Purification of circular RNA aptamer-based

Abstract

This specification provides a construct and method for isolating a circular RNA comprising, in order from 5' to 3', a first portion of an aptamer, a polyribonucleotide containing one or more sequences of interest, and a linear polyribonucleotide containing a second portion of the aptamer, wherein intramolecular ligation at the 5' and 3' ends of the construct forms a circular RNA comprising a complete aptamer that is fully continuous across the ligation sites, and the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA. In certain embodiments, the circular RNA is a vaccine.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 518,717, filed on August 10, 2023, the entire content of which is incorporated herein by reference.

[0002] Statement Regarding Research Sponsored by Federal Government Funds Not applicable.

[0003] Incorporation by Reference of Materials Recorded on a Compact Disk The sequence listing in an XML file named _, _ KB in size, created on _, and submitted to the United States Patent and Trademark Office via the Patent Center is incorporated herein by reference.

Background Art

[0004] The background will be described in relation to nucleic acid purification without limiting the scope of the present disclosure.

[0005] The development and introduction of mRNA vaccines, such as COVID vaccines, have demonstrated the importance of this vaccination approach and have spurred technological innovations to enhance the efficiency of mRNA vaccines and reduce reactogenicity. An important new approach uses circular RNA (circRNA), which lacks ends and thus does not stimulate innate immunity and inflammatory responses, resulting in reduced reactogenicity and improved expression of the desired antigen. The lack of ends provides a second advantage to circRNA: these RNAs are more stable because they cannot be degraded by exonucleases and, as a result, express proteins over a longer period.

[0006] A key challenge in the development of circRNAs as vaccine candidates, and in other applications where circRNAs are desired, is the efficient purification of circRNAs from their linear precursors. This challenge is particularly difficult for large RNA molecules (>1000 nt). [Overview of the project] [Problems that the invention aims to solve]

[0007] Despite numerous advances, current purification methods involving chromatography and exonuclease treatment result in low yields and / or low enrichment. This can lead to significant linear RNA carryover, potentially greatly increasing the potential toxicity of circRNA-based vaccines. Novel methods are needed to purify circRNA that can be used at scale for vaccine production or in any other use where circRNA purity provides a critical advantage by minimizing side effects. [Means for solving the problem]

[0008] As embodied and broadly described herein, the disclosure relates to a construct for isolating circRNA, comprising a linear polyribonucleotide containing halves of two aptamers at the 5' and 3' ends of the polyribonucleotide, and a polyribonucleotide containing one or more sequences of interest between the halves of the two aptamer sequences, wherein intramolecular ligation of the 5' and 3' ends of the construct forms a circRNA containing a completely continuous aptamer across the ligation site, and the complete aptamer formed by the ligation is present within the circRNA but not in the non-ligated form of the RNA. The presence of the complete aptamer within the circRNA provides a binding site for aptamer ligands used in affinity purification of the circRNA. Embodiments of the disclosed invention include ligating the 5' and 3' ends of a polyribonucleotide construct using the aforementioned methods for ligating RNA ends, including ligation catalyzed by ribozymes or protein enzymes, or chemically induced ligation such as click chemistry. In one embodiment, a complete aptamer is subjected to a specific low molecular weight ligand, polymer ligand, or polypeptide ligand with <10 μM K d So, K d Selectively binds at concentrations of <1, <2, <3, <4, <5, <6, <7, <8, <9, or <10 μM. Examples of aptamer ligands that may be used in the present invention as described herein include, but are not limited to, streptavidin, cephadex, streptomycin, 4,4'-methylenedianiline (MDA), imidazole, glutathione, polyhistidine, bacteriophage-coated protein MS2, bacteriophage-coated protein PP7, Csy4 protein, or other proteins or bacteriophage-coated proteins that bind to RNA structures.

[0009] In another embodiment, the reaction to produce a complete aptamer and circRNA, which is an intramolecular ligation of the two ends of a polyribonucleotide, is carried out by a natural or artificial ribozyme. In another embodiment, the reaction to produce a complete aptamer and circRNA, which is an intramolecular ligation of the two ends of a polyribonucleotide, is carried out by a natural or artificial ribozyme embedded in the polyribonucleotide sequence. In another embodiment, the complete aptamer binds to an aptamer ligand immobilized on a substrate. In another embodiment, the circRNA can induce the expression of one or more proteins when introduced into eukaryotic cells. In another embodiment, the circRNA is a vaccine. In another embodiment, the circRNA is captured using an aptamer ligand, where the ligand is ligated to a substrate, and the substrate is a gel, beads, agarose, polyacrylamide, UV-curable polymer, PEG-based hydrogel, emulsion bead-nucleic acid conjugate, polymer bead-nucleic acid conjugate, or a combination thereof. In another embodiment, the substrate is contained within a container, tube, syringe, microcontainer, spin column, flow cell, or a combination thereof. In yet another embodiment, the circRNA is isolated and released after elution with a molecule selected from RNA denaturants, including, but not limited to, biotin, streptavidin, cephadex, dextran, streptomycin, 4,4'-methylenedianiline (MDA), imidazole, glutathione, dithiothreitol (DTT), polyhistidine, bacteriophage coat protein MS2, bacteriophage coat protein PP7, Csy4 protein, or other proteins or bacteriophage coat proteins that bind to the RNA structure, polypeptides or other molecules or polymers capable of replacing the original target molecule used to bind to the aptamer, or, but not limited to, TRIzol, urea, phenol, EDTA, or guanidinium isothiocyanate.

[0010] As embodied and broadly described herein, one aspect of the present disclosure relates to a construct for isolating circRNA, comprising, in the order of 5' to 3' direction, a 3' splice site recognition sequence, a 5' splice site recognition sequence, a splicing ribozyme, a first half of an aptamer, a polyribonucleotide containing one or more sequences of interest, and a linear polyribonucleotide containing a second half of the aptamer. By incubating the construct under conditions in which the splicing ribozyme can catalyze a splicing reaction at a splice site embedded within the construct, the two halves of the aptamer sequences are ligated together to produce circRNA containing a fully continuous aptamer sequence and a polyribonucleotide sequence of interest, while the splicing ribozyme is released as a byproduct of the cyclization reaction and excluded from the circRNA. The complete aptamer within the circRNA, although not present in the linear form of the RNA, provides a binding site for aptamer ligands used for affinity purification of circRNA.

[0011] As embodied and broadly described herein, one aspect of the present disclosure relates to a construct for isolating circRNA comprising, in the order of 5' to 3' direction, half of a ribozyme sequence, half of an aptamer, a polyribonucleotide containing one or more sequences of interest, a second half of the aptamer, and a linear polyribonucleotide containing a second half of the ribozyme sequence. By incubating the construct under conditions in which a splicing ribozyme sequence embedded within the construct can catalyze a splicing reaction at a splicing site embedded within the construct, the two aptamer sequences are ligated together to produce circRNA containing a fully continuous aptamer sequence and a polyribonucleotide sequence of interest, while the splicing ribozyme is released as a byproduct of the cyclization reaction and excluded from the circRNA. The complete aptamer within the circRNA, although not present in the linear form of the RNA, provides a binding site for aptamer ligands used in affinity purification of circRNA.

[0012] As embodied and broadly described herein, one aspect of the present disclosure relates to a construct for isolating circRNA, comprising: a first half of an aptamer, a polyribonucleotide containing one or more sequences of interest, a linear polyribonucleotide containing a second half of an aptamer, the sprint oligo sequence thereof being complementary to each half of each aptamer sequence, and capable of ligation by a ligation enzyme such as an RNA ligase or DNA ligase, wherein intramolecular ligation of the 5' and 3' ends of the linear RNA construct generates a circRNA containing an aptamer that is fully continuous at a ligation junction, and the complete aptamer within the circRNA provides a binding site for an aptamer ligand used for affinity purification of the circRNA.

[0013] As embodied and broadly described herein, the present disclosure relates to a construct for isolating circRNA, comprising: a first portion of an aptamer, a polyribonucleotide containing one or more sequences of interest, and a linear polyribonucleotide containing a second portion of the aptamer, in order from 5' to 3' directions, wherein intramolecular ligations at the 5' and 3' ends of the construct form a circular RNA comprising a complete aptamer that is fully continuous across the ligation sites, and the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA.

[0014] As embodied and broadly described herein, the present disclosure relates to a construct for isolating circRNA, comprising: a linear polyribonucleotide splicing and ligation comprising, in order from 5' to 3' direction, a 3' splice site recognition sequence, a 5' splice site recognition sequence, a splicing ribozyme, a first portion of an aptamer, a polyribonucleotide containing one or more sequences of interest, and a second portion of an aptamer, wherein the first and second portions of the aptamer ligate to produce a complete aptamer and a circular RNA comprising a polyribonucleotide containing one or more sequences of interest, the splicing ribozyme being released as a byproduct of RNA cyclization and excluded from the circular RNA, and the complete aptamer within the circular RNA providing a binding site for affinity purification of the circular RNA.

[0015] As embodied and broadly described herein, the present disclosure relates to a construct for isolating circRNA, comprising: a linear polyribonucleotide comprising, in order from 5' to 3', a first portion of a ribozyme sequence, a first portion of an aptamer, a polyribonucleotide containing one or more sequences of interest, a second portion of an aptamer, and a second portion of a ribozyme, wherein splicing of the first and second portions of the ribozyme releases the ribozyme, forming a circular RNA with a complete aptamer at the splice junction, the complete aptamer within the circular RNA providing a binding site for affinity purification of the circular RNA.

[0016] As embodied and widely described herein, the present disclosure relates to a construct for isolating circRNA, comprising: a first portion of an aptamer, a polyribonucleotide containing one or more sequences of interest, and a linear polyribonucleotide comprising a second portion of an aptamer, the sprint oligo sequence thereof being complementary to the first and second aptamer portions to induce ligation by a ligation enzyme, the ligation of the linear polyribonucleotide forming a circular RNA comprising a complete aptamer that is fully continuous at a ligation junction, and the complete aptamer within the circular RNA providing a binding site for affinity purification of the circular RNA. In one embodiment, the complete aptamer binds to streptavidin, cephadex, streptomycin, MDA(4,4'-methylenedianiline), imidazole, His tag, MS2, PP7, Csy4, or glutathione. In another embodiment, the complete aptamer binds to a molecule immobilized on a substrate. In another embodiment, the circular RNA expresses one or more proteins. In another embodiment, the circular RNA is a vaccine. In another embodiment, splicing is trans-splicing by a trans-splicing ribozyme or a group I intronic ribozyme. In another embodiment, ligation is by a ligase selected from T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 cleavage type, RrtcB ligase, and TS2126 RNA ligase 1. In another embodiment, the circular RNA is isolated and released by elution with a small molecule selected from streptavidin, cephadex, streptomycin, MDA(4,4'-methylenedianiline), imidazole, His tag, MS2, PP7, Csy4, or glutathione. In another embodiment, substrates such as gels, columns, beads, agarose, polymers, polyacrylamides, UV-curable polymers, PEG-based hydrogels, emulsion bead-nucleic acid conjugates, polymer bead-nucleic acid conjugates, or combinations thereof are used to capture circular RNA.In another embodiment, the substrate is contained in a container which is a tube, syringe, microcontainer, spin column, flow cell, or a combination thereof.

[0017] As embodied and broadly described herein, the disclosure relates to a method for isolating circular RNA from the above-described constructs, comprising reacting the constructs under conditions that form circular RNA containing a complete aptamer, and isolating the circular RNA by capturing the aptamer with a ligand that specifically binds to the aptamer. In one embodiment, the complete aptamer binds to streptavidin, cephadex, streptomycin, MDA(4,4'-methylenedianiline), imidazole, His tag, MS2, PP7, Csy4, or glutathione. In another embodiment, the complete aptamer binds to a molecule immobilized on a substrate. In another embodiment, the circular RNA expresses one or more proteins. In another embodiment, the circular RNA is a vaccine. In another embodiment, splicing is trans-splicing by a trans-splicing ribozyme or a group I intronic ribozyme. In another embodiment, ligation is performed by a ligase selected from T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 cleavage type, RrtcB ligase, and TS2126 RNA ligase 1. In yet another embodiment, a substrate is used to capture circular RNA, which is a gel, column, beads, agarose, polymer, polyacrylamide, UV-curable polymer, PEG-based hydrogel, emulsion bead-nucleic acid conjugate, polymer bead-nucleic acid conjugate, or a combination thereof. In yet another embodiment, the substrate is contained in a container, which is a tube, syringe, microcontainer, spin column, flow cell, or a combination thereof.In another embodiment, circular RNA is isolated and released by elution with a molecule selected from RNA denaturants, including but not limited to biotin, streptavidin, cephadex, dextran, streptomycin, 4,4'-methylenedianiline (MDA), imidazole, glutathione, dithiothreitol (DTT), polyhistidine, bacteriophage coat protein MS2, bacteriophage coat protein PP7, Csy4 protein, or other proteins that bind to the RNA structure, or polypeptides or other molecules or polymers that can replace the original target molecule used to bind to the bacteriophage coat protein aptamer, or RNA denaturants, including but not limited to TRIzol, urea, phenol, EDTA, or guanidinium isothiocyanate.

[0018] As embodied and broadly described herein, the present disclosure relates to a method for preparing and isolating a circular RNA vaccine, comprising: preparing one of the above-described constructs, wherein one or more sequences of interest are viral antigens; reacting the constructs under conditions under which circular RNA is formed; and isolating the circular RNA by capturing the aptamers with ligands that specifically bind to the aptamers.

[0019] As embodied and broadly described herein, the disclosure relates to a method for expressing a protein of interest in any of the constructs described above, comprising isolating a circular RNA by capturing an aptamer with a ligand that specifically binds to the aptamer, and incubating the circular RNA under conditions in which the protein of interest is expressed from the circular RNA.

[0020] To better understand the features and benefits of this disclosure, a detailed description of this disclosure is provided here, along with the attached diagrams. [Brief explanation of the drawing]

[0021] [Figure 1]This is a diagram showing an overview of the aptamer purification scheme. This figure shows the steps used to purify circRNA using aptamer affinity purification and trans-splicing circularization. [Figure 2] This is a diagram showing an EX Thermo Fisher agarose gel of circRNA after aptamer purification. The RNA in lane 1 was purified using the aptamer method combined with RNase R treatment, and a 90% pure circRNA product as shown in lanes 4 and 5 was generated. The upper band is circRNA, and the lower band is linear precursor, nicked circRNA, or intron RNA.

Modes for Carrying Out the Invention

[0022] Although the implementation and use of various aspects of the present disclosure will be discussed in detail below, it should be understood that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific situations. The specific aspects discussed herein are merely illustrative of specific methods for implementing and using the present disclosure and do not limit the scope of the present disclosure.

[0023] To facilitate understanding of the present disclosure, some terms are defined below. The terms defined herein have meanings generally understood by those skilled in the art related to the present disclosure. Terms such as "a", "an", and "the" are not intended to refer to only a single entity but include general classes for which specific examples may be used for illustration. The terms herein are used to describe specific aspects of the present disclosure, but their use does not limit the present disclosure except as outlined in the claims.

[0024] An aptamer is typically a single-stranded oligonucleotide 20 to 100 nucleotides in length that adopts a specific three-dimensional structure capable of binding to a target molecule with high affinity and specificity. The binding properties of an aptamer are conferred by its unique sequence and the resulting secondary and tertiary structures, such as hairpins, bulges, loops, and G-quadruplexes. RNA aptamers are identified through an in vitro selection process called SELEX (Systematic Evolution of Ligands by Exponential enrichment), which involves iterative rounds of binding, partitioning, and amplification from a diverse library of randomized sequences. Once selected, RNA aptamers can be chemically modified to increase their stability against nuclease degradation for use as research tools, diagnostic agents, or therapeutic approaches that bind to proteins, peptides, small molecules, and even entire cells or tissues with antibody-like performance.

[0025] Isolated and purified ligand-binding aptamers are identified, their nucleotide base sequences are determined, and in the context of the present disclosure, they are then split at the opposite ends of the construct. After the ends of the construct are ligated and a circRNA is formed, the halves of the two aptamers are linked to each other to form a complete aptamer.

[0026] As used herein, the term "synthesizing" refers to chemical methods known in the art for generating a desired nucleotide sequence, including cases where the desired sequence contains regions with randomized sequences. Typically, such sequences are generated with an automated DNA or RNA synthesizer programmed with the desired sequence. Such programming can include combinations of defined sequences and random nucleotides.

[0027] As used herein, a "random combinatorial oligonucleotide library" refers to a large number of oligonucleotides of different sequences where the insertion of a given base at a given location within the sequence is random.

[0028] As used herein, “polymerase chain reaction (PCR) primer nucleotide sequence” refers to a defined nucleotide sequence that forms an oligonucleotide used to anneal to a homologous or closely related sequence in order to form a double helix necessary for the polymerase enzyme to initiate extension.

[0029] As used herein, the term “amplify” refers to duplicating an array once or multiple times. With respect to libraries, amplify refers to duplicating at least a majority of the individual members of the library in large quantities.

[0030] As used herein, the term “modified” is used herein to describe oligonucleotides or libraries in which one or more of the four constituent nucleotide bases of the oligonucleotide are typically analogs or esters of nucleotides that make up the DNA or RNA backbone, and such modification confers enhanced nuclease resistance.

[0031] As used herein, “enzymatic” amplification refers to the replication of oligonucleotides using a nucleotide polymerase enzyme such as DNA polymerase or RNA polymerase. If amplification involves repetitive replication cycles, such as using a “polymerase chain reaction,” the polymerase is a thermostable polymerase, such as DNA polymerase from Thermus aquaticus.

[0032] As used herein, the term “contact” in the context of target selection is defined as incubating an oligoribonucleotide library with a target molecule.

[0033] As used herein, the term "aptamer ligand" refers to a molecule that binds to an aptamer selectively and specifically with high affinity.

[0034] As used herein, the term “purify” in the context of circRNA enrichment and isolation refers to the separation of the circRNA / aptamer / ligand complex under conditions in which weakly bound oligoribonucleotides are removed. In one embodiment, the circRNA / aptamer / ligand complex is retained on the substrate, while oligoribonucleotides that do not contain a complete aptamer and are unable to bind to the aptamer ligand are washed away.

[0035] As used herein, “ribozyme” refers to RNA that has catalytic properties to induce an esterification reaction resulting in splicing at a specific nucleotide position within an RNA sequence.

[0036] As used herein, “natural ribozyme” refers to RNA catalysts found in nature.

[0037] As used herein, “artificial ribozyme” refers to an RNA catalyst generated by novel design and subsequently synthesized, or an RNA catalyst having a sequence derived from a natural ribozyme but with less than 30% homology to the original natural ribozyme, while still capable of performing double-step esterification reactions resulting in RNA splicing and RNA cyclization. Such synthetic derivatives may be generated by rational design or in vitro evolution of natural ribozymes with randomized sequences in a selective region.

[0038] Vaccines / Immunogens. The present invention includes vaccines for both active and passive immunity. Immunogenic compositions suitable for use as vaccines include the circRNA of the present invention. The circRNA is prepared by the methods disclosed herein. The circRNA vaccines disclosed herein are less reactive than linear RNA; that is, circRNA does not tend to evoke an innate immune response.

[0039] In one example, circRNA may be used as part of a vaccine to control the development of a Th1 or Th2 subset in a subject or patient. In addition to in vivo modulation, circRNA may be used ex vivo to modify cells in vitro and then administered to the subject. In one example, circRNA may contain one or more antigens derived from a target pathogen. A circRNA vaccine may contain more than one circRNA insert to target more than one antigen or to target a modification of the same antigen.

[0040] As used herein, the term “complete aptamer” refers to the ligation of an aptamer split into two parts (which may or may not be of equal length), but when an intervening sequence (e.g., an intron) is removed from the two junctions, or when intramolecular ligation is performed, a contiguous polynucleotide containing the complete aptamer sequence is reconstructed. The thus reconstructed aptamer can then bind to a specific ligand with high affinity. Non-limiting examples of ligands that can bind to the aptamer with high affinity and specificity include streptavidin, cephadex, streptomycin, 4,4'-methylenedianiline (MDA), imidazole, glutathione, polyhistidine, bacteriophage-coated protein MS2, bacteriophage-coated protein PP7, Csy4 protein, or other proteins or bacteriophage-coated proteins that bind to RNA structures.

[0041] Complete aptamers bind to molecules that can be immobilized on a substrate. Non-limiting examples of substrates that can be used to bind complete aptamers on circRNA include gels, beads, agarose, polyacrylamide, UV-curable polymers, PEG-based hydrogels, emulsion bead-nucleic acid conjugates, polymer bead-nucleic acid conjugates, or combinations thereof. In another embodiment, the substrate is contained within a container, tube, syringe, microcontainer, spin column, flow cell, or combination thereof. The substrate may be placed, for example, within a container, which could be a tube, syringe, microcontainer, spin column, flow cell, or combination thereof.

[0042] As used herein, the terms “identical” or “identity” percent mean two or more sequences or subsequences, whether RNA or DNA sequences, that are identical or have a specified percentage of identical nucleotide residues (i.e., approximately 60% identity across a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity when compared and aligned to the maximum correspondence across a comparison window or specified region). In one embodiment, the aptamer sequence has approximately 60% identity across specific nucleotide (nucleotide:nt) regions, or 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity across these regions, which are approximately 10nt, 20nt, 30nt, 40nt, 50nt, 60nt, 70nt, 80nt , containing aptamer sequences of 90nt, 100nt, 110nt, 120nt, 130nt, 140nt, 150nt, 160nt, 170nt, 180nt, 190nt, 200nt, 210nt, 220nt, 230nt, 240nt, 250nt, 260nt, 270nt, 280nt, 290nt, 300nt, 325nt, 350nt, 375nt, 400nt, 450nt, or approximately 500nt.

[0043] In the context of nucleotides, the terms “sequence identity,” “sequence identity percentage,” or “identical percentage” refer to nucleotides of two sequences that are identical when aligned to the maximum correspondence, whether in DNA or RNA sequences. The length of the sequence identity comparison may, optionally, span the entire length of the nucleotide sequence or a fragment of the nucleotide sequence. Preferably, the fragment is at least about 8 nt in length and may be up to about 700 nt. In one embodiment, an aptamer has a sequence identity of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or higher compared to known aptamer sequences, such as those disclosed herein. In one embodiment, the aptamer has sequence identity of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or higher compared to known aptamer sequences such as those disclosed herein. In one embodiment, the aptamer has sequence identity of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or less, or 95% or higher compared to known aptamer sequences such as those disclosed herein.

[0044] The terms "substantial homology" or "substantial similarity," when referring to a nucleotide sequence or fragment thereof, indicate that, when optimally aligned with another nucleotide sequence (or its complementary strand) with appropriate nucleotide insertions or deletions, the aligned sequence is identical in at least about 95, 96, 97, 98, or 99% of its nucleotide sequence. Preferably, the homology extends over the full-length sequence or over nucleotide sequences, such as RNA or DNA sequences.

[0045] In one embodiment, the time used for the aptamer to purify circRNA is approximately 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

[0046] In one embodiment, by using an aptamer to purify circRNA, the amount of circRNA obtained is approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, and 42% of the circRNA obtained compared to circRNA not purified using the aptamer of the present invention disclosed herein. 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72% It increases by approximately 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or approximately 100%.

[0047] In one embodiment, by using an aptamer to purify circRNA, the amount of circRNA obtained is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, compared to circRNA not purified using the aptamer of the present invention disclosed herein. At least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 4 6%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, less It will increase by at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0048] In one embodiment, purification of circRNA using an aptamer results in a half-life of circRNA in the formulation of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.

[0049] In further embodiments, purification of circRNA using aptamers extends the half-life of circRNA in nanoparticles to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.

[0050] In further embodiments, purification of circRNA using aptamers extends the intracellular half-life of circRNA after administration to an organism to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.

[0051] Subsequently, the circRNA containing the complete aptamer may be isolated and released from the substrate after elution with a molecule selected from RNA denaturants, including but not limited to biotin, streptavidin, cephadex, dextran, streptomycin, 4,4'-methylenedianiline (MDA), imidazole, glutathione, dithiothreitol (DTT), polyhistidine, bacteriophage-coated protein MS2, bacteriophage-coated protein PP7, Csy4 protein, or other proteins or bacteriophage-coated proteins that bind to the RNA structure, polypeptides or other molecules or polymers capable of replacing the original target molecule used to bind to the aptamer, or but not limited to TRIzol, urea, phenol, EDTA, or guanidinium isothiocyanate.

[0052] This invention solves the problem of circRNA isolation by using affinity chromatography that is more specific to circRNA than to its linear precursor. Furthermore, this method can be generalized to all circRNAs produced by reverse splicing or other ligation methods and should be independent of the circRNA sequence and length.

[0053] The methods described herein, for example, use permuted group I intron constructs to produce circRNA containing an open reading frame (ORF) and elements necessary to mediate translation (e.g., internal ribosome entry site: IRES) by splicing (reverse splicing). However, this method is also compatible with alternative cyclization methods (e.g., oligosprint-mediated ligation).

[0054] Development of circRNA aptamer purification - using trans-splicing to incorporate aptamer sequences.

[0055] Developing aptamer purification for circRNA required overcoming several technical challenges. One of the main problems hindering the use of aptamers in circRNA purification is that the complete aptamer sequence, transcribed into the precursor constructor RNA, is contained in both the linear precursor and the circRNA, making it impossible to use aptamers for selective circRNA purification. To overcome this problem, a strategy was devised to split the aptamer into two halves within the linear precursor RNA, so that after the cyclization reaction, only the complete aptamer sequence with ligand-binding ability is formed within the circRNA. This strategy allows the circRNA aptamer to gain high affinity for the ligand, while the other half of the linear RNA sequence cannot bind to the ligand.

[0056] One embodiment of the method disclosed herein uses a modified version of a self-splicing group I intron that allows two RNAs to be spliced ​​together when a specific guide sequence is used. By making a specific sequence change in the guide sequence, it was found that an aptamer could be positioned at the splice site of circRNA (Figure 1), thereby enabling the use of the original aptamer strategy in the context of trans-splicing. This method was tested and found to yield circRNA with a purity of 90%, exceeding what could be achieved by conventional purification methods, when used in combination with other methods (Figure 2). Using this innovative method, high-purity circRNA was purified with minimal chemical processing.

[0057] Figure 1 shows an overview of the aptamer purification scheme. This figure illustrates the steps used to purify circRNA using aptamer affinity purification and trans-splicing cyclization. The gene of interest and IRES are formed, and a portion of the aptamer is flanked at each end, and then a ribozyme is flanked at one end of the RNA. The ribozyme is then cleaved, and the RNA is cyclized to form a complete aptamer. Next, the aptamer containing the circRNA is captured by beads bound with aptamer ligands in this example, and the ribozyme and other RNA contaminants are separated from the beads. Finally, the purified circRNA is eluted from the beads, for example, by molecular substitution.

[0058] Figure 2 shows an EX agarose gel (ThermoFisher, USA) showing circRNA after aptamer purification. RNA in lane 1 was purified using the aptamer method disclosed herein in combination with RNase R treatment, yielding 90% circRNA products shown in lanes 4 and 5. The upper bands are circRNA, and the lower bands are linear precursors, nicked circRNA, or intronic RNA. Non-limiting examples of aptamers that may be used in the present invention include those listed in Table 1, showing the aptamer name, sequence, and specific ligand.

[0059] [Table 1]

[0060] Further small molecule binding aptamers may mimic the following: anthracycline, tetracycline, benzoylmethylecgonine, thalidomide, diclofenac, somatropin, insulin, vasopressin, gonadriverine, estradiol, aflatoxin, anatoxin, atrazine, benzylguanine, brevetoxin-2, bromacil, cadmium, danfloxacin, fumonisin, kanamycin, ketamine, lysergamin, malathion, methamphetamine, N-methylmesoporphyrin, ochratoxin, okadic acid. (Acid), organophosphate insecticides, oxytetracycline, palladium, polychlorinated bufunyl, progesterone, T-2 toxin, tebuconazole, inabenefide, mefenacet, trambmycin, or zearalenone, these are listed in Ruscito and DeRosa, Small-Molecule Binding Aptamers: Selection Strategies, Characterization, and Applications, Front. Chem., May 10, 2016, Sec. Chemical Biology, Vol. 4-2016 | doi.org / 10.3389 / fchem.2016.00014, and Strehlitz et al., "Aptamers for pharmaceuticals and their application in environmental analytics," Bioanal Rev. 2012;4(1): pp. 1-30, December 17, 2011. As taught in doi:10.1007 / s12566-011-0026-1, the sequence is incorporated herein by reference.

[0061] Example 1. A construct for isolating a circular RNA, wherein intramolecular ligations at the 5' and 3' ends of the construct, comprising a first portion of an aptamer, a polyribonucleotide containing one or more sequences of interest, and a linear polyribonucleotide containing a second portion of the aptamer, in the order of 5' to 3' directions, form a circular RNA containing a complete aptamer that is fully continuous across the ligation site, and the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA.

[0062] Example 2. A construct for isolating circular RNA, wherein splicing and ligation of a linear polyribonucleotide, comprising a 3' splice site recognition sequence, a 5' splice site recognition sequence, a splicing ribozyme, a first portion of an aptamer, a polyribonucleotide containing one or more sequences of interest, and a second portion of an aptamer, in the order of 5' to 3' direction, ligates the first and second portions of the aptamer to produce a complete aptamer and a circular RNA containing one or more sequences of interest, the splicing ribozyme is released as a byproduct of RNA cyclization and excluded from the circular RNA, and the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA.

[0063] Example 3. A construct for isolating a circular RNA, comprising, in order from 5' to 3' direction, a first portion of a ribozyme sequence, a first portion of an aptamer, a polyribonucleotide containing one or more sequences of interest, a second portion of an aptamer, and a second portion of a ribozyme, wherein splicing of the first and second portions of the ribozyme releases the ribozyme, forming a circular RNA with a complete aptamer at the splice junction, and the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA.

[0064] Example 4. A construct for isolating a circular RNA, comprising, in order, a first portion of an aptamer, a polyribonucleotide containing one or more sequences of interest, and a linear polyribonucleotide comprising a second portion of an aptamer, wherein its sprint oligo sequence is complementary to the first and second aptamer portions to induce ligation by a ligation enzyme, the ligation of the linear polyribonucleotide forms a circular RNA containing a complete aptamer that is fully continuous at a ligation junction, and the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA.

[0065] Example 5. A construct described in any one of Examples 1-4, wherein the complete aptamer binds to streptavidin, cefadex, streptomycin, MDA(4,4'-methylenedianiline), imidazole, His tag, MS2, PP7, Csy4, or glutathione.

[0066] Example 6. A construct according to any one of Examples 1-5, wherein a complete aptamer binds to a molecule immobilized on a substrate.

[0067] Example 7. A construct according to any one of Examples 1-6, wherein a circular RNA expresses one or more proteins.

[0068] Example 8. A construct described in any one of Examples 1-7, wherein the circular RNA is the vaccine.

[0069] Example 9. A construct according to any one of Examples 1 to 3, wherein the splicing is trans-splicing by a trans-splicing ribozyme or a group I intronic ribozyme.

[0070] Example 10. The construct described in Example 4, wherein ligation is performed by a ligase selected from T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 cleavage type, RrtcB ligase, and TS2126 RNA ligase 1.

[0071] Example 11. A construct according to any one of Examples 1 to 10, wherein a substrate is used to capture circular RNA, and is a gel, column, beads, agarose, polymer, polyacrylamide, UV-curable polymer, PEG-based hydrogel, emulsion bead-nucleic acid conjugate, polymer bead-nucleic acid conjugate, or a combination thereof.

[0072] Example 12. The construct described in Example 11, wherein the substrate is contained in a container that is a tube, syringe, microcontainer, spin column, flow cell, or a combination thereof.

[0073] Example 13. A construct described in any one of Examples 1-11, wherein circular RNA is isolated by elution with a small molecule selected from streptavidin, cephadex, streptomycin, MDA(4,4'-methylenedianiline), imidazole, His tag, MS2, PP7, Csy4, or glutathione, and then released.

[0074] Example 14. A method for isolating circular RNA from a construct according to any one of claims 1 to 4, comprising reacting the construct under conditions that would form circular RNA containing a complete aptamer, and isolating the circular RNA by capturing the aptamer with a ligand that specifically binds to the aptamer.

[0075] Example 15. The method according to Example 14, wherein the complete aptamer binds to streptavidin, cefadex, streptomycin, MDA(4,4'-methylenedianiline), imidazole, His tag, MS2, PP7, Csy4, or glutathione.

[0076] Example 16. The method according to Example 14 or Example 15, wherein a complete aptamer binds to a molecule immobilized on a substrate.

[0077] Example 17. The method according to any one of Examples 14-16, wherein a circular RNA expresses one or more proteins.

[0078] Example 18. The method according to any one of Examples 14-17, wherein the circular RNA is the vaccine.

[0079] Example 19. The method according to any one of Examples 14-18, wherein the splicing is trans-splicing by a trans-splicing ribozyme or a group I intronic ribozyme.

[0080] Example 20. The method according to any one of Examples 14-19, wherein ligation is performed by a ligase selected from T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 cleavage type, RrtcB ligase, and TS2126 RNA ligase 1.

[0081] Example 21. The method according to any one of Examples 14-20, wherein a substrate is used to capture circular RNA, the substrate being a gel, column, beads, agarose, polymer, polyacrylamide, UV-curable polymer, PEG-based hydrogel, emulsion bead-nucleic acid conjugate, polymer bead-nucleic acid conjugate, or a combination thereof.

[0082] Example 22. The method according to any one of Examples 14-21, wherein the substrate is in a container that is a tube, syringe, microcontainer, spin column, flow cell, or a combination thereof.

[0083] Example 23. The method according to any one of Examples 14-22, wherein circular RNA is isolated and released after elution with a polypeptide or another molecule or polymer capable of replacing the original target molecule used to bind to an RNA structure, bacteriophage coat protein MS2, bacteriophage coat protein PP7, Csy4 protein, or other protein or bacteriophage coat protein, aptamer, or RNA denaturant, but not limited to TRIzol, urea, phenol, EDTA, or guanidinium isothiocyanate.

[0084] Example 24. A method for preparing and isolating a circular RNA vaccine, comprising: preparing a construct described in any one of Examples 1 to 4, wherein one or more target sequences are viral antigens; reacting the construct under conditions under which circular RNA is formed; and isolating the circular RNA by capturing an aptamer with a ligand that specifically binds to the aptamer.

[0085] Example 25. A method for expressing a target protein in a construct described in any one of Examples 1 to 4, comprising isolating a circular RNA by capturing an aptamer with a ligand that specifically binds to the aptamer, and incubating the circular RNA under conditions in which the target protein is expressed from the circular RNA.

[0086] Any aspect of the disclosure discussed herein is intended to be applicable with respect to any method, kit, reagent, or composition of the present disclosure, and vice versa. Furthermore, the compositions of the present disclosure may be used to achieve the methods of the present disclosure.

[0087] It will be understood that certain embodiments described herein are illustrative and not limit the disclosure. The main features of the disclosure can be used in various embodiments without departing from the scope of the disclosure. Those skilled in the art will recognize, or can verify simply by using common experiments, numerous procedures that are equivalent to certain procedures described herein. Such equivalents are deemed to be within the scope of the disclosure and are covered by the claims.

[0088] All publications and patent applications referenced herein represent the level of skill of a person skilled in the art relating to this disclosure. All publications and patent applications are incorporated herein by reference to the same extent that each individual publication or patent application is specifically indicated to be incorporated by reference.

[0089] The use of the words “a” or “an” in the claims and / or specification in combination with the term “comprising” may mean “one,” but also coincide with the meanings of “one or more,” “at least one,” and “one or more.” The use of the term “or” in the claims is used to mean “and / or” unless it is explicitly indicated that it refers only to alternatives, or that the alternatives are mutually exclusive, however this disclosure supports definitions that refer only to alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes variations in device-specific errors, the methods used to determine the value, or variations that exist between the subjects of study.

[0090] As used herein and in the claims, the words “comprising” (and all forms of “comprising,” such as “comprise” and “comprises”), “having” (and all forms of “having,” such as “have” and “has”), “including” (and all forms of “including,” such as “includes” and “include”), or “containing” (and all forms of “containing,” such as “contains” and “contain”) are either comprehensive or open-ended and do not preclude any additional, undescribed elements or method steps. In any embodiment of the compositions and methods provided herein, “comprising” may be replaced with “essentially consisting of” or “consisting of.” As used herein, the phrase “essentially consisting of” requires that it not substantially affect the features or functions of the claimed invention, as well as any specified integer or step. As used herein, the term "consists of" is used to indicate the existence of only the integer elements described (e.g., features, elements, characteristics, methods / process steps, or limitations) or a group of integer elements (e.g., multiple features, elements, characteristics, methods / process steps, or limitations).

[0091] As used herein, the term “or any combination thereof” refers to all permutations and combinations of the items listed before that term. For example, “A, B, C, or any combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and also to include BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, where the order is important in the particular context. Continuing this example, explicitly included are combinations that involve repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A person skilled in the art will understand that, unless otherwise clearly indicated by the context, there is typically no limit to the number of items or terms in any combination.

[0092] Where used herein, approximate words such as “about,” “substantial,” or “effectively,” when modified in this specification, refer to a state that, while not necessarily absolute or complete, is considered by those skilled in the art to be close enough to guarantee that the state exists. The extent to which the description may vary depends on the degree of modification and whether those skilled in the art can still perceive that the modified feature still possesses the required characteristics and capabilities of the unmodified feature. Generally, as discussed above, numerical values ​​modified by approximate words such as “about” in this specification may vary by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15% from the stated value.

[0093] Furthermore, section headings in this specification are provided to maintain consistency with proposals under 37 CFR 1.77 or to provide other constituent clues. These headings are not intended to limit or characterize any disclosures described in any claims that may arise from this disclosure. Specifically, for example, the heading “Field of Invention” refers to a so-called technical field, but such claims should not be limited by the wording under this heading that describes the so-called technical field. Furthermore, the description of the technology in the “Background of Invention” section should not be construed as an acknowledgment that the technology is prior art to any disclosure in this disclosure. Also, the “Summary” should not be considered a characterization of this disclosure as described in the published claims. Furthermore, any reference to the singular “invention” in this disclosure should not be used to assert that this disclosure has only one point of novelty. Multiple inventions may be described in accordance with the limitations of multiple claims arising from this disclosure, and such claims define the invention(s) and their equivalents that are thereby protected. In any case, the scope of such claims should be considered on a basis of their own merit in light of this disclosure, but should not be limited by the headings described herein.

[0094] All compositions and / or methods disclosed and claimed herein can be prepared and carried out in light of this disclosure without requiring undue experimentation. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the compositions and / or methods, as well as to the steps or order of steps of the methods described herein, without departing from the concepts, spirit, and scope of this disclosure. All such similar substitutes and modifications that are obvious to those skilled in the art shall be deemed to be within the spirit, scope, and concepts of this disclosure as defined by the appended claims.

[0095] To assist the Patent Office and any person reading any patent issued pursuant to this application in interpreting the claims attached herein, the applicant notes that, with respect to any of the attached claims, unless the terms “means for” or “steps for” are expressly used in a particular claim, the applicant does not intend to invoke § 112(6), § 112(f), or any equivalent existing as of the filing date of this specification.

[0096] In each claim, each dependent claim may be dependent on both the independent claim and all prior dependent claims, provided that the prior claim provides adequate prior art with respect to the terminology or elements of the claim.

Claims

1. In the 5' to 3' direction, intramolecular ligations at the 5' and 3' ends of the construct, comprising a first portion of the aptamer, a polyribonucleotide containing one or more target sequences, and a linear polyribonucleotide containing a second portion of the aptamer, form a circular RNA containing a complete aptamer that is fully continuous across the ligation sites. For isolating circular RNA, the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA. Structure.

2. The linear polyribonucleotide comprises, in order from 5' to 3', a 3' splice site recognition sequence, a 5' splice site recognition sequence, a splicing ribozyme, a first portion of an aptamer, a polyribonucleotide containing one or more target sequences, and a second portion of an aptamer. The splicing and ligation of the linear polyribonucleotides link the first and second portions of the aptamer to produce a circular RNA comprising a complete aptamer and one or more polyribonucleotides containing the desired sequence. Splicing ribozymes are released as a byproduct of RNA cyclization and excluded from the circular RNA. For isolating circular RNA, the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA. Structure.

3. The linear polyribonucleotide comprises, in order from 5' to 3', a first portion of a ribozyme sequence, a first portion of an aptamer, a polyribonucleotide containing one or more target sequences, a second portion of an aptamer, and a linear polyribonucleotide containing the second portion of a ribozyme. Splicing of the first and second portions of the ribozyme releases the ribozyme, forming a circular RNA containing the complete aptamer at the splice junction. For isolating circular RNA, the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA. Structure.

4. The following components are included in order: a first portion of the aptamer, a polyribonucleotide containing one or more target sequences, and a linear polyribonucleotide containing a second portion of the aptamer. The sprint oligo sequence is complementary to the first and second aptamer regions so as to induce ligation by the ligation enzyme. The ligation of linear polyribonucleotides forms a circular RNA containing a complete aptamer that is fully continuous at the ligation junction, and the complete aptamer within the circular RNA provides a binding site for affinity purification of the circular RNA, for isolating the circular RNA. Structure.

5. A construct according to any one of claims 1 to 4, wherein the complete aptamer is bound to streptavidin, cefadex, streptomycin, MDA (4,4'-methylenedianiline), imidazole, His tag, MS2, PP7, Csy4, or glutathione.

6. The construct according to any one of claims 1 to 5, wherein a complete aptamer binds to a molecule immobilized on a substrate.

7. A construct according to any one of claims 1 to 6, wherein a circular RNA expresses one or more proteins.

8. A construct according to any one of claims 1 to 7, wherein the circular RNA is the vaccine.

9. The construct according to any one of claims 1 to 3, wherein the splicing is trans-splicing by a trans-splicing ribozyme or a group I intron ribozyme.

10. The construct according to claim 4, wherein ligation is performed by a ligase selected from T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 cleavage type, RrtcB ligase, and TS2126 RNA ligase 1.

11. The construct according to any one of claims 1 to 10, wherein circular RNA is isolated by elution with a small molecule selected from streptavidin, cephadex, streptomycin, MDA (4,4'-methylenedianiline), imidazole, His tag, MS2, PP7, Csy4, or glutathione, and then released.

12. A construct according to any one of claims 1 to 11, wherein a substrate is used to capture circular RNA, the substrate being a gel, column, beads, agarose, polymer, polyacrylamide, UV-curable polymer, PEG-based hydrogel, emulsion bead-nucleic acid conjugate, polymer bead-nucleic acid conjugate, or a combination thereof.

13. The construct according to claim 12, wherein the substrate is contained in a container which is a tube, syringe, microcontainer, spin column, flow cell, or a combination thereof.

14. A method for isolating circular RNA from a construct according to any one of claims 1 to 4, Reacting the construct under conditions that form a circular RNA containing the complete aptamer, Isolating circular RNA by capturing aptamers with ligands that specifically bind to aptamers, and Methods that include...

15. The method according to claim 14, wherein the complete aptamer binds to streptavidin, cefadex, streptomycin, MDA (4,4'-methylenedianiline), imidazole, His tag, MS2, PP7, Csy4, or glutathione.

16. The method according to claim 14 or 15, wherein a complete aptamer binds to a molecule immobilized on a substrate.

17. The method according to any one of claims 14 to 16, wherein a circular RNA expresses one or more proteins.

18. The method according to any one of claims 14 to 17, wherein the circular RNA is the vaccine.

19. The method according to any one of claims 14 to 18, wherein the splicing is trans-splicing by a trans-splicing ribozyme or a group I intron ribozyme.

20. The method according to any one of claims 14 to 19, wherein ligation is performed by a ligase selected from T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase 2 cleavage type, RrtcB ligase, and TS2126 RNA ligase 1.

21. The method according to any one of claims 14 to 20, wherein a substrate is used to capture circular RNA, the substrate being a gel, column, beads, agarose, polymer, polyacrylamide, UV-curable polymer, PEG-based hydrogel, emulsion bead-nucleic acid conjugate, polymer bead-nucleic acid conjugate, or a combination thereof.

22. The method according to any one of claims 14 to 21, wherein the substrate is contained in a container which is a tube, syringe, microcontainer, spin column, flow cell, or a combination thereof.

23. The method according to any one of claims 14 to 22, wherein circular RNA is isolated and released after elution with a molecule selected from RNA denaturants, including but not limited to TRIZOL, urea, phenol, EDTA, or guanidinium isothiocyanate, and is then released.

24. A method for producing and isolating a circular RNA vaccine, To prepare a construct according to any one of claims 1 to 4, wherein one or more target sequences are viral antigens, Reacting the construct under conditions that form circular RNA, Isolating circular RNA by capturing aptamers with ligands that specifically bind to aptamers, and Methods that include...

25. A method for expressing a target protein using a construct according to any one of claims 1 to 4, Isolating circular RNA by capturing the aptamer with a ligand that specifically binds to the aptamer, Incubating circular RNA under conditions in which the target protein is expressed from circular RNA and Methods that include...

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