.

172. 172. The method of claim 171, wherein the chromatography is high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), ion exchange chromatography (IEC), or size exclusion chromatography-high performance liquid chromatography (SEC-HPLC).

173. The method of any one of claims 120 to 172, further comprising concentrating the circular RNA molecules.

174. 174. The method of claim 173, wherein the enrichment step is carried out by incubation with a kinase.

175. 175. The method of claim 174, wherein the kinase comprises polynucleotide kinase (PNK).

176. 174. The method of claim 173, wherein the concentration step is carried out by incubation with a phosphatase.

177. 177. The method of any one of claims 173 to 176, wherein the concentrating step is carried out by incubation with one or more ribonucleases.

178. 178. The method of claim 177, wherein the one or more ribonucleases comprise RNase R and / or a 5' phosphate-dependent exonuclease.

179. 179. The method of any one of claims 120 to 178, wherein in the population of RNA molecules produced by the method, ligation of the 5' end to the 3' end occurs in the presence of an RNA ligase in at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the RNA molecules in the population.

180. 180. The method of any one of claims 120 to 179, wherein in the population of RNA molecules produced by said method, at least 70%, 80%, 90% or 95% of the RNA molecules in said population are circular RNA molecules.

181. 181. The method of any one of claims 120 to 180, wherein the RNA molecule comprises modified nucleosides.

182. The modified nucleoside is pseudouridine or N 1 The method of claim 181, comprising -methyl methylpseudouridine.

183. An RNA molecule produced by the method of any one of claims 120 to 182.

184. A truncated RNA molecule produced by the method of any one of claims 124 to 182.

185. 185. The RNA molecule of claim 183 or 184, wherein the RNA molecule is a linear RNA molecule.

186. A circular RNA molecule produced by the method of any one of claims 129 to 182.

187. A composition comprising an RNA molecule according to any one of claims 1 to 57, 183 and 185.

188. 186. A composition comprising the truncated RNA molecule of any one of claims 7 to 57, 184 and 185.

189. A composition comprising a circular RNA molecule according to any one of claims 14 to 57 and 186.

190. A composition comprising a combination according to any one of claims 58 to 112.

191. The composition according to any one of claims 187 to 190, which is a pharmaceutical composition.

192. 192. The composition of claim 191, comprising a pharmaceutically acceptable excipient.

193. 193. The composition of any one of claims 187 to 192, comprising a lipid nanoparticle (LNP).

194. 193. A method of vaccinating a subject, the method comprising administering an RNA molecule according to any one of claims 1 to 57 and 183 to 186, a combination according to any one of claims 58 to 112, an RNA molecule produced by the method of any one of claims 120 to 182, or a composition according to any one of claims 187 to 193.

195. 193. A method of treating a disease or disorder in a subject, the method comprising administering an RNA molecule of any one of claims 1 to 57 and 183 to 186, a combination of any one of claims 58 to 112, an RNA molecule produced by the method of any one of claims 120 to 182, or a composition of any one of claims 187 to 193.

196. 194. An RNA molecule according to any one of claims 1 to 57 and 183 to 186, a combination according to any one of claims 58 to 112, an RNA molecule produced by the method of any one of claims 120 to 182, or a composition according to any one of claims 187 to 193 for use in vaccinating a subject, wherein said RNA molecule or composition is administered to said subject.

197. 194. An RNA molecule according to any one of claims 1 to 57 and 183 to 186, a combination according to any one of claims 58 to 112, an RNA molecule made by a method according to any one of claims 120 to 182, or a composition according to any one of claims 187 to 193 for use in the treatment of a disease or disorder in a subject, wherein said RNA molecule or composition is administered to said subject.

198. 194. Use of an RNA molecule according to any one of claims 1 to 57 and 183 to 186, a combination according to any one of claims 58 to 112, an RNA molecule produced by a method according to any one of claims 120 to 182, or a composition according to any one of claims 187 to 193 in the manufacture of a medicament for vaccination of a subject, wherein said medicament is administered to said subject.

199. 194. Use of an RNA molecule according to any one of claims 1 to 57 and 183 to 186, a combination according to any one of claims 58 to 112, an RNA molecule produced by a method according to any one of claims 120 to 182, or a composition according to any one of claims 187 to 193 in the manufacture of a medicament for the treatment of a disease or disorder in a subject, wherein the medicament is administered to the subject.

200. 182. The RNA molecule of any one of claims 1 to 57, the combination of any one of claims 58 to 112, the system of claims 114 to 119, or the method of any one of claims 120 to 182, wherein the RNA molecule comprises an internal ribosome entry site (IRES).

201. 201. The RNA molecule, combination, system or method of claim 200, wherein the IRES comprises an IRES derived from Coxsackievirus B3, Coxsackievirus B1, encephalomyocarditis virus, Epstein-Barr virus nuclear antigen 1, enterovirus serotype EV-B107, enterovirus serotype EV-D94, echovirus E11, coronavirus 19, coxsackievirus A20, poliovirus serotype 3, simian V4 virus, human rhinovirus A1, hepatitis C virus, human rhinovirus A21, human rhinovirus B17, human rhinovirus, human rhinovirus B37, human rhinovirus B92, human rhinovirus B3, or human rhinovirus C54.

202. 202. The RNA molecule, combination, system or method of claim 200 or 201, wherein the IRES has at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity, or at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence identity, to SEQ ID NO: 14, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107.

203. 203. The RNA molecule, combination, system or method of any one of claims 200 to 202, wherein the RNA molecule comprises a binding site for poly(A) binding protein (PABP).

204. 204. The RNA molecule, combination, system or method of any one of claims 200 to 203, wherein the RNA molecule comprises a binding site for poly(C) binding protein (PCBP).

205. 205. The RNA molecule, combination, system or method of any one of claims 200 to 204, wherein the RNA molecule comprises an eIF4G binding site.

206. The RNA molecule, combination, system or method of any one of claims 200 to 205, wherein the RNA molecule comprises LVT-18 (SEQ ID NO: 109), LVT-20 (SEQ ID NO: 111), LVT-22 (SEQ ID NO: 113) or LVT14 / eIF4G (SEQ ID NO: 115).

207. 207. The RNA molecule, combination, system or method of any one of claims 200 to 206, wherein the RNA molecule has reduced immunogenicity.

208. 208. The RNA molecule, combination, system or method of any one of claims 200 to 207, wherein said reduced immunogenicity comprises reduced detection by Toll-like receptors (TLRs).

209. 209. The RNA molecule, combination, system or method of any one of claims 200 to 208, wherein said reduced immunogenicity comprises reduced detection by retinoic acid inducible gene I (RIG-I).

210. 210. The RNA molecule, combination, system or method of any one of claims 200 to 209, wherein said reduced immunogenicity comprises reduced detection by melanoma differentiation associated protein 5 (MDA5).

211. 211. The RNA molecule, combination, system or method of any one of claims 207 to 210, wherein the RNA molecule has reduced immunogenicity compared to an RNA molecule not having an IRES sequence.

212. The RNA molecule, combination, system or method of any one of claims 207 to 210, wherein the RNA molecule has reduced immunogenicity compared to an RNA molecule that does not have any of a PABP-binding site, a PCBP-binding site or an eIF4G-binding site.

213. 213. The RNA molecule, combination, system or method of any one of claims 200 to 212, wherein the RNA molecule is circular RNA and exhibits increased protein expression compared to the corresponding non-circular mRNA.

214. 214. The RNA molecule, combination, system or method of claim 213, wherein said RNA molecule exhibits high protein expression in vivo.

215. 215. The RNA molecule, combination, system or method of claim 213 or 214, wherein the RNA molecule exhibits high protein expression ex vivo.

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