Positive selection methods and systems for purifying circular nucleic acids

Abstract

Disclosed herein are compositions comprising an oligonucleotide positive selection probe for purifying circular nucleic acids, wherein the circular nucleic acids are produced by self-splicing of a precursor nucleic acid, wherein the positive selection probe is at least 60% complementary to at least a portion of the circular nucleic acid produced by self-splicing of the precursor nucleic acid, the at least a portion of the circular nucleic acid spanning a spliced portion of the circular nucleic acid, the precursor nucleic acid lacking a sequence at least 60% complementary to the positive selection probe. Also disclosed herein is a method for purifying circular nucleic acids from a sample, wherein the circular nucleic acids are produced by self-splicing of a precursor nucleic acid, the method comprising: (i) contacting a sample comprising the circular nucleic acids produced by self-splicing of the precursor nucleic acid and a positive selection probe that is at least 60% complementary to at least a portion of the circular nucleic acids with the positive selection probe immobilized on a solid surface under conditions that allow the circular nucleic acids produced by self-splicing of the precursor nucleic acid to bind to the positive selection probe; and (ii) collecting the portion bound to the positive selection probe on the solid surface.

Description

[0001] 1. Relevant Applications

[0002] This application has no related priority claims.

[0003] 2. Merging into the sequence list by reference

[0004] The contents of the electronic sequence list TPG03610-sequencing list.xml (file size: 6.17MB; created on January 10, 2024) are incorporated herein by reference in their entirety. 3. Technical Field

[0006] This application relates to the field of molecular biology, specifically to methods and systems for purifying circular nucleic acids. Circular nucleic acids to be purified using the methods provided herein can be generated through the self-splicing of precursor nucleic acids. 4. Background Technology

[0008] Circular RNAs (circRNAs) are a class of RNA molecules formed by linking ends together, and have been shown to possess a variety of biological functions in recent years. (Yang et al., Cell Research, 27(5):626-641(2017); Abe et al., Scientific Reports, 5:16435(2015); Gao et al., Nature Cell Biology, 23(3):278-291(2021); Pamudurti et al., Molecular Cell, 66(1):9-21(2017)). Compared with linear RNAs, circRNAs have better stability, thus providing a promising new platform for RNA drugs.

[0009] Purity is a critical factor in RNA therapy because contaminants often lead to immunogenicity. While methods for purifying RNA are available, efficient methods and systems for purifying RNA, especially circRNA, remain lacking. The compositions, methods, and systems presented in this article address this need and offer relevant advantages. 5. Overview of the Invention

[0011] 1. A composition comprising an oligonucleotide negative probe for purifying circular nucleic acids, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid comprising an oligonucleotide negative tag, wherein the negative tag is at least 90% complementary to the negative probe, wherein the negative tag is removed during the self-splicing of the precursor nucleic acid, preferably the negative tag is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the negative probe, more preferably 100% complementary.

[0012] 2. The composition according to claim 1, wherein the precursor nucleic acid includes the negative selection tag at its 5' end; and / or the precursor nucleic acid includes the negative selection tag at its 3' end.

[0013] When both the 5' end and the 3' end of the negative selection label are present, the 5' end negative selection label and the 3' end negative selection label can be the same or different.

[0014] 3. The composition according to claim 1 or 2, wherein the circular nucleic acid is a circular RNA (circRNA), preferably the precursor nucleic acid is RNA having group II intron self-splicing activity.

[0015] 4. The composition according to claim 3, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0016] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0017] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0018] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0019] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0020] 5. The composition according to claim 3, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a linker sequence; (d) a target sequence; (e) a linker sequence; (f) exon fragment 1 (E1); and (g) a 5' intron fragment; wherein:

[0021] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0022] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0023] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0024] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0025] 6. The composition according to claim 3, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; and (g) a 3' homologous arm; wherein:

[0026] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0027] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0028] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0029] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0030] 7. The composition according to claim 3, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a linker sequence; (e) a target sequence; (f) a linker sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0031] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0032] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0033] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0034] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0035] 8. The composition according to any one of claims 1-7, wherein the length of the negative tag and the negative probe is 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides; preferably, the length of the negative tag and the negative probe is 20-30 nucleotides.

[0036] 9. The composition according to any one of claims 1-8, wherein the negative probe is an RNA probe; or the negative probe is a DNA probe.

[0037] 10. The composition according to any one of claims 1-9, wherein the negative label has a polynucleotide sequence comprising SEQ ID NO: n, and the negative probe has a polynucleotide sequence comprising SEQ ID NO: n+1100, wherein n is an integer from 135 to 1234; or wherein the negative label has a polynucleotide sequence comprising SEQ ID NO: m, and the negative probe has a polynucleotide sequence comprising SEQ ID NO: m+1210, wherein m is an integer from 2335 to 3544 or from 4755 to 5964.

[0038] 11. The composition according to any one of items 1-9, wherein the negative label has a polynucleotide sequence comprising any one of SEQ ID NO:7189-7202; or the negative probe has a polynucleotide sequence comprising any one of SEQ ID NO:7175-7188.

[0039] 12. The composition according to claim 11, wherein the negative label and negative probe of the composition are selected from any of the following combinations:

[0040] (a) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7189, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7175;

[0041] (b) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7190, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7176;

[0042] (c) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7191, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7177;

[0043] (d) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7192, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7178;

[0044] (e) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7193, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7179;

[0045] (f) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7194, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7180;

[0046] (g) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7195, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7181;

[0047] (h) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7196, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7182;

[0048] (i) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7197, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7183;

[0049] (j) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7198, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7184;

[0050] (k) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7199, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7185;

[0051] (l) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7200, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7186;

[0052] (m) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7201, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7187;

[0053] (n) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7202, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7188.

[0054] 13. The composition according to any one of items 4-7, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II introns of the unpaired region into two fragments.

[0055] 14. The composition according to claim 13, wherein the 5' intron fragment and the 3' intron fragment are obtained by cleaving group II introns at the ring region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0056] The 5' and 3' intron segments are obtained by cutting group II introns in linear regions between domain 1 and domain 2, between domain 2 and domain 3, between domain 3 and domain 4, between domain 4 and domain 5, or between domain 5 and domain 6.

[0057] 15. The composition according to claim 13 or 14, wherein the group II intron comprises one or more nucleotide modifications relative to its wild-type form, and said modifications are selected from one or more deletions, substitutions, and additions.

[0058] Preferably, the modification comprises one or more EBS sequences of group II introns, wherein the EBS sequences are complementary to one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0059] 16. The composition according to claim 15, wherein the modification is a modification of two EBS sequences (e.g., EBS1 and EBS3) of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0060] 17. The composition according to claim 15, wherein the modification is a modification of two EBS sequences (e.g., EBS1' and EBS3') of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0061] 18. The composition according to claim 15 or 16, wherein the modification is a modification of the EBS1 and / or δ sequence of group II introns, or a modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides.

[0062] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to regions of corresponding length in the target sequence. Optionally, the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein 100% of the nucleotides of the EBS1 and / or δ sequence and its upstream sequence are complementary to regions of corresponding length in the target sequence.

[0063] 19. The composition according to claim 14, wherein the modification comprises partial or complete deletion of domain 4, such as deletion of the protein (IEP) sequence encoded by an intron in domain 4, preferably complete deletion of domain 4; or

[0064] The modification includes the omission of the open reading frame (ORF).

[0065] 20. The composition according to claim 16, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence, preferably the near-scarless circRNA having a scar region of length equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0066] 21. The composition according to claim 17, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0067] 22. The composition according to any one of items 3-21, wherein the group II intron is a group II intron derived from a microorganism, preferably the group II intron is Cte1, and more preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO:33-41.

[0068] 23. The composition according to any one of claims 4-22, wherein the 3' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO: 42-52; and / or

[0069] The 5' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:75-88.

[0070] 24. The composition according to any one of items 4-23, wherein E1 and / or E2 is 0-20 nucleotides in length, preferably 0-10 nucleotides in length, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length;

[0071] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NO:53-63 and SEQ ID NO:7211-7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NO:64-74 and SEQ ID NO:7213.

[0072] 25. The composition according to item 24, wherein E1, E2, or both are 0 nucleotides in length.

[0073] 26. The composition according to any one of claims 4-25, wherein the target sequence is a non-coding sequence selected from the group consisting of: spacer sequences of SEQ ID NO:4-6, polyA sequences, poly-AC sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences, and protein binding sites.

[0074] 27. The composition according to any one of claims 6-25, wherein the target sequence comprises: an internal ribosome entry site (IRES)-like polynucleotide sequence or a native IRES sequence, and a protein-coding sequence, preferably the protein-coding sequence encoding a therapeutic product.

[0075] 28. The composition according to any one of claims 1-27, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0076] 29. The composition according to claim 28, wherein at least one of the modified RNA nucleotide and / or modified nucleoside is m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), Y (pseudouridine), m1A (1-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine), Am (2'-O-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxyn-valine) Acylcarbamoyl adenosine), ms2hn6A (2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine), Ar(p)(2'-O-ribosyl adenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), (5-formylcytidine), m5Cm (5,2'-O-dimethylcytidine), ac4Cm (N4-acetyl-2'-O-methylcytidine), k2C (lysine), m! G(1-methylguanosine), m2G(N2-methylguanosine), m7G(7-methylguanosine), Gm(2'-O-methylguanosine), m22G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(huaitin), oayW(peroxyhuaitin), OH yW (hydroxyhuaitoside), OHyW* (insufficiently modified hydroxyhuaitoside), imG (huaitoside), mimG (methylhuaitoside), Q (pigmentoside), oQ (epoxypigmentoside), galQ (galactosylpigmentoside), manQ (mannosylpigmentoside), preQo (7-cyano-7-deazoguanosine), preQi (7-aminomethyl-7-deazoguanosine), G+ (archopurinoside), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), nmm5U (5-carbamoylmethyluridine), nmm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2'-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6,2'-O-dimethyladenosine), m6 2Am (N6,N6,O-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m'Gm (l,2'-O-dimethylguanosine), m'Am (l,2'-O-dimethyladenosine), rm 5U (5-taurate methyluridine), τm5s2U (5-taurate methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), or ac6A (N6-acetyladenosine).

[0077] 30. The composition according to any one of claims 1-27, wherein the precursor RNA comprises unmodified RNA nucleotides and / or unmodified nucleosides; or the precursor RNA does not comprise modified RNA nucleotides and / or modified nucleosides.

[0078] 31. The composition according to any one of claims 1-30, further comprising a solid surface,

[0079] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

[0080] Further preferably, the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads, or silica beads, or

[0081] Further preferably, the solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion base materials, or an integral support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

[0082] 32. The composition according to claim 31, wherein the anion probe is fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation, preferably covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0083] 33. The composition according to claim 31 or 32, wherein the solid surface is a magnetic bead.

[0084] Preferably, the magnetic beads are functionally modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specific groups, or combinations of two or more of the above, more preferably NHS-modified magnetic beads, or

[0085] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0086] Preferably, the magnetic beads are magnetic beads with a diameter of 0.1μm to 10μm, more preferably magnetic beads with a diameter of 0.5μm to 5μm, more preferably magnetic beads with a diameter of 0.8μm to 1.2μm, and more preferably magnetic beads with a diameter of 1μm.

[0087] 34. The composition according to claim 31 or 32, wherein the solid surface is an agarose chromatography packing material.

[0088] Preferably, the diameter of the chromatography packing material is 5 mm or more, and the height is 20 cm or more; more preferably, the diameter of the chromatography packing material is 10 mm or more, and the height is 30 cm or more; even more preferably, the diameter of the chromatography packing material is 16 mm, and the height is 40 cm.

[0089] Further, the agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, an agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, agarose chromatography packing material with CNBr-modified surface, agarose chromatography packing material with affinity or specificity groups, or a combination of two or more of the above, and is more preferably an NHS-modified agarose chromatography packing material.

[0090] 35. The composition according to any one of claims 31-34, wherein the 3' end of the anion probe is covalently fixed to the solid surface; or the 5' end of the anion probe is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0091] 36. The composition according to any one of claims 31-35, wherein the 3' end and / or 5' end of the anion probe is further modified with a group selected from amino, carboxyl, NHS-carboxyl, and thiol.

[0092] 37. The composition according to any one of claims 1-36, wherein the minimum free energy (MFE) value of the negative tag is in the range of -2 to 2, and the change in MFE value (ΔMFE) of the precursor nucleic acid resulting from the inclusion of the tag is in the range of -2 to 2.

[0093] 38. The composition according to item 37, wherein the MFE value of the negative label is approximately 0.

[0094] 39. The composition according to item 37 or 38, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag is in the range of 0-2, preferably the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag is about 0.

[0095] 40. A negative selection kit for purifying circular nucleic acids, comprising: a composition containing an oligonucleotide negative selection probe for purifying circular nucleic acids as described in any one of claims 1-39, a binding buffer, and an elution buffer.

[0096] 41. The kit according to claim 40, wherein the binding solution comprises a salt, said salt comprising a cation selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na+ K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably, the concentration of the salt in the binding solution is 50 mM-5 M.

[0097] Further preferably, the salt is a cation of Li. + Or Na + Salts.

[0098] 42. The kit according to claim 41, wherein the pH range of the binding buffer is 4.0-9.0, preferably 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, or 8.5-9.0;

[0099] Preferably, the binding solution further includes a buffering substance, a chelating agent, and optionally a stabilizer.

[0100] 43. The kit according to item 41 or 42, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl, preferably further comprising EDTA and Tris-HCl, and the pH range of the binding solution is 6.0-8.0, preferably 7.5.

[0101] 44. The kit according to any one of claims 40-43, wherein the concentration of salts in the eluent is 0M, and the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3-CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably the salt being a cation of Li. + Or Na + Salts,

[0102] Preferably, the eluent further includes buffering substances, chelating agents, and optionally stabilizers.

[0103] 45. The kit according to claim 44, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.0, preferably 7.5; or

[0104] The eluent has a pH range of 2.0-4.0, preferably a pH range of 2.5-3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5-3.0.

[0105] 46. ​​A negative selection kit for purifying circular nucleic acids, comprising: magnetic beads coupled with a negative selection probe, an elution buffer, and a binding buffer, wherein the negative selection probe is at least 90% complementary to a negative selection tag, the circular nucleic acid being generated by the self-splicing of a precursor nucleic acid containing an oligonucleotide negative selection tag, wherein the negative selection tag is removed during the self-splicing of the precursor nucleic acid, preferably the negative selection tag being at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the negative selection probe, more preferably 100% complementary.

[0106] 47. The kit according to claim 46, wherein the negative probe and negative label are the negative probe and negative label involved in any one of claims 1-39, or

[0107] The eluent and binding fluid are the eluent and binding fluids involved in any of items 40-45.

[0108] 48. The kit according to item 46 or 47, wherein the magnetic beads with the immobilized anion probe are reusable.

[0109] 49. A method for purifying circular nucleic acids from a sample, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, and wherein the precursor nucleic acid has an oligonucleotide negative select tag, the oligonucleotide negative select tag being removed during the self-splicing process, the method comprising:

[0110] (i) Under conditions that allow the negative label to bind to the negative probe, contact a sample with an oligonucleotide negative probe that is at least 90% complementary to the negative label, wherein the negative probe is immobilized on a solid surface; and

[0111] (ii) Collect the unbound portion of the sample.

[0112] Preferably, the negative label and the negative probe are at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary, and more preferably 100% complementary.

[0113] 50. The method of claim 49, wherein the precursor nucleic acid includes the negative select tag at its 5' end; or the precursor nucleic acid includes the negative select tag at its 3' end.

[0114] When both the 5' end and the 3' end of the negative selection label are present, the 5' end negative selection label and the 3' end negative selection label can be the same or different.

[0115] 51. The method according to item 49 or 50, wherein the circular nucleic acid is a circular RNA (circRNA), preferably the precursor nucleic acid is RNA having group II intron self-splicing activity.

[0116] 52. The method according to claim 51, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked together: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0117] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0118] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0119] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0120] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0121] 53. The method according to claim 51, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a adapter sequence; (d) a target sequence; (e) an adapter sequence; (f) exon fragment 1 (E1); and (g) a 5' intron fragment; wherein:

[0122] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0123] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0124] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0125] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0126] 54. The method according to claim 51, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; and (g) a 3' homologous arm; wherein:

[0127] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0128] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0129] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0130] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0131] 55. The method according to claim 51, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a adapter sequence; (e) a target sequence; (f) an adapter sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0132] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0133] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0134] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0135] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0136] 56. The method according to any one of claims 49-55, wherein the length of the negative tag and the negative probe is 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides; preferably, the length of the negative tag and the negative probe is 20-30 nucleotides.

[0137] 57. The method according to any one of claims 49-56, wherein the negative probe is an RNA probe; or the negative probe is a DNA probe.

[0138] 58. The method according to any one of claims 49-57, wherein the negative label has a polynucleotide sequence comprising SEQ ID NO:n, and the negative probe has a polynucleotide sequence comprising SEQ ID NO:n+1100, wherein n is an integer from 135 to 1234; or wherein the negative label has a polynucleotide sequence comprising SEQ ID NO:m, and the negative probe has a polynucleotide sequence comprising SEQ ID NO:m+1210, wherein m is an integer from 2335 to 3544 or from 4755 to 5964.

[0139] 59. The method according to any one of claims 49-58, wherein the negative label has a polynucleotide sequence comprising any one of SEQ ID NO:7189-7202; or the negative probe has a polynucleotide sequence comprising any one of SEQ ID NO:7175-7188.

[0140] 60. The method of claim 59, wherein the negative label and the negative probe are selected from any of the following combinations:

[0141] (a) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7189, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7175;

[0142] (b) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7190, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7176;

[0143] (c) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7191, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7177;

[0144] (d) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7192, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7178;

[0145] (e) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7193, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7179;

[0146] (f) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7194, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7180;

[0147] (g) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7195, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7181;

[0148] (h) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7196, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7182;

[0149] (i) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7197, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7183;

[0150] (j) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7198, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7184;

[0151] (k) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7199, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7185;

[0152] (l) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7200, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7186;

[0153] (m) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7201, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7187;

[0154] (n) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7202, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7188.

[0155] 61. The method according to any one of items 49-60, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II introns of the unpaired region into two fragments.

[0156] 62. The method according to claim 61, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting group II introns at the loop region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0157] The 5' and 3' intron segments are obtained by cutting group II introns in linear regions between domain 1 and domain 2, between domain 2 and domain 3, between domain 3 and domain 4, between domain 4 and domain 5, or between domain 5 and domain 6.

[0158] 63. The method according to claim 61 or 62, wherein the group II intron comprises one or more nucleotide modifications relative to its wild-type form, and the modifications are selected from one or more deletions, substitutions, and additions.

[0159] Preferably, the modification comprises one or more EBS sequences of group II introns, wherein the EBS sequences are complementary to one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0160] 64. The method according to item 63, wherein the modification is a modification of two EBS sequences (such as EBS1 and EBS3) of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0161] 65. The method according to claim 63, wherein the modification is a modification of two EBS sequences (such as EBS1' and EBS3') of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0162] 66. The method according to claim 64 or 65, wherein the modification is a modification of the EBS1 and / or δ sequence of group II introns, or a modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides.

[0163] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to regions of corresponding length in the target sequence. Optionally, the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein 100% of the nucleotides of the EBS1 and / or δ sequence and its upstream sequence are complementary to regions of corresponding length in the target sequence.

[0164] 67. The method according to claim 66, wherein the modification includes partial or complete deletion of domain 4, such as deletion of the protein (IEP) sequence encoded by an intron in domain 4, preferably complete deletion of domain 4; or

[0165] The modification includes the omission of the open reading frame (ORF).

[0166] 68. The method according to claim 64, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence, preferably the near-scarless circRNA having a scar region of length equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0167] 69. The method of claim 65, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0168] 70. The method according to any one of claims 51-69, wherein the group II intron is a group II intron derived from a microorganism, preferably the group II intron is Cte1, and more preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO:33-41.

[0169] 71. The method according to any one of claims 52-70, wherein the 3' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:42-52; and / or

[0170] The 5' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:75-88.

[0171] 72. The method according to any one of items 52-71, wherein the length of E1 and / or E2 is 0-20 nucleotides, preferably 0-10 nucleotides, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides;

[0172] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NO:53-63 and SEQ ID NO:7211-7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NO:64-74 and SEQ ID NO:7213.

[0173] 73. The method according to item 72, wherein E1, E2, or both are 0 nucleotides in length.

[0174] 74. The method according to any one of claims 52-73, wherein the target sequence is a non-coding sequence selected from the group consisting of: spacer sequences of SEQ ID NO:4-6, polyA sequences, poly-AC sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences, and protein binding sites.

[0175] 75. The method according to any one of claims 52-73, wherein the target sequence comprises: an internal ribosome entry site (IRES)-like polynucleotide sequence or a natural IRES sequence, and a protein-coding sequence, preferably the protein-coding sequence encoding a therapeutic product.

[0176] 76. The method according to any one of claims 49-75, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0177] 77. The method according to claim 76, wherein at least one of the modified RNA nucleotide and / or modified nucleoside is m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), Y (pseudouridine), m1A (1-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine), Am (2'-O-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxyn-valine) Acylcarbamoyl adenosine), ms2hn6A (2-methylthio-N6-hydroxyn-valine carbamoyl adenosine), Ar(p)(2'-O-ribosyl adenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), (5-formylcytidine), m5Cm (5,2'-O-dimethylcytidine), ac4Cm (N4-acetyl-2'-O-methylcytidine), k2C (lysine), m! G (1-methylguanosine), m2G (N2-methylguanosine), m7G (7-methylguanosine), Gm (2'-O-methylguanosine), m2 2G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(huaitoside), oayW(peroxyhuaitoside), OHyW(hydroxyhuaitoside), OHyW*(undermodified hydroxyhuaitoside), imG(huaitoside), mimG(methylhuaitoside), Q(huaitoside), oQ(epoxyhuaitoside), galQ(galactosyl-huaitoside), manQ(mannosyl-huaitoside), preQo(7-cyano-7-deazoguanosine), preQi(7-aminomethyl-7-deazoguanosine), G+(archopurinol), D(dihydrouridine), m5Um(5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), nmm5U (5-carbamoylmethyluridine), nmm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2'-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6,2'-O-dimethyladenosine), m6 2Am (N6,N6,O-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m'Gm (l,2'-O-dimethylguanosine), m'Am (l,2'-O-dimethyladenosine), rm 5U (5-taurate methyluridine), τm5s2U (5-taurate methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), or ac6A (N6-acetyladenosine).

[0178] 78. The method according to any one of claims 52-73, wherein the precursor RNA comprises unmodified RNA nucleotides and / or unmodified nucleosides; or the precursor RNA does not comprise modified RNA nucleotides and / or modified nucleosides.

[0179] 79. The method according to any one of claims 52-78, further comprising a solid surface.

[0180] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

[0181] Further preferably, the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads, or silica beads, or

[0182] Further preferably, the solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion base materials, or an integral support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

[0183] 80. The method according to claim 79, wherein the anion probe is fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation, preferably covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0184] 81. The method according to item 79 or 80, wherein the solid surface is a magnetic bead.

[0185] Preferably, the magnetic beads are functionally modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specific groups, or combinations of two or more of the above, more preferably NHS-modified magnetic beads, or

[0186] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0187] Preferably, the magnetic beads are magnetic beads with a diameter of 0.1μm to 10μm, more preferably magnetic beads with a diameter of 0.5μm to 5μm, more preferably magnetic beads with a diameter of 0.8μm to 1.2μm, and more preferably magnetic beads with a diameter of 1μm.

[0188] 82. The method according to claim 79 or 80, wherein the solid surface is a chromatography packing material containing agarose, preferably the diameter of the chromatography packing material is 5 mm or more and the height is 20 cm or more, more preferably the diameter of the chromatography packing material is 10 mm or more and the height is 30 cm or more, and even more preferably the diameter of the chromatography packing material is 16 mm and the height is 40 cm.

[0189] Further, the agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, an agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, agarose chromatography packing material with CNBr-modified surface, agarose chromatography packing material with affinity or specificity groups, or a combination of two or more of the above, and is more preferably an NHS-modified agarose chromatography packing material.

[0190] 83. The method according to any one of claims 79-82, wherein the 3' end of the anion probe is covalently fixed to the solid surface; or the 5' end of the anion probe is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0191] 84. The method according to any one of claims 79-83, wherein the 3' end and / or 5' end of the anion probe is further modified with a group selected from: amino, carboxyl, NHS-carboxyl, mercapto.

[0192] 85. The composition according to any one of claims 49-84, wherein the minimum free energy (MFE) value of the negative tag is in the range of -2 to 2, and the change in MFE value (ΔMFE) of the precursor nucleic acid resulting from the inclusion of the tag is in the range of -2 to 2.

[0193] 86. The composition according to claim 85, wherein the MFE value of the negative label is approximately 0.

[0194] 87. The composition according to claim 85 or 86, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag is in the range of 0-2, preferably the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag is about 0.

[0195] 88. The method according to any one of claims 49-87, wherein a sample comprising the circular nucleic acid to be purified is contacted with the negative probe at a pH of 4.0-9.0, preferably 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, or 8.5-9.0; or

[0196] The sample is in contact with the cathode probe at a temperature of 0℃-60℃; or

[0197] The sample is contacted with the anion probe in a binding solution, the binding solution comprising salts, preferably further comprising buffering substances, chelating agents, optionally including stabilizers, and more preferably the salts comprising cations selected from the group consisting of Ba. 2+ Ca 2+ Mg2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably, the concentration of the salt in the binding solution is 50 mM-5 M.

[0198] Further preferably, the salt is a cation of Li. + Or Na + Salts.

[0199] 89. The method according to claim 88, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl, preferably further comprising EDTA and Tris-HCl, and the pH range of the binding solution is 6.0-8.0, preferably 7.5.

[0200] Preferably, the method further includes the step of using an eluent to elute a portion of the anion probe bound to the solid surface to recover the solid surface coupled with the anion probe for reuse.

[0201] 90. The method according to any one of claims 49-89, wherein the concentration of salts in the eluent is 0M, and the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 +and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably the salt being a cation of Li. + Or Na + Salts,

[0202] Preferably, the eluent further includes buffering substances, chelating agents, and optionally stabilizers.

[0203] 91. The method according to claim 90, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.0, preferably 7.5; or

[0204] The eluent has a pH range of 2.0-4.0, preferably a pH range of 2.5-3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5-3.0.

[0205] 92. The method according to any one of items 49-91, wherein the method is scalable and may include a continuous flow process, preferably performed using a liquid chromatograph (LC) or an enzyme-linked immunosorbent assay (ELISA) reader, and more preferably the LC is a rapid liquid chromatograph (FPLC) or a high-performance liquid chromatograph (HPLC).

[0206] 93. A method for purifying circular nucleic acids from a sample, comprising the following steps:

[0207] Couple the magnetic bead to the negative selection probe;

[0208] The IVT reaction solution containing the circular nucleic acid obtained from in vitro transcription was incubated with magnetic beads coupled with a negative selection probe.

[0209] The magnetic beads are collected and eluted to recover the magnetic beads coupled with the anion selection probe;

[0210] Collect the unbound parts.

[0211] The negative probe is at least 90% complementary to the negative tag, the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid containing an oligonucleotide negative tag, wherein the negative tag is removed during the self-splicing of the precursor nucleic acid, preferably the negative tag is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the negative probe, and more preferably 100% complementary.

[0212] 94. The method according to item 93, wherein the IVT reaction solution is incubated with magnetic beads coupled with an anion-selective probe in a binding solution, and the magnetic beads coupled with an anion-selective probe are eluted and recovered using an elution buffer.

[0213] 95. The method according to claim 94, wherein the eluent and binding solution are the eluent and binding solution involved in any one of claims 88-91, and more preferably the anion probe and anion label are the anion probe and anion label involved in any one of claims 49-92.

[0214] 96. Circular nucleic acids purified by the method according to any one of items 49-95, or by the composition comprising oligonucleotide negative probes for purifying circular nucleic acids according to any one of items 1-39, or by the kit according to any one of items 40-48.

[0215] 97. A composition having the circular nucleic acid of claim 96, wherein the cyclization percentage (PC) of the composition is at least 90%, at least 95%, at least 98%, or at least 99%; or

[0216] The total cyclic purity of the composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, and above 95%, preferably with an polymer content below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, and below 1.5%.

[0217] Further preferably, the size of the circular nucleic acid is 1000nt-5000nt.

[0218] 98. A composition for generating circular nucleic acids, comprising:

[0219] Linearized plasmids used to generate precursor nucleic acids;

[0220] The generated precursor nucleic acid;

[0221] The generated circular nucleic acid;

[0222] Non-circular precursor nucleic acids;

[0223] Residual impurities from in vitro transcription reaction, wherein the precursor nucleic acid and circular nucleic acid are any of the precursor nucleic acids and circular nucleic acids involved in items 1-48.

[0224] The preferred composition has a total cyclic purity ranging from 20% to 50% and a polymer content ranging from 10% to 20%.

[0225] 99. The composition according to claim 98, used as a sample by any one of claims 49-96, or purified as a sample by any one of claims 1-39 using a composition comprising an oligonucleotide negative probe for purifying circular nucleic acids, or purified using any one of claims 40-48.

[0226] 100. The composition according to claim 99, wherein the purified cyclic nucleic acid composition has a total cyclic purity of ≥90.0%, ≥91%, ≥92%, ≥93%, ≥94%, or ≥95%.

[0227] The preferred polymer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, and below 1.5%.

[0228] Further preferably, the size of the circular nucleic acid is 1000nt-5000nt.

[0229] 101. A composition comprising a first oligonucleotide negative probe and a second oligonucleotide negative probe for purifying circular nucleic acids, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid comprising a first oligonucleotide negative tag and a second oligonucleotide negative tag, wherein the first and second negative tags are at least 90% complementary to the first and second negative probes, respectively, and are removed during the self-splicing of the precursor nucleic acid, preferably the first and second negative tags are at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the first and second negative probes, more preferably 100% complementary.

[0230] 102. The composition according to claim 101, wherein the precursor nucleic acid includes the first negative tag at its 5' end and the second negative tag at its 3' end, the first negative tag and the second negative tag may be the same or different.

[0231] 103. The composition according to claim 101 or 102, wherein the circular nucleic acid is a circular RNA (circRNA), preferably the precursor nucleic acid is RNA having group II intron self-splicing activity.

[0232] 104. The composition according to claim 103, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked together: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0233] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0234] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0235] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0236] (4) The target sequence is absent, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0237] Further preferably, the precursor RNA from 5' to 3' includes the following operatively linked elements: a first negative tag; (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); (e) a 5' intron fragment; and a second negative tag.

[0238] 105. The composition according to claim 103, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a linker sequence; (d) a target sequence; (e) a linker sequence; (f) exon fragment 1 (E1); and (g) a 5' intron fragment; wherein:

[0239] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0240] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0241] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0242] (4) The target sequence is absent, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0243] Further preferably, the precursor RNA comprises the following operatively linked elements from 5' to 3': a first negative tag; (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a adapter sequence; (d) a target sequence; (e) an adapter sequence; (f) exon fragment 1 (E1); (g) a 5' intron fragment; and a second negative tag.

[0244] 106. The composition according to claim 103, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; and (g) a 3' homologous arm; wherein:

[0245] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0246] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0247] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0248] (4) The target sequence is absent, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0249] Further preferably, the precursor RNA comprises the following operatively linked elements from 5' to 3': a first negative tag; (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; (g) a 3' homologous arm; and a second negative tag.

[0250] 107. The composition according to claim 103, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a linker sequence; (e) a target sequence; (f) a linker sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0251] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0252] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0253] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0254] (4) The target sequence is absent, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0255] Further preferably, the precursor RNA comprises the following operatively linked elements from 5' to 3': a first negative tag; (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a adapter sequence; (e) a target sequence; (f) an adapter sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; (i) a 3' homologous arm; and a second negative tag.

[0256] 108. The composition according to any one of claims 101-107, wherein the lengths of the first negative label, the second negative label, the first negative probe, and the second negative probe are 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides, respectively; preferably, the lengths of the first negative label, the second negative label, the first negative probe, and the second negative probe are 20-30 nucleotides, respectively.

[0257] 109. The composition according to any one of claims 101-108, wherein the first negative probe and the second negative probe are RNA probes; or the first negative probe and the second negative probe are DNA probes.

[0258] 110. The composition according to any one of items 101-109, wherein,

[0259] When the first negative label and negative probe pair are the same as the second negative label and negative probe pair, the first negative label and the second negative label have a polynucleotide sequence comprising SEQ ID NO:n, and the first negative probe and the second negative probe have a polynucleotide sequence comprising SEQ ID NO:n+1100, where n is an integer from 135 to 1234; or

[0260] When the first negative label and negative probe pair are different from the second negative label and negative probe pair, the first negative label has a polynucleotide sequence containing SEQ ID NO: n; the first negative probe has a polynucleotide sequence containing SEQ ID NO: n+1210; the second negative label has a polynucleotide sequence containing SEQ ID NO: n+2420; the second negative probe has a polynucleotide sequence containing SEQ ID NO: n+3630; where n is an integer from 2335 to 3544.

[0261] 111. The composition according to any one of claims 101-109, wherein the first negative label and negative probe pair are the same as the second negative label and negative probe pair, the first negative label and the second negative label having a polynucleotide sequence comprising any one of SEQ ID NO:7189-7202; or the first negative probe and the second negative probe having a polynucleotide sequence comprising any one of SEQ ID NO:7175-7188.

[0262] 112. The composition according to claim 111, wherein the first cathode label and the first cathode probe of the composition are selected from any of the following combinations, and the second cathode label and the second cathode probe of the composition are selected from any of the following combinations.

[0263] (a) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7189, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7175;

[0264] (b) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7190, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7176;

[0265] (c) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7191, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7177;

[0266] (d) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7192, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7178;

[0267] (e) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7193, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7179;

[0268] (f) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7194, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7180;

[0269] (g) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7195, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7181;

[0270] (h) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7196, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7182;

[0271] (i) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7197, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7183;

[0272] (j) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7198, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7184;

[0273] (k) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7199, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7185;

[0274] (l) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7200, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7186;

[0275] (m) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7201, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7187;

[0276] (n) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7202, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7188.

[0277] 113. The composition according to any one of items 104-107, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II introns of the unpaired region into two fragments.

[0278] 114. The composition according to claim 113, wherein the 5' intron fragment and the 3' intron fragment are obtained by cleaving group II introns at the ring region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0279] The 5' and 3' intron segments are obtained by cutting group II introns in linear regions between domain 1 and domain 2, between domain 2 and domain 3, between domain 3 and domain 4, between domain 4 and domain 5, or between domain 5 and domain 6.

[0280] 115. The composition according to claim 113 or 114, wherein the group II introns comprise one or more nucleotide modifications relative to their wild-type form, and said modifications are selected from one or more deletions, substitutions, and additions.

[0281] Preferably, the modification comprises one or more EBS sequences of group II introns, wherein the EBS sequences are complementary to one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0282] 116. The composition according to claim 115, wherein the modification is a modification of two EBS sequences (such as EBS1 and EBS3) of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0283] 117. The composition according to claim 115, wherein the modification is a modification of two EBS sequences (e.g., EBS1' and EBS3') of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0284] 118. The composition according to claim 115 or 116, wherein the modification is a modification of the EBS1 and / or δ sequence of group II introns, or a modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides.

[0285] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to regions of corresponding length in the target sequence. Optionally, the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein 100% of the nucleotides of the EBS1 and / or δ sequence and its upstream sequence are complementary to regions of corresponding length in the target sequence.

[0286] 119. The composition according to claim 114, wherein the modification comprises partial or complete deletion of domain 4, such as deletion of the protein (IEP) sequence encoded by an intron in domain 4, preferably complete deletion of domain 4; or

[0287] The modification includes the omission of the open reading frame (ORF).

[0288] 120. The composition according to claim 116, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence, preferably the near-scarless circRNA having a scar region of length equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0289] 121. The composition according to claim 117, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0290] 122. The composition according to any one of claims 103-121, wherein the group II intron is a group II intron derived from a microorganism, preferably the group II intron is Cte1, and more preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0291] 123. The composition according to any one of claims 104-122, wherein the 3' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:42-52; and / or

[0292] The 5' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:75-88.

[0293] 124. The composition according to any one of claims 104-123, wherein E1 and / or E2 are 0-20 nucleotides in length, preferably 0-10 nucleotides in length, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length;

[0294] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NO:53-63 and SEQ ID NO:7211-7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NO:64-74 and SEQ ID NO:7213.

[0295] 125. The composition according to item 124, wherein E1, E2, or both are 0 nucleotides in length.

[0296] 126. The composition according to any one of claims 104-125, wherein the target sequence is a non-coding sequence selected from the group consisting of: spacer sequences of SEQ ID NO:4-6, polyA sequences, poly-AC sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences, and protein binding sites.

[0297] 127. The composition according to any one of claims 106-15, wherein the target sequence comprises: an internal ribosome entry site (IRES)-like polynucleotide sequence or a natural IRES sequence, and a protein-coding sequence, preferably the protein-coding sequence encoding a therapeutic product.

[0298] 128. The composition according to any one of claims 101-127, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0299] 129. The composition according to claim 128, wherein at least one of the modified RNA nucleotide and / or modified nucleoside is m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), Y (pseudouridine), m1A (1-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine), Am (2'-O-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxyn-valine) Acylcarbamoyl adenosine), ms2hn6A (2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine), Ar(p)(2'-O-ribosyl adenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), (5-formylcytidine), m5Cm (5,2'-O-dimethylcytidine), ac4Cm (N4-acetyl-2'-O-methylcytidine), k2C (lysine), m! G(1-methylguanosine), m2G(N2-methylguanosine), m7G(7-methylguanosine), Gm(2'-O-methylguanosine), m22G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(huaitin), oayW(peroxyhuaitin), OH yW (hydroxyhuaitoside), OHyW* (insufficiently modified hydroxyhuaitoside), imG (huaitoside), mimG (methylhuaitoside), Q (pigmentoside), oQ (epoxypigmentoside), galQ (galactosylpigmentoside), manQ (mannosylpigmentoside), preQo (7-cyano-7-deazoguanosine), preQi (7-aminomethyl-7-deazoguanosine), G+ (archopurinoside), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), nmm5U (5-carbamoylmethyluridine), nmm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2'-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6,2'-O-dimethyladenosine), m6 2Am (N6,N6,O-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m'Gm (l,2'-O-dimethylguanosine), m'Am (l,2'-O-dimethyladenosine), rm 5U (5-taurate methyluridine), τm5s2U (5-taurate methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), or ac6A (N6-acetyladenosine).

[0300] 130. The composition according to any one of claims 101-127, wherein the precursor RNA comprises unmodified RNA nucleotides and / or unmodified nucleosides; or the precursor RNA does not comprise modified RNA nucleotides and / or modified nucleosides.

[0301] 131. The composition according to any one of claims 101-130, further comprising a solid surface,

[0302] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

[0303] Further preferably, the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads, or silica beads, or

[0304] Further preferably, the solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion base materials, or an integral support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

[0305] 132. The composition according to claim 131, wherein the first and second anionic probes are fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation, preferably covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0306] 133. The composition according to claim 131 or 132, wherein the solid surface is a magnetic bead.

[0307] Preferably, the magnetic beads are functionally modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specific groups, or combinations of two or more of the above, more preferably NHS-modified magnetic beads, or

[0308] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0309] Preferably, the magnetic beads are magnetic beads with a diameter of 0.1μm to 10μm, more preferably magnetic beads with a diameter of 0.5μm to 5μm, more preferably magnetic beads with a diameter of 0.8μm to 1.2μm, and more preferably magnetic beads with a diameter of 1μm.

[0310] 134. The composition according to claim 131 or 132, wherein the solid surface is an agarose chromatography packing material.

[0311] Preferably, the diameter of the chromatography packing material is 5 mm or more, and the height is 20 cm or more; more preferably, the diameter of the chromatography packing material is 10 mm or more, and the height is 30 cm or more; even more preferably, the diameter of the chromatography packing material is 16 mm, and the height is 40 cm.

[0312] Further, the agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, an agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, agarose chromatography packing material with CNBr-modified surface, agarose chromatography packing material with affinity or specificity groups, or a combination of two or more of the above, and is more preferably an NHS-modified agarose chromatography packing material.

[0313] 135. The composition according to any one of claims 131-134, wherein the 3' ends of the first and second anion probes are covalently fixed to the solid surface; or the 5' ends of the first and second anion probes are covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0314] 136. The composition according to any one of claims 131-135, wherein the 3' end and / or 5' end of the first and second anionic probes are further modified with groups selected from: amino, carboxyl, NHS-carboxyl, and thiol.

[0315] 137. The composition according to any one of claims 101-136, wherein the minimum free energy (MFE) values ​​of the first negative tag and the second negative tag are in the range of -2 to 2, and the change in MFE value (ΔMFE) of the precursor nucleic acid resulting from the inclusion of the tags is in the range of -2 to 2.

[0316] 138. The composition according to claim 137, wherein the MFE values ​​of the first and second negative labels are approximately 0.

[0317] 139. The composition according to item 137 or 138, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the first negative label and the second negative label is in the range of 0-2, preferably the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the first negative label and the second negative label is about 0.

[0318] 140. A negative selection kit for purifying circular nucleic acids, comprising: a composition containing an oligonucleotide negative selection probe for purifying circular nucleic acids as described in any one of claims 101-139, a binding buffer, and an elution buffer.

[0319] 141. The kit according to claim 140, wherein the binding solution comprises a salt, said salt comprising a cation selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably, the concentration of the salt in the binding solution is 50 mM-5 M.

[0320] Further preferably, the salt is a cation of Li. + Or Na + Salts.

[0321] 142. The kit according to claim 141, wherein the pH range of the binding buffer is 4.0-9.0, preferably 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, or 8.5-9.0;

[0322] Preferably, the binding solution further includes a buffering substance, a chelating agent, and optionally a stabilizer.

[0323] 143. The kit according to item 141 or 142, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl, preferably further comprising EDTA and Tris-HCl, and the pH range of the binding solution is 6.0-8.0, preferably 7.5.

[0324] 144. The kit according to any one of claims 140-143, wherein the concentration of salts in the eluent is 0M, and the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K+ 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably the salt being a cation of Li. + Or Na + Salts,

[0325] Preferably, the eluent further includes buffering substances, chelating agents, and optionally stabilizers.

[0326] 145. The kit according to claim 144, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.0, preferably 7.5; or

[0327] The eluent has a pH range of 2.0-4.0, preferably a pH range of 2.5-3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5-3.0.

[0328] 146. A negative selection kit for purifying circular nucleic acids, comprising: magnetic beads coupled with a negative selection probe, an elution buffer, and a binding buffer, wherein the negative selection probe is at least 90% complementary to a negative selection tag, the circular nucleic acid being generated by the self-splicing of a precursor nucleic acid containing an oligonucleotide negative selection tag, wherein the negative selection tag is removed during the self-splicing of the precursor nucleic acid, preferably the negative selection tag being at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the negative selection probe, more preferably 100% complementary.

[0329] 147. The kit according to claim 146, wherein the negative probe and negative label are the negative probe and negative label involved in any of claims 101-139, or

[0330] The eluent and binding fluid are the eluent and binding fluids involved in any of items 140-145.

[0331] 148. The kit according to item 146 or 147, wherein the magnetic beads with the immobilized anion probe are reusable.

[0332] 149. A method for purifying circular nucleic acids from a sample, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, and wherein the precursor nucleic acid has a first oligonucleotide negative selector tag and a second oligonucleotide negative selector tag, the first negative selector tag and the second negative selector tag being removed during the self-splicing process, the method comprising:

[0333] (i) Under conditions allowing the first cathode label to bind with the first cathode probe, and the second cathode label to bind with the second cathode probe, the sample is contacted with the first cathode probe and the second cathode probe, respectively, which are at least 90% complementary to the first cathode label and the second cathode probe, wherein the first cathode probe and the second cathode probe are fixed to a solid surface; and

[0334] (ii) Collect the unbound portion of the sample.

[0335] Preferably, the first and second negative labels are at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the first and second negative probes, respectively, and more preferably 100% complementary.

[0336] 150. The method according to claim 149, wherein the precursor nucleic acid includes the first negative label at its 5' end and the second negative label at its 3' end, the first negative label and the second negative label may be the same or different.

[0337] 151. The method according to item 149 or 150, wherein the circular nucleic acid is a circular RNA (circRNA), preferably the precursor nucleic acid is RNA having group II intron self-splicing activity.

[0338] 152. The method according to claim 151, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked together: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0339] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0340] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0341] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0342] (4) The target sequence is absent, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0343] Further preferably, the precursor RNA from 5' to 3' includes the following operatively linked elements: a first negative tag; (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); (e) a 5' intron fragment; and a second negative tag.

[0344] 153. The method according to claim 151, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a adapter sequence; (d) a target sequence; (e) an adapter sequence; (f) exon fragment 1 (E1); and (g) a 5' intron fragment; wherein:

[0345] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0346] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0347] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0348] (4) The target sequence is absent, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0349] Further preferably, the precursor RNA comprises the following operatively linked elements from 5' to 3': a first negative tag; (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a adapter sequence; (d) a target sequence; (e) an adapter sequence; (f) exon fragment 1 (E1); (g) a 5' intron fragment; and a second negative tag.

[0350] 154. The method according to claim 151, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; and (g) a 3' homologous arm; wherein:

[0351] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0352] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0353] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0354] (4) The target sequence is absent, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0355] Further preferably, the precursor RNA comprises the following operatively linked elements from 5' to 3': a first negative tag; (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; (g) a 3' homologous arm; and a second negative tag.

[0356] 155. The method according to claim 151, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a adapter sequence; (e) a target sequence; (f) an adapter sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0357] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0358] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0359] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0360] (4) The target sequence is absent, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0361] Further preferably, the precursor RNA comprises the following operatively linked elements from 5' to 3': a first negative tag; (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a adapter sequence; (e) a target sequence; (f) an adapter sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; (i) a 3' homologous arm; and a second negative tag.

[0362] 156. The method according to any one of claims 149-155, wherein the lengths of the first negative label, the second negative label, the first negative probe, and the second negative probe are 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides, respectively; preferably, the lengths of the first negative label, the second negative label, the first negative probe, and the second negative probe are 20-30 nucleotides, respectively.

[0363] 157. The method according to any one of claims 149-156, wherein the first negative probe and the second negative probe are RNA probes; or the first negative probe and the second negative probe are DNA probes.

[0364] 158. The method according to any one of items 149-157, wherein,

[0365] When the first negative label and negative probe pair are the same as the second negative label and negative probe pair, the first negative label and the second negative label have a polynucleotide sequence comprising SEQ ID NO:n, and the first negative probe and the second negative probe have a polynucleotide sequence comprising SEQ ID NO:n+1100, where n is an integer from 135 to 1234; or

[0366] When the first negative label and negative probe pair are different from the second negative label and negative probe pair, the first negative label has a polynucleotide sequence containing SEQ ID NO: n; the first negative probe has a polynucleotide sequence containing SEQ ID NO: n+1210; the second negative label has a polynucleotide sequence containing SEQ ID NO: n+2420; the second negative probe has a polynucleotide sequence containing SEQ ID NO: n+3630; where n is an integer from 2335 to 3544.

[0367] 159. The method according to any one of claims 49-58, wherein the first negative label and negative probe pair are the same as the second negative label and negative probe pair, the first negative label and the second negative label having a polynucleotide sequence comprising any one of SEQ ID NO:7189-7202; or the first negative probe and the second negative probe having a polynucleotide sequence comprising any one of SEQ ID NO:7175-7188.

[0368] 160. The method according to claim 159, wherein the first cathode label and the first cathode probe of the composition are selected from any of the following combinations, and the second cathode label and the second cathode probe of the composition are selected from any of the following combinations.

[0369] (a) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7189, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7175;

[0370] (b) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7190, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7176;

[0371] (c) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7191, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7177;

[0372] (d) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7192, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7178;

[0373] (e) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7193, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7179;

[0374] (f) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7194, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7180;

[0375] (g) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7195, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7181;

[0376] (h) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7196, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7182;

[0377] (i) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7197, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7183;

[0378] (j) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7198, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7184;

[0379] (k) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7199, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7185;

[0380] (l) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7200, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7186;

[0381] (m) The polynucleotide sequence of the first or second negative select tag is shown in SEQ ID NO:7201, and the polynucleotide sequence of the first or second negative select probe is shown in SEQ ID NO:7187;

[0382] (n) The polynucleotide sequence of the first or second negative tag is shown in SEQ ID NO:7202, and the polynucleotide sequence of the first or second negative probe is shown in SEQ ID NO:7188.

[0383] 161. The method according to any one of items 149-160, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II introns of the unpaired region into two fragments.

[0384] 162. The method according to claim 161, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting group II introns at the loop region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0385] The 5' and 3' intron segments are obtained by cutting group II introns in linear regions between domain 1 and domain 2, between domain 2 and domain 3, between domain 3 and domain 4, between domain 4 and domain 5, or between domain 5 and domain 6.

[0386] 163. The method according to claim 161 or 162, wherein the group II intron comprises one or more nucleotide modifications relative to its wild-type form, and the modifications are selected from one or more deletions, substitutions, and additions.

[0387] Preferably, the modification comprises one or more EBS sequences of group II introns, wherein the EBS sequences are complementary to one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0388] 164. The method according to claim 163, wherein the modification is a modification of two EBS sequences (such as EBS1 and EBS3) of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0389] 165. The method according to claim 163, wherein the modification is a modification of two EBS sequences (such as EBS1' and EBS3') of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0390] 166. The method according to claim 164 or 165, wherein the modification is a modification of the EBS1 and / or δ sequence of group II introns, or a modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides.

[0391] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to regions of corresponding length in the target sequence. Optionally, the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein 100% of the nucleotides of the EBS1 and / or δ sequence and its upstream sequence are complementary to regions of corresponding length in the target sequence.

[0392] 167. The method according to claim 166, wherein the modification includes partial or complete deletion of domain 4, such as deletion of the protein (IEP) sequence encoded by an intron in domain 4, preferably complete deletion of domain 4; or

[0393] The modification includes the omission of the open reading frame (ORF).

[0394] 168. The method according to claim 164, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence, preferably the near-scarless circRNA having a scar region of length equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0395] 169. The method of claim 165, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0396] 170. The method according to any one of claims 151-169, wherein the group II intron is a group II intron derived from a microorganism, preferably the group II intron is Cte1, and more preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0397] 171. The method according to any one of claims 152-170, wherein the 3' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO: 42-52; and / or

[0398] The 5' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:75-88.

[0399] 172. The method according to any one of items 152-171, wherein the length of E1 and / or E2 is 0-20 nucleotides, preferably 0-10 nucleotides, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides;

[0400] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NO:53-63 and SEQ ID NO:7211-7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NO:64-74 and SEQ ID NO:7213.

[0401] 173. The method according to item 172, wherein E1, E2, or both are 0 nucleotides in length.

[0402] 174. The method according to any one of claims 152-173, wherein the target sequence is a non-coding sequence selected from the group consisting of: spacer sequences of SEQ ID NO:4-6, polyA sequences, poly-AC sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences, and protein binding sites.

[0403] 175. The method according to any one of claims 152-173, wherein the target sequence comprises: an internal ribosome entry site (IRES)-like polynucleotide sequence or a natural IRES sequence, and a protein-coding sequence, preferably the protein-coding sequence encoding a therapeutic product.

[0404] 176. The method according to any one of claims 149-175, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0405] 177. The method according to claim 176, wherein at least one of the modified RNA nucleotide and / or modified nucleoside is m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), Y (pseudouridine), m1A (1-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine), Am (2'-O-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxyn-valine) Acylcarbamoyl adenosine), ms2hn6A (2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine), Ar(p)(2'-O-ribosyl adenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), (5-formylcytidine), m5Cm (5,2'-O-dimethylcytidine), ac4Cm (N4-acetyl-2'-O-methylcytidine), k2C (lysine), m! G(1-methylguanosine), m2G(N2-methylguanosine), m7G(7-methylguanosine), Gm(2'-O-methylguanosine), m22G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(huaitin), oayW(peroxyhuaitin), OH yW (hydroxyhuaitoside), OHyW* (insufficiently modified hydroxyhuaitoside), imG (huaitoside), mimG (methylhuaitoside), Q (pigmentoside), oQ (epoxypigmentoside), galQ (galactosylpigmentoside), manQ (mannosylpigmentoside), preQo (7-cyano-7-deazoguanosine), preQi (7-aminomethyl-7-deazoguanosine), G+ (archopurinoside), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), nmm5U (5-carbamoylmethyluridine), nmm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2'-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6,2'-O-dimethyladenosine), m6 2Am (N6,N6,O-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m'Gm (l,2'-O-dimethylguanosine), m'Am (l,2'-O-dimethyladenosine), rm 5U (5-taurate methyluridine), τm5s2U (5-taurate methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), or ac6A (N6-acetyladenosine).

[0406] 178. The method according to any one of claims 152-173, wherein the precursor RNA comprises unmodified RNA nucleotides and / or unmodified nucleosides; or the precursor RNA does not comprise modified RNA nucleotides and / or modified nucleosides.

[0407] 179. The method according to any one of claims 152-178, further comprising a solid surface,

[0408] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

[0409] Further preferably, the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads, or silica beads, or

[0410] Further preferably, the solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion base materials, or an integral support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

[0411] 180. The method according to claim 179, wherein the first and second anionic probes are fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation, preferably covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0412] 181. The method according to item 179 or 180, wherein the solid surface is a magnetic bead.

[0413] Preferably, the magnetic beads are functionally modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specific groups, or combinations of two or more of the above, more preferably NHS-modified magnetic beads, or

[0414] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0415] Preferably, the magnetic beads are magnetic beads with a diameter of 0.1μm to 10μm, more preferably magnetic beads with a diameter of 0.5μm to 5μm, more preferably magnetic beads with a diameter of 0.8μm to 1.2μm, and more preferably magnetic beads with a diameter of 1μm.

[0416] 182. The method according to claim 179 or 180, wherein the solid surface is a chromatography packing material containing agarose, preferably the diameter of the chromatography packing material is 5 mm or more and the height is 20 cm or more, more preferably the diameter of the chromatography packing material is 10 mm or more and the height is 30 cm or more, and even more preferably the diameter of the chromatography packing material is 16 mm and the height is 40 cm.

[0417] Further, the agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, an agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, agarose chromatography packing material with CNBr-modified surface, agarose chromatography packing material with affinity or specificity groups, or a combination of two or more of the above, and is more preferably an NHS-modified agarose chromatography packing material.

[0418] 183. The method according to any one of claims 179-182, wherein the 3' ends of the first cathode probe and the second cathode probe are covalently fixed to the solid surface; or the 5' ends of the first cathode probe and the second cathode probe are covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0419] 184. The method according to any one of claims 179-183, wherein the 3' end and / or 5' end of the first and second anion probes are further modified with groups selected from: amino, carboxyl, NHS-carboxyl, and thiol.

[0420] 185. The method according to any one of claims 149-184, wherein the minimum free energy (MFE) values ​​of the first negative label and the second negative label are in the range of -2 to 2, and the change in MFE value (ΔMFE) of the precursor nucleic acid resulting from the inclusion of the label is in the range of -2 to 2.

[0421] 186. The method according to item 185, wherein the MFE values ​​of the first negative label and the second negative label are approximately 0.

[0422] 187. The method according to item 185 or 186, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the first negative label and the second negative label is in the range of 0-2, preferably the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the first negative label and the second negative label is about 0.

[0423] 188. The method according to any one of claims 149-187, wherein a sample comprising the purifying circular nucleic acid is contacted with the first and second negative probes at a pH of 4.0-9.0, preferably 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, or 8.5-9.0; or

[0424] The sample is in contact with the first and second cathode probes at a temperature of 0℃-60℃; or

[0425] The sample is contacted with the first and second anionic probes in a binding solution. The binding solution comprises salts, preferably further comprising buffering substances, chelating agents, and optionally stabilizers. More preferably, the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably, the concentration of the salt in the binding solution is 50 mM-5 M.

[0426] Further preferably, the salt is a cation of Li. + Or Na + Salts.

[0427] 189. The method according to claim 188, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl, preferably further comprising EDTA and Tris-HCl, and the pH range of the binding solution is 6.0-8.0, preferably 7.5.

[0428] Preferably, the method further includes the step of using an eluent to elute a portion of the first and second anionic probes bound to the solid surface to recover the solid surface coupled with the first and second anionic probes for reuse.

[0429] 190. The method according to any one of claims 149-189, wherein the concentration of salts in the eluent is 0 M, and the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably the salt being a cation of Li. + Or Na + Salts,

[0430] Preferably, the eluent further includes buffering substances, chelating agents, and optionally stabilizers.

[0431] 191. The method according to claim 190, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.0, preferably 7.5; or

[0432] The eluent has a pH range of 2.0-4.0, preferably a pH range of 2.5-3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5-3.0.

[0433] 192. The method according to any one of items 149-191, wherein the method is scalable and may include a continuous flow process, preferably performed using a liquid chromatograph (LC) or an enzyme-linked immunosorbent assay (ELISA) reader, and more preferably the LC is a rapid liquid chromatograph (FPLC) or a high-performance liquid chromatograph (HPLC).

[0434] 193. A method for purifying circular nucleic acids from a sample, comprising the following steps:

[0435] Couple the magnetic bead to the first and second cathode probes;

[0436] The IVT reaction solution containing the circular nucleic acid obtained by in vitro transcription was incubated with magnetic beads coupled with a first negative probe and a second negative probe.

[0437] The magnetic beads are collected and eluted to recover the magnetic beads coupled with the first and second anion selection probes;

[0438] Collect the unbound parts.

[0439] The first and second negative select tags are at least 90% complementary to the first and second negative select probes, respectively. The circular nucleic acid is generated by the self-splicing of a precursor nucleic acid containing a first oligonucleotide negative select tag and a second oligonucleotide negative select tag. The negative select tags are removed during the self-splicing of the precursor nucleic acid. Preferably, the first and second negative select tags are at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the first and second negative select probes, respectively. More preferably, they are 100% complementary.

[0440] 194. The method according to claim 193, wherein the IVT reaction solution is incubated with magnetic beads coupled with a first anion probe and a second anion probe in a binding solution, and the magnetic beads coupled with the first anion probe and the second anion probe are eluted and recovered using an elution solution.

[0441] 195. The method according to claim 194, wherein the eluent and binding solution are the eluent and binding solution involved in any one of claims 188-191, and more preferably the anion probe and anion label are the first anion probe and the second anion probe involved in any one of claims 149-195, and the first anion label and the second anion label.

[0442] 196. Circular nucleic acid purified by the method according to any one of items 149-195 or by using the composition comprising a first oligonucleotide negative probe and a second oligonucleotide negative probe for purifying circular nucleic acid according to any one of items 101-139 or by using the kit according to any one of items 140-148.

[0443] 197. A composition having the circular nucleic acid of claim 196, wherein the cyclization percentage (PC) of the composition is at least 90%, at least 95%, at least 98%, or at least 99%; or

[0444] The total cyclic purity of the composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, and above 95%, preferably with an polymer content below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, and below 1.5%.

[0445] Further preferably, the size of the circular nucleic acid is 1000nt-5000nt.

[0446] 198. A composition for generating circular nucleic acids, comprising:

[0447] Linearized plasmids used to generate precursor nucleic acids;

[0448] The generated precursor nucleic acid;

[0449] The generated circular nucleic acid;

[0450] Non-circular precursor nucleic acids;

[0451] Residual impurities from in vitro transcription reactions, wherein the precursor nucleic acid and circular nucleic acid are the precursor nucleic acid and circular nucleic acid involved in any of items 101-148.

[0452] The preferred composition has a total cyclic purity ranging from 20% to 50% and a polymer content ranging from 10% to 20%.

[0453] 199. The composition according to claim 198, used as a sample by any one of claims 149-196, or as a sample purified by any one of claims 101-139 using a composition comprising a first oligonucleotide negative probe and a second oligonucleotide negative probe for purifying circular nucleic acids, or using a kit according to any one of claims 140-148.

[0454] 200. The composition according to claim 199, wherein the purified cyclic nucleic acid composition has a total cyclic purity of ≥90.0%, ≥91%, ≥92%, ≥93%, ≥94%, or ≥95%.

[0455] The preferred polymer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, and below 1.5%.

[0456] Further preferably, the size of the circular nucleic acid is 1000nt-5000nt.

[0457] 201. A composition comprising an oligonucleotide positive probe for purifying circular nucleic acids, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, wherein the positive probe is at least 60% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the splice portion of the circular nucleic acid, and the precursor nucleic acid does not contain a sequence that is at least 60% complementary to the positive probe.

[0458] 202. The composition according to claim 201, wherein the positive probe is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, more preferably 100% complementary, and the precursor nucleic acid does not contain any sequence complementary to the positive probe at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, more preferably 100% complementary.

[0459] 203. The composition according to item 201 or 202, wherein the circular nucleic acid is a circular RNA (circRNA), preferably the precursor nucleic acid is RNA having group II intron self-splicing activity.

[0460] 204. The composition according to claim 203, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0461] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0462] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0463] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0464] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0465] 205. The composition according to claim 203, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a linker sequence; (d) a target sequence; (e) a linker sequence; (f) exon fragment 1 (E1); and (g) a 5' intron fragment; wherein:

[0466] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0467] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0468] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0469] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0470] 206. The composition according to claim 203, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; and (g) a 3' homologous arm; wherein:

[0471] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0472] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0473] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0474] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0475] 207. The composition according to claim 203, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a adapter sequence; (e) a target sequence; (f) an adapter sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0476] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0477] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0478] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0479] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0480] 208. The composition according to any one of items 201-207, wherein the cation-selective probe has a length of 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides, preferably 20-30 nucleotides.

[0481] 209. The composition according to any one of items 201-208, wherein the positively selected probe is an RNA probe or a DNA probe.

[0482] 210. The composition according to any one of claims 204-209, wherein the anode probe comprises a first anode probe and optionally a second anode probe.

[0483] The first positively selected probe is at least 60% complementary to the linker fragment formed by E1 and E2 of the circular nucleic acid, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, and the complementarity between the first positively selected probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%; or

[0484] The first positive probe is at least 60% complementary to at least a portion of the link formed by the circularization of the 3' and 5' ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, and the complementarity between the first positive probe and any portion of the uncirculated target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0485] 211. The composition according to any one of items 201-210, wherein the positive probe has a polynucleotide sequence comprising any one of SEQ ID NO. 7203-7206 and SEQ ID NO. 7222-7225.

[0486] 212. The composition according to claim 210, wherein the positive selector comprises a first positive selector and a second positive selector, the second positive selector being at least 60% complementary to any portion of any two side sequences adjacent to both ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, the first positive selector being less than 60% complementary to any portion of the uncircularized target sequence, preferably less than 70%, 80%, 90%, or 95%, and more preferably the sequence of the second positive selector is selected from TT, GATT, or TTTC.

[0487] 213. The composition according to any one of items 204-207, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II introns of the unpaired region into two fragments.

[0488] 214. The composition according to claim 213, wherein the 5' intron fragment and the 3' intron fragment are obtained by cleaving group II introns at the ring region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0489] The 5' and 3' intron segments are obtained by cutting group II introns in linear regions between domain 1 and domain 2, between domain 2 and domain 3, between domain 3 and domain 4, between domain 4 and domain 5, or between domain 5 and domain 6.

[0490] 215. The composition according to claim 213 or 214, wherein the group II intron comprises one or more nucleotide modifications relative to its wild-type form, and said modifications are selected from one or more deletions, substitutions, and additions.

[0491] Preferably, the modification comprises one or more EBS sequences of group II introns, wherein the EBS sequences are complementary to one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0492] 216. The composition according to claim 215, wherein the modification is a modification of two EBS sequences (e.g., EBS1 and EBS3) of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0493] 217. The composition according to claim 215, wherein the modification is a modification of two EBS sequences (e.g., EBS1' and EBS3') of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0494] 218. The composition according to claim 215 or 216, wherein the modification is a modification of the EBS1 and / or δ sequence of group II introns, or a modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides.

[0495] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to regions of corresponding length in the target sequence. Optionally, the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein 100% of the nucleotides of the EBS1 and / or δ sequence and its upstream sequence are complementary to regions of corresponding length in the target sequence.

[0496] 219. The composition according to claim 214, wherein the modification comprises partial or complete deletion of domain 4, such as deletion of the protein (IEP) sequence encoded by an intron in domain 4, preferably complete deletion of domain 4; or

[0497] The modification includes the omission of the open reading frame (ORF).

[0498] 220. The composition according to claim 216, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence, preferably the near-scarless circRNA having a scar region of length equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0499] 221. The composition according to claim 217, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0500] 222. The composition according to any one of claims 203-221, wherein the group II intron is a group II intron derived from a microorganism, preferably the group II intron is Cte1, and more preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0501] 223. The composition according to any one of claims 204-222, wherein the 3' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:42-52; and / or

[0502] The 5' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:75-88.

[0503] 224. The composition according to any one of items 204-223, wherein E1 and / or E2 is 0-20 nucleotides in length, preferably 0-10 nucleotides in length, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length;

[0504] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NO:53-63 and SEQ ID NO:7211-7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NO:64-74 and SEQ ID NO:7213.

[0505] 225. The composition according to item 224, wherein E1, E2, or both are 0 nucleotides in length.

[0506] 226. The composition according to any one of claims 204-225, wherein the target sequence is a non-coding sequence selected from the group consisting of: spacer sequences of SEQ ID NO:4-6, polyA sequences, poly-AC sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences, and protein binding sites.

[0507] 227. The composition according to any one of claims 206-225, wherein the target sequence comprises: an internal ribosome entry site (IRES)-like polynucleotide sequence or a native IRES sequence, and a protein-coding sequence, preferably the protein-coding sequence encoding a therapeutic product.

[0508] 228. The composition according to any one of claims 201-227, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0509] 229. The composition according to claim 228, wherein at least one of the modified RNA nucleotide and / or modified nucleoside is m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), Y (pseudouridine), m1A (1-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine), Am (2'-O-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxyn-valine) Acylcarbamoyl adenosine), ms2hn6A (2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine), Ar(p)(2'-O-ribosyl adenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), (5-formylcytidine), m5Cm (5,2'-O-dimethylcytidine), ac4Cm (N4-acetyl-2'-O-methylcytidine), k2C (lysine), m! G(1-methylguanosine), m2G(N2-methylguanosine), m7G(7-methylguanosine), Gm(2'-O-methylguanosine), m22G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(huaitin), oayW(peroxyhuaitin), OH yW (hydroxyhuaitoside), OHyW* (insufficiently modified hydroxyhuaitoside), imG (huaitoside), mimG (methylhuaitoside), Q (pigmentoside), oQ (epoxypigmentoside), galQ (galactosylpigmentoside), manQ (mannosylpigmentoside), preQo (7-cyano-7-deazoguanosine), preQi (7-aminomethyl-7-deazoguanosine), G+ (archopurinoside), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), nmm5U (5-carbamoylmethyluridine), nmm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2'-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6,2'-O-dimethyladenosine), m6 2Am (N6,N6,O-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m'Gm (l,2'-O-dimethylguanosine), m'Am (l,2'-O-dimethyladenosine), rm 5U (5-taurate methyluridine), τm5s2U (5-taurate methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), or ac6A (N6-acetyladenosine).

[0510] 230. The composition according to any one of claims 201-227, wherein the precursor RNA comprises unmodified RNA nucleotides and / or unmodified nucleosides; or the precursor RNA does not comprise modified RNA nucleotides and / or modified nucleosides.

[0511] 231. The composition according to any one of claims 201-230, further comprising a solid surface,

[0512] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

[0513] Further preferably, the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads, or silica beads, or

[0514] Further preferably, the solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion base materials, or an integral support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

[0515] 232. The composition according to claim 231, wherein the cation-selective probe is fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation, preferably covalent fixation is achieved by covalent linkage of NH2 and NHS-modified carboxyl groups.

[0516] 233. The composition according to item 231 or 232, wherein the solid surface is a magnetic bead.

[0517] Preferably, the magnetic beads are functionally modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specific groups, or combinations of two or more of the above, more preferably NHS-modified magnetic beads, or

[0518] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0519] Preferably, the magnetic beads are magnetic beads with a diameter of 0.1μm to 10μm, more preferably magnetic beads with a diameter of 0.5μm to 5μm, more preferably magnetic beads with a diameter of 0.8μm to 1.2μm, and more preferably magnetic beads with a diameter of 1μm.

[0520] 234. The composition according to claim 231 or 232, wherein the solid surface is an agarose chromatography packing material.

[0521] Preferably, the diameter of the chromatography packing material is 5 mm or more, and the height is 20 cm or more; more preferably, the diameter of the chromatography packing material is 10 mm or more, and the height is 30 cm or more; even more preferably, the diameter of the chromatography packing material is 16 mm, and the height is 40 cm.

[0522] Further, the agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, an agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, agarose chromatography packing material with CNBr-modified surface, agarose chromatography packing material with affinity or specificity groups, or a combination of two or more of the above, and is more preferably an NHS-modified agarose chromatography packing material.

[0523] 235. The composition according to any one of items 231-234, wherein the 3' end of the anode probe is covalently fixed to the solid surface; or the 5' end of the anode probe is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0524] 236. The composition according to any one of claims 231-235, wherein the 3' end and / or 5' end of the anodic probe is further modified with a group selected from amino, carboxyl, NHS-carboxyl, and thiol.

[0525] 237. The composition according to claim 235, wherein the anode probe comprises a second anode probe, a first anode probe and a second anode probe connected in sequence, wherein the 3' end of one of the second anode probes is covalently fixed to the solid surface; or the 5' end of another second anode probe is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved by covalent connection of NH2 and NHS modified carboxyl groups.

[0526] 238. The composition according to claim 236, wherein the anode probe comprises a second anode probe, a first anode probe and a second anode probe connected in sequence, wherein the 3' end of one second anode probe and / or the 5' end of the other second anode probe is further modified with a group selected from: amino, carboxyl, NHS-carboxyl, mercapto.

[0527] 239. The composition according to item 237 or 238, wherein the positive probe has a polynucleotide sequence comprising any one of SEQ ID NO. 7205-7206.

[0528] 240. A positive selection kit for purifying circular nucleic acids, comprising: a composition containing an oligonucleotide positive selection probe for purifying circular nucleic acids as described in any one of claims 201-239, a binding solution, and an elution solution.

[0529] 241. The kit according to claim 240, wherein the binding solution comprises a salt, the salt comprising a cation selected from the group consisting of: Ba 2+ Ca 2+ Mg2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably, the concentration of the salt in the binding solution is 50 mM-5 M.

[0530] Further preferably, the salt is a cation of Li. + Or Na + Salts.

[0531] 242. The kit according to claim 241, wherein the pH range of the binding buffer is 4.0-9.0, preferably 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, or 8.5-9.0;

[0532] Preferably, the binding solution further includes a buffering substance, a chelating agent, and optionally a stabilizer.

[0533] 243. The kit according to item 241 or 242, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl, preferably further comprising EDTA and Tris-HCl, and the pH range of the binding solution is 6.0-8.0, preferably 7.5.

[0534] 244. The kit according to any one of claims 240-243, wherein the concentration of salts in the eluent is 0M, and the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably the salt being a cation of Li. + Or Na + Salts,

[0535] Preferably, the eluent further includes buffering substances, chelating agents, and optionally stabilizers.

[0536] 245. The kit according to claim 244, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.0, preferably 7.5; or

[0537] The eluent has a pH range of 2.0-4.0, preferably a pH range of 2.5-3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5-3.0.

[0538] 246. A positive selection kit for purifying circular nucleic acids, comprising: magnetic beads coupled with a positive selection probe, an elution buffer, and a binding buffer, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, the positive selection probe is at least 60% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the splice portion of the circular nucleic acid, and the precursor nucleic acid does not contain a sequence at least 60% complementary to the positive selection probe.

[0539] 247. The kit according to item 246, wherein the positive probe is a positive probe as described in any of items 201-239, or the elution buffer and binding buffer are elution buffers and binding buffers as described in any of items 240-245.

[0540] 248. The kit according to item 246 or 247, wherein the magnetic bead with the immobilized anode probe is reusable.

[0541] 249. A method for purifying circular nucleic acids from a sample, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, the method comprising:

[0542] (i) Under conditions allowing the circular nucleic acid generated by the self-splicing of the precursor nucleic acid to bind to the positive probe, a sample comprising the circular nucleic acid generated by the self-splicing of the precursor nucleic acid is brought into contact with a positive probe that is at least 60% complementary to at least a portion of the circular nucleic acid, wherein the positive probe is fixed to a solid surface; and

[0543] (ii) Collect the portion of the anode probe that has bonded to the solid surface.

[0544] At least a portion of the circular nucleic acid spans the splice region of the circular nucleic acid, and the precursor nucleic acid does not contain a sequence that is at least 60% complementary to the positive probe.

[0545] 250. The method according to claim 249, wherein the positive probe is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, more preferably 100% complementary, and the precursor nucleic acid does not contain a sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the positive probe, more preferably 100% complementary.

[0546] 251. The method according to item 249 or 250, wherein the circular nucleic acid is a circular RNA (circRNA), preferably the precursor nucleic acid is RNA having group II intron self-splicing activity.

[0547] 252. The method according to claim 251, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked together: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0548] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0549] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0550] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0551] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0552] 253. The method according to claim 251, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a adapter sequence; (d) a target sequence; (e) an adapter sequence; (f) exon fragment 1 (E1); and (g) a 5' intron fragment; wherein:

[0553] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0554] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0555] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0556] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0557] 254. The method according to claim 251, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; and (g) a 3' homologous arm; wherein:

[0558] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0559] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0560] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0561] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0562] 255. The method according to claim 251, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a adapter sequence; (e) a target sequence; (f) an adapter sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0563] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0564] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0565] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0566] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0567] 256. The method according to any one of items 249-255, wherein the length of the positive probe is 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides, preferably 20-30 nucleotides.

[0568] 257. The method according to any one of items 249-256, wherein the positively selected probe is an RNA probe or a DNA probe.

[0569] 258. The method according to any one of claims 251-257, wherein the anode probe comprises a first anode probe and optionally a second anode probe.

[0570] The first positively selected probe is at least 60% complementary to the linker fragment formed by E1 and E2 of the circular nucleic acid, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, and the complementarity between the first positively selected probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%; or

[0571] The first positive probe is at least 60% complementary to at least a portion of the link formed by the circularization of the 3' and 5' ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, and the complementarity between the first positive probe and any portion of the uncirculated target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0572] 259. The method according to any one of claims 249-258, wherein the positive probe has a polynucleotide sequence comprising any one of SEQ ID NO. 7203-7206 and SEQ ID NO. 7222-7225.

[0573] 260. The method according to claim 259, wherein the positive selection probe comprises a first positive selection probe and a second positive selection probe, the second positive selection probe being at least 60% complementary to any portion of any two sides of the sequence adjacent to both ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, the first positive selection probe being less than 60% complementary to any portion of the uncirculated target sequence, preferably less than 70%, 80%, 90%, or 95%, and more preferably the sequence of the second positive selection probe is selected from TT, GATT, or TTTC.

[0574] 261. The method according to any one of items 249-260, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II introns of the unpaired region into two fragments.

[0575] 262. The method according to claim 261, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting group II introns at the loop region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0576] The 5' and 3' intron segments are obtained by cutting group II introns in linear regions between domain 1 and domain 2, between domain 2 and domain 3, between domain 3 and domain 4, between domain 4 and domain 5, or between domain 5 and domain 6.

[0577] 263. The method according to claim 261 or 262, wherein the group II intron comprises one or more nucleotide modifications relative to its wild-type form, and the modifications are selected from one or more deletions, substitutions, and additions.

[0578] Preferably, the modification comprises one or more EBS sequences of group II introns, wherein the EBS sequences are complementary to one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0579] 264. The method according to item 263, wherein the modification is a modification of two EBS sequences (such as EBS1 and EBS3) of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0580] 265. The method according to item 263, wherein the modification is a modification of two EBS sequences (such as EBS1' and EBS3') of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0581] 266. The method according to claim 264 or 265, wherein the modification is a modification of the EBS1 and / or δ sequence of group II introns, or a modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides.

[0582] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to regions of corresponding length in the target sequence. Optionally, the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein 100% of the nucleotides of the EBS1 and / or δ sequence and its upstream sequence are complementary to regions of corresponding length in the target sequence.

[0583] 267. The method according to claim 266, wherein the modification includes partial or complete deletion of domain 4, such as deletion of the protein (IEP) sequence encoded by an intron in domain 4, preferably complete deletion of domain 4; or

[0584] The modification includes the omission of the open reading frame (ORF).

[0585] 268. The method according to claim 264, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence, preferably the near-scarless circRNA having a scar region of length equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0586] 269. The method of claim 265, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0587] 270. The method according to any one of claims 251-269, wherein the group II intron is a group II intron derived from a microorganism, preferably the group II intron is Cte1, and more preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0588] 271. The method according to any one of claims 252-270, wherein the 3' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO: 42-52; and / or

[0589] The 5' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:75-88.

[0590] 272. The method according to any one of items 252-271, wherein the length of E1 and / or E2 is 0-20 nucleotides, preferably 0-10 nucleotides, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides;

[0591] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NO:53-63 and SEQ ID NO:7211-7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NO:64-74 and SEQ ID NO:7213.

[0592] 273. The method according to item 272, wherein E1, E2, or both are 0 nucleotides in length.

[0593] 274. The method according to any one of claims 252-273, wherein the target sequence is a non-coding sequence selected from the group consisting of: spacer sequences of SEQ ID NO:4-6, polyA sequences, poly-AC sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitch, ribozymes other than autosplicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences, and protein binding sites.

[0594] 275. The method according to any one of claims 252-273, wherein the target sequence comprises: an internal ribosome entry site (IRES)-like polynucleotide sequence or a natural IRES sequence, and a protein-coding sequence, preferably the protein-coding sequence encoding a therapeutic product.

[0595] 276. The method according to any one of claims 249-275, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0596] 277. The method according to claim 276, wherein at least one of the modified RNA nucleotide and / or modified nucleoside is m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), Y (pseudouridine), m1A (1-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine), Am (2'-O-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxyn-valine) Acylcarbamoyl adenosine), ms2hn6A (2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine), Ar(p)(2'-O-ribosyl adenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), (5-formylcytidine), m5Cm (5,2'-O-dimethylcytidine), ac4Cm (N4-acetyl-2'-O-methylcytidine), k2C (lysine), m! G(1-methylguanosine), m2G(N2-methylguanosine), m7G(7-methylguanosine), Gm(2'-O-methylguanosine), m22G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(huaitin), oayW(peroxyhuaitin), OH yW (hydroxyhuaitoside), OHyW* (insufficiently modified hydroxyhuaitoside), imG (huaitoside), mimG (methylhuaitoside), Q (pigmentoside), oQ (epoxypigmentoside), galQ (galactosylpigmentoside), manQ (mannosylpigmentoside), preQo (7-cyano-7-deazoguanosine), preQi (7-aminomethyl-7-deazoguanosine), G+ (archopurinoside), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), nmm5U (5-carbamoylmethyluridine), nmm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2'-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6,2'-O-dimethyladenosine), m6 2Am (N6,N6,O-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m'Gm (l,2'-O-dimethylguanosine), m'Am (l,2'-O-dimethyladenosine), rm 5U (5-taurate methyluridine), τm5s2U (5-taurate methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), or ac6A (N6-acetyladenosine).

[0597] 278. The method according to any one of claims 252-273, wherein the precursor RNA comprises unmodified RNA nucleotides and / or unmodified nucleosides; or the precursor RNA does not comprise modified RNA nucleotides and / or modified nucleosides.

[0598] 279. The method according to any one of items 252-278, further comprising a solid surface,

[0599] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

[0600] Further preferably, the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads, or silica beads, or

[0601] Further preferably, the solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion base materials, or an integral support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

[0602] 280. The method according to claim 279, wherein the cation-selective probe is fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation, preferably covalent fixation is achieved by covalent linkage of NH2 and NHS-modified carboxyl groups.

[0603] 281. The method according to item 279 or 280, wherein the solid surface is a magnetic bead.

[0604] Preferably, the magnetic beads are functionally modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specific groups, or combinations of two or more of the above, more preferably NHS-modified magnetic beads, or

[0605] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0606] Preferably, the magnetic beads are magnetic beads with a diameter of 0.1μm to 10μm, more preferably magnetic beads with a diameter of 0.5μm to 5μm, more preferably magnetic beads with a diameter of 0.8μm to 1.2μm, and more preferably magnetic beads with a diameter of 1μm.

[0607] 282. The method according to claim 279 or 280, wherein the solid surface is a chromatography packing material containing agarose, preferably the diameter of the chromatography packing material is 5 mm or more and the height is 20 cm or more, more preferably the diameter of the chromatography packing material is 10 mm or more and the height is 30 cm or more, and even more preferably the diameter of the chromatography packing material is 16 mm and the height is 40 cm.

[0608] Further, the agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, an agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, agarose chromatography packing material with CNBr-modified surface, agarose chromatography packing material with affinity or specificity groups, or a combination of two or more of the above, and is more preferably an NHS-modified agarose chromatography packing material.

[0609] 283. The method according to any one of items 279-282, wherein the 3' end of the anode probe is covalently fixed to the solid surface; or the 5' end of the anode probe is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS modified carboxyl groups.

[0610] 284. The method according to any one of claims 279-283, wherein the 3' end and / or 5' end of the anodic probe is further modified with a group selected from: amino, carboxyl, NHS-carboxyl, mercapto.

[0611] 285. The method according to claim 283, wherein the anode probe comprises a second anode probe, a first anode probe and a second anode probe connected in sequence, wherein the 3' end of one second anode probe is covalently fixed to the solid surface; or the 5' end of another second anode probe is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved by covalent connection of NH2 and NHS modified carboxyl groups.

[0612] 286. The method according to claim 284, wherein the cation selector comprises a second cation selector, a first cation selector, and a second cation selector connected in sequence, wherein the 3' end of one second cation selector and / or the 5' end of the other second cation selector is further modified with a group selected from: amino, carboxyl, NHS-carboxyl, mercapto.

[0613] 287. The method according to claim 285 or 286, wherein the positive probe has a polynucleotide sequence comprising any one of SEQ ID NO. 7205-7206.

[0614] 288. The method according to any one of claims 249-287, wherein a sample comprising the purifying circular nucleic acid is contacted with the positivistic probe at a pH of 4.0-9.0, preferably 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, or 8.5-9.0; or

[0615] The sample is in contact with the anode probe at a temperature of 0℃-60℃; or

[0616] The sample is in contact with the cation-selected probe in a binding solution, the binding solution comprising salts, preferably further comprising buffering substances, chelating agents, optionally including stabilizers, and more preferably comprising cations selected from the group consisting of Ba. 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably, the concentration of the salt in the binding solution is 50 mM-5 M.

[0617] Further preferably, the salt is a cation of Li. + Or Na + Salts.

[0618] 289. The method according to claim 288, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl, preferably further comprising EDTA and Tris-HCl, and the pH range of the binding solution is 6.0-8.0, preferably 7.5.

[0619] Preferably, the method further includes the step of using an eluent to elute a portion of the anode probe bound to the solid surface to recover the solid surface coupled with the anode probe for reuse.

[0620] 290. The method according to any one of claims 249-289, wherein the concentration of salts in the eluent is 0 M, and the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably the salt being a cation of Li. + Or Na + Salts,

[0621] Preferably, the eluent further includes buffering substances, chelating agents, and optionally stabilizers.

[0622] 291. The method according to claim 290, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.0, preferably 7.5; or

[0623] The eluent has a pH range of 2.0-4.0, preferably a pH range of 2.5-3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5-3.0.

[0624] 292. The method according to any one of items 249-291, wherein the method is scalable and may include a continuous flow process, preferably performed using a liquid chromatograph (LC) or an enzyme-linked immunosorbent assay (ELISA) reader, and more preferably the LC is a rapid liquid chromatograph (FPLC) or a high-performance liquid chromatograph (HPLC).

[0625] 293. A method for purifying circular nucleic acids from a sample, comprising the following steps:

[0626] Couple the magnetic bead with the anode probe;

[0627] The IVT reaction solution containing the circular nucleic acid obtained by in vitro transcription was incubated with magnetic beads coupled with a positively selected probe.

[0628] The magnetic beads are collected and eluted to obtain the eluted circular nucleic acid, and the magnetic beads coupled with the positive probe are recovered.

[0629] The circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, wherein the positive probe is at least 60% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the splice portion of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive probe.

[0630] 294. The method according to claim 293, wherein the IVT reaction solution is incubated with magnetic beads coupled with a positive probe in a binding solution, the magnetic beads are eluted with an elution buffer to obtain the eluted circular nucleic acid, and the magnetic beads coupled with the positive probe are recovered.

[0631] 295. The method according to claim 294, wherein the eluent and binding solution are the eluent and binding solution involved in any one of claims 288-291, and more preferably the anode probe is the anode probe involved in any one of claims 249-292.

[0632] 296. Circular nucleic acid purified by the method according to any one of items 249-295, or by using the composition comprising an oligonucleotide positive probe for purifying circular nucleic acid according to any one of items 201-239, or by using the kit according to any one of items 240-248.

[0633] 297. A composition having the circular nucleic acid of claim 296, wherein the cyclization percentage (PC) of the composition is at least 90%, at least 95%, at least 98%, or at least 99%; or

[0634] The total cyclic purity of the composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, and above 95%, preferably with an polymer content below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, and below 1.5%.

[0635] Further preferably, the size of the circular nucleic acid is 1000nt-5000nt.

[0636] 298. A composition for generating circular nucleic acids, comprising:

[0637] Linearized plasmids used to generate precursor nucleic acids;

[0638] The generated precursor nucleic acid;

[0639] The generated circular nucleic acid;

[0640] Non-circular precursor nucleic acids;

[0641] Residual impurities from in vitro transcription reactions, wherein the precursor nucleic acid and circular nucleic acid are the precursor nucleic acid and circular nucleic acid involved in any of items 201-248.

[0642] The preferred composition has a total cyclic purity ranging from 20% to 50% and a polymer content ranging from 10% to 20%.

[0643] 299. The composition according to item 298, used as a sample by any one of items 249-296, or purified as a sample by any one of items 201-239 using a composition comprising an oligonucleotide positive probe for purifying circular nucleic acids, or purified using any one of items 240-248.

[0644] 300. The composition according to claim 299, wherein the purified cyclic nucleic acid composition has a total cyclic purity of ≥90.0%, ≥91%, ≥92%, ≥93%, ≥94%, or ≥95%.

[0645] The preferred polymer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, and below 1.5%.

[0646] Further preferably, the size of the circular nucleic acid is 1000nt-5000nt.

[0647] 301. A composition comprising oligonucleotide probes for purifying circular nucleic acids, wherein the probes comprise negatively selected probes and positively selected probes.

[0648] The circular nucleic acid is generated by the self-splicing of a precursor nucleic acid containing an oligonucleotide negative selector tag, wherein the negative selector tag is at least 90% complementary to the negative selector probe, and wherein the negative selector tag is removed during the self-splicing of the precursor nucleic acid. Preferably, the negative selector tag is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the negative selector probe, more preferably 100% complementary.

[0649] The positive probe is at least 60% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the splice portion of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive probe.

[0650] 302. The composition according to claim 301, wherein the precursor nucleic acid includes the negative select tag at its 5' end; and / or the precursor nucleic acid includes the negative select tag at its 3' end.

[0651] When both the 5' end and the 3' end negative selection label are present, the 5' end negative selection label and the 3' end negative selection label can be the same or different.

[0652] Preferably, the positive probe is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, more preferably 100% complementary, and the precursor nucleic acid does not contain any sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the positive probe, more preferably 100% complementary.

[0653] 303. The composition according to claim 301 or 302, wherein the circular nucleic acid is a circular RNA (circRNA), preferably the precursor nucleic acid is RNA having group II intron self-splicing activity.

[0654] 304. The composition according to claim 303, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked together: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0655] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0656] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0657] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0658] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0659] 305. The composition according to claim 303, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a adapter sequence; (d) a target sequence; (e) an adapter sequence; (f) exon fragment 1 (E1); and (g) a 5' intron fragment; wherein:

[0660] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0661] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0662] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0663] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0664] 306. The composition according to claim 303, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; and (g) a 3' homologous arm; wherein:

[0665] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0666] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0667] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0668] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0669] 307. The composition according to claim 303, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a linker sequence; (e) a target sequence; (f) a linker sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0670] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0671] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0672] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0673] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0674] 308. The composition according to any one of claims 301-307, wherein the length of the negative tag and the negative probe is 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides; preferably, the length of the negative tag and the negative probe is 20-30 nucleotides;

[0675] The length of the positive selection probe is 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides, preferably 20-30 nucleotides.

[0676] 309. The composition according to any one of claims 301-308, wherein the negative probe is an RNA probe; or the negative probe is a DNA probe; and the positive probe is an RNA probe or a DNA probe.

[0677] 310. The composition according to any one of claims 301-309, wherein the negative select tag has a polynucleotide sequence comprising SEQ ID NO: n, and the negative select probe has a polynucleotide sequence comprising SEQ ID NO: n+1100, wherein n is an integer from 135 to 1234; or wherein the negative select tag has a polynucleotide sequence comprising SEQ ID NO: m, and the negative select probe has a polynucleotide sequence comprising SEQ ID NO: m+1210, wherein m is an integer from 2335 to 3544 or from 4755 to 5964.

[0678] Preferably, the positive selection probe includes a first positive selection probe and an optional second positive selection probe.

[0679] The first positively selected probe is at least 60% complementary to the linker fragment formed by E1 and E2 of the circular nucleic acid, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, and the complementarity between the first positively selected probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%; or

[0680] The first positive probe is at least 60% complementary to at least a portion of the link formed by the circularization of the 3' and 5' ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, and the complementarity between the first positive probe and any portion of the uncirculated target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0681] 311. The composition according to any one of claims 301-309, wherein the negative label has a polynucleotide sequence comprising any one of SEQ ID NO: 7189-7202; or the negative probe has a polynucleotide sequence comprising any one of SEQ ID NO: 7175-7188.

[0682] Preferably, the positive selection probe has a polynucleotide sequence comprising any one of SEQ ID NO.7203-7206 and SEQ ID NO.7222-7225.

[0683] 312. The composition according to claim 311, wherein the negative label and negative probe of the composition are selected from any of the following combinations:

[0684] (a) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7189, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7175;

[0685] (b) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7190, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7176;

[0686] (c) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7191, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7177;

[0687] (d) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7192, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7178;

[0688] (e) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7193, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7179;

[0689] (f) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7194, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7180;

[0690] (g) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7195, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7181;

[0691] (h) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7196, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7182;

[0692] (i) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7197, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7183;

[0693] (j) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7198, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7184;

[0694] (k) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7199, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7185;

[0695] (l) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7200, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7186;

[0696] (m) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7201, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7187;

[0697] (n) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7202, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7188.

[0698] Preferably, the positive selection probe includes a first positive selection probe and a second positive selection probe. The second positive selection probe is at least 60% complementary to any portion of any two sequences on the outer sides near both ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary. The complementarity between the first positive selection probe and any portion of the uncirculated target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%. More preferably, the sequence of the second positive selection probe is selected from TT, GATT, or TTTC.

[0699] 313. The composition according to any one of items 304-307, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II introns of the unpaired region into two fragments.

[0700] 314. The composition according to claim 313, wherein the 5' intron fragment and the 3' intron fragment are obtained by cleaving group II introns at the ring region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0701] The 5' and 3' intron segments are obtained by cutting group II introns in linear regions between domain 1 and domain 2, between domain 2 and domain 3, between domain 3 and domain 4, between domain 4 and domain 5, or between domain 5 and domain 6.

[0702] 315. The composition according to claim 313 or 314, wherein the group II introns comprise one or more nucleotide modifications relative to their wild-type form, and said modifications are selected from one or more deletions, substitutions, and additions.

[0703] Preferably, the modification comprises one or more EBS sequences of group II introns, wherein the EBS sequences are complementary to one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0704] 316. The composition according to claim 315, wherein the modification is a modification of two EBS sequences (such as EBS1 and EBS3) of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0705] 317. The composition according to claim 315, wherein the modification is a modification of two EBS sequences (e.g., EBS1' and EBS3') of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0706] 318. The composition according to claim 315 or 316, wherein the modification is a modification of the EBS1 and / or δ sequence of group II introns, or a modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides.

[0707] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to regions of corresponding length in the target sequence. Optionally, the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein 100% of the nucleotides of the EBS1 and / or δ sequence and its upstream sequence are complementary to regions of corresponding length in the target sequence.

[0708] 319. The composition according to claim 314, wherein the modification comprises partial or complete deletion of domain 4, such as deletion of the protein (IEP) sequence encoded by an intron in domain 4, preferably complete deletion of domain 4; or

[0709] The modification includes the omission of the open reading frame (ORF).

[0710] 320. The composition according to claim 316, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence, preferably the near-scarless circRNA having a scar region of length equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0711] 321. The composition according to claim 317, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0712] 322. The composition according to any one of claims 303-321, wherein the group II intron is a group II intron derived from a microorganism, preferably the group II intron is Cte1, and more preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0713] 323. The composition according to any one of claims 304-322, wherein the 3' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:42-52; and / or

[0714] The 5' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:75-88.

[0715] 324. The composition according to any one of items 304-323, wherein E1 and / or E2 are 0-20 nucleotides in length, preferably 0-10 nucleotides in length, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length;

[0716] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NO:53-63 and SEQ ID NO:7211-7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NO:64-74 and SEQ ID NO:7213.

[0717] 325. The composition according to claim 324, wherein E1, E2, or both are 0 nucleotides in length.

[0718] 326. The composition according to any one of claims 304-325, wherein the target sequence is a non-coding sequence selected from the group consisting of: spacer sequences of SEQ ID NO:4-6, polyA sequences, poly-AC sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences, and protein binding sites.

[0719] 327. The composition according to any one of claims 306-325, wherein the target sequence comprises: an internal ribosome entry site (IRES)-like polynucleotide sequence or a native IRES sequence, and a protein-coding sequence, preferably the protein-coding sequence encoding a therapeutic product.

[0720] 328. The composition according to any one of claims 301-327, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0721] 329. The composition according to claim 328, wherein at least one of the modified RNA nucleotide and / or modified nucleoside is m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), Y (pseudouridine), m1A (1-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine), Am (2'-O-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxyn-valine) Acylcarbamoyl adenosine), ms2hn6A (2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine), Ar(p)(2'-O-ribosyl adenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), (5-formylcytidine), m5Cm (5,2'-O-dimethylcytidine), ac4Cm (N4-acetyl-2'-O-methylcytidine), k2C (lysine), m! G(1-methylguanosine), m2G(N2-methylguanosine), m7G(7-methylguanosine), Gm(2'-O-methylguanosine), m22G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(huaitin), oayW(peroxyhuaitin), OH yW (hydroxyhuaitoside), OHyW* (insufficiently modified hydroxyhuaitoside), imG (huaitoside), mimG (methylhuaitoside), Q (pigmentoside), oQ (epoxypigmentoside), galQ (galactosylpigmentoside), manQ (mannosylpigmentoside), preQo (7-cyano-7-deazoguanosine), preQi (7-aminomethyl-7-deazoguanosine), G+ (archopurinoside), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), nmm5U (5-carbamoylmethyluridine), nmm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2'-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6,2'-O-dimethyladenosine), m6 2Am (N6,N6,O-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m'Gm (l,2'-O-dimethylguanosine), m'Am (l,2'-O-dimethyladenosine), rm 5U (5-taurate methyluridine), τm5s2U (5-taurate methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), or ac6A (N6-acetyladenosine).

[0722] 330. The composition according to any one of claims 301-327, wherein the precursor RNA comprises unmodified RNA nucleotides and / or unmodified nucleosides; or the precursor RNA does not comprise modified RNA nucleotides and / or modified nucleosides.

[0723] 331. The composition according to any one of claims 301-330, further comprising a solid surface,

[0724] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

[0725] Further preferably, the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads, or silica beads, or

[0726] Further preferably, the solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion base materials, or an integral support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

[0727] 332. The composition according to claim 331, wherein the anion probe is fixed on the solid surface by physical adsorption, covalent fixation, or affinity fixation, preferably covalent fixation is achieved by covalent linkage of NH2 and NHS-modified carboxyl groups.

[0728] The cation selection probe is fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation, preferably by covalent connection of NH2 and NHS modified carboxyl groups.

[0729] 333. The composition according to claim 331 or 332, wherein the solid surface is a magnetic bead.

[0730] Preferably, the magnetic beads are functionally modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specific groups, or combinations of two or more of the above, more preferably NHS-modified magnetic beads, or

[0731] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0732] Preferably, the magnetic beads are magnetic beads with a diameter of 0.1μm to 10μm, more preferably magnetic beads with a diameter of 0.5μm to 5μm, more preferably magnetic beads with a diameter of 0.8μm to 1.2μm, and more preferably magnetic beads with a diameter of 1μm.

[0733] 334. The composition according to claim 331 or 332, wherein the solid surface is an agarose chromatography packing material.

[0734] Preferably, the diameter of the chromatography packing material is 5 mm or more, and the height is 20 cm or more; more preferably, the diameter of the chromatography packing material is 10 mm or more, and the height is 30 cm or more; even more preferably, the diameter of the chromatography packing material is 16 mm, and the height is 40 cm.

[0735] Further, the agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, an agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, agarose chromatography packing material with CNBr-modified surface, agarose chromatography packing material with affinity or specificity groups, or a combination of two or more of the above, and is more preferably an NHS-modified agarose chromatography packing material.

[0736] 335. The composition according to any one of claims 331-334, wherein the 3' end of the anion probe is covalently fixed to the solid surface; or the 5' end of the anion probe is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved through the covalent connection of NH2 and NHS-modified carboxyl groups.

[0737] The 3' end of the anode probe is covalently fixed to the solid surface; or the 5' end of the anode probe is covalently fixed to the solid surface. Preferably, the solid surface is a magnetic bead, and the covalent fixation is preferably achieved through the covalent connection of NH2 and NHS-modified carboxyl groups.

[0738] The anion probe and the cation probe are fixed on different solid surfaces.

[0739] 336. The composition according to any one of claims 331-335, wherein the 3' end and / or 5' end of the anion probe is further modified with a group selected from: amino, carboxyl, NHS-carboxyl, mercapto.

[0740] The 3' and / or 5' ends of the cation-selective probe are further modified with groups selected from the following: amino, carboxyl, NHS-carboxyl, and thiol.

[0741] 337. The composition according to any one of claims 301-336, wherein the minimum free energy (MFE) value of the negative tag ranges from -2 to 2, and the change in MFE value (ΔMFE) of the precursor nucleic acid resulting from the inclusion of the tag ranges from -2 to 2.

[0742] Preferably, the anode probe includes a second anode probe, a first anode probe, and a second anode probe connected in sequence, wherein the 3' end of one second anode probe is covalently fixed to the solid surface; or the 5' end of another second anode probe is covalently fixed to the solid surface. Preferably, the solid surface is a magnetic bead, and the covalent fixation is preferably achieved through the covalent connection of NH2 and NHS modified carboxyl groups.

[0743] 338. The composition according to claim 337, wherein the MFE value of the negative label is approximately 0.

[0744] Preferably, the cation selection probe comprises a second cation selection probe, a first cation selection probe, and a second cation selection probe connected in sequence, wherein the 3' end of one second cation selection probe and / or the 5' end of the other second cation selection probe are further modified with groups selected from the following: amino, carboxyl, NHS-carboxyl, and thiol.

[0745] 339. The composition according to claim 337 or 338, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag is in the range of 0-2, preferably approximately 0.

[0746] Preferably, the positive selection probe has a polynucleotide sequence comprising any one of SEQ ID NO.7205-7206.

[0747] 340. A negative selection kit for purifying circular nucleic acids, comprising: a composition containing oligonucleotide probes for purifying circular nucleic acids as described in any one of claims 301-339, a binding buffer, and an elution buffer.

[0748] 341. The kit according to claim 340, wherein the binding solution comprises a salt, the salt comprising a cation selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br- NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably, the concentration of the salt in the binding solution is 50 mM-5 M.

[0749] Further preferably, the salt is a cation of Li. + Or Na + Salts.

[0750] 342. The kit according to claim 341, wherein the pH range of the binding buffer is 4.0-9.0, preferably 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, or 8.5-9.0;

[0751] Preferably, the binding solution further includes a buffering substance, a chelating agent, and optionally a stabilizer.

[0752] 343. The kit according to item 341 or 342, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl, preferably the binding solution further comprises EDTA and Tris-HCl, and the pH range of the binding solution is 6.0-8.0, preferably 7.5.

[0753] 344. The kit according to any one of claims 340-343, wherein the concentration of salts in the eluent is 0M, and the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably the salt being a cation of Li.+ Or Na + Salts,

[0754] Preferably, the eluent further includes buffering substances, chelating agents, and optionally stabilizers.

[0755] 345. The kit according to claim 344, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.0, preferably 7.5; or

[0756] The eluent has a pH range of 2.0-4.0, preferably a pH range of 2.5-3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5-3.0.

[0757] 346. A kit for purifying circular nucleic acids, comprising: magnetic beads coupled with a negative probe, magnetic beads coupled with a positive probe, elution buffer, and binding buffer.

[0758] The negative selection probe is at least 90% complementary to the negative selection tag. The circular nucleic acid is generated by the self-splicing of a precursor nucleic acid containing an oligonucleotide negative selection tag, wherein the negative selection tag is removed during the self-splicing of the precursor nucleic acid. Preferably, the negative selection tag is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the negative selection probe, and more preferably 100% complementary.

[0759] The positive probe is at least 60% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the splice portion of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive probe.

[0760] 347. The kit according to claim 346, wherein the negative probe and negative label are the negative probe, negative label, and positive probe as described in any one of claims 301-339, or

[0761] The eluent and binding fluid are the eluent and binding fluids involved in any of items 340-345.

[0762] 348. The kit according to item 346 or 347, wherein the magnetic bead with the anion probe immobilized is reusable, and the magnetic bead with the anode probe immobilized is reusable.

[0763] 349. A method for purifying circular nucleic acids from a sample, the circular nucleic acids being generated by the self-splicing of a precursor nucleic acid, wherein the precursor nucleic acid has an oligonucleotide negative select tag, the oligonucleotide negative select tag being removed during the self-splicing process, the method comprising:

[0764] (i) Under conditions that allow the negative label to bind to the negative probe, the sample is brought into contact with an oligonucleotide negative probe that is at least 60% complementary to the negative label, wherein the negative probe is fixed on a solid surface;

[0765] (ii) Collect the unbound portion of the sample;

[0766] (iii) Under conditions allowing the circular nucleic acid generated by the self-splicing of the precursor nucleic acid to bind to the positive probe, a sample comprising the circular nucleic acid generated by the self-splicing of the precursor nucleic acid is brought into contact with a positive probe that is at least 60% complementary to at least a portion of the circular nucleic acid, wherein the positive probe is fixed to a solid surface; and

[0767] (iv) Collect the portion of the anode probe that has bonded to the solid surface.

[0768] At least a portion of the circular nucleic acid spans the splice region of the circular nucleic acid, and the precursor nucleic acid does not contain a sequence that is at least 60% complementary to the positive probe.

[0769] Preferably, the negative label and the negative probe are at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary, and more preferably 100% complementary.

[0770] 350. The method of claim 349, wherein the precursor nucleic acid includes the negative select tag at its 5' end; or the precursor nucleic acid includes the negative select tag at its 3' end.

[0771] When both the 5' end and the 3' end negative selection label are present, the 5' end negative selection label and the 3' end negative selection label can be the same or different.

[0772] Preferably, the positive probe is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, more preferably 100% complementary. The precursor nucleic acid does not contain any sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the positive probe, more preferably 100% complementary.

[0773] 351. The method according to item 349 or 350, wherein the circular nucleic acid is a circular RNA (circRNA), preferably the precursor nucleic acid is RNA having group II intron self-splicing activity.

[0774] 352. The method according to claim 351, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked together: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0775] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0776] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0777] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0778] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0779] 353. The method according to claim 351, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a adapter sequence; (d) a target sequence; (e) an adapter sequence; (f) exon fragment 1 (E1); and (g) a 5' intron fragment; wherein:

[0780] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0781] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0782] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0783] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0784] 354. The method according to claim 351, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; and (g) a 3' homologous arm; wherein:

[0785] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0786] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0787] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0788] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0789] 355. The method according to claim 351, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a adapter sequence; (e) a target sequence; (f) an adapter sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0790] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0791] (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides;

[0792] (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and

[0793] (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0794] 356. The method according to any one of claims 349-355, wherein the length of the negative tag and the negative probe is 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides; preferably, the length of the negative tag and the negative probe is 20-30 nucleotides.

[0795] 357. The method according to any one of claims 349-356, wherein the negative probe is an RNA probe; or the negative probe is a DNA probe, and the positive probe is an RNA probe or a DNA probe.

[0796] 358. The method according to any one of claims 349-357, wherein the negative select tag has a polynucleotide sequence comprising SEQ ID NO: n, and the negative select probe has a polynucleotide sequence comprising SEQ ID NO: n+1100, wherein n is an integer from 135 to 1234; or wherein the negative select tag has a polynucleotide sequence comprising SEQ ID NO: m, and the negative select probe has a polynucleotide sequence comprising SEQ ID NO: m+1210, wherein m is an integer from 2335 to 3544 or from 4755 to 5964.

[0797] Preferably, the positive selection probe includes a first positive selection probe and an optional second positive selection probe.

[0798] The first positively selected probe is at least 60% complementary to the linker fragment formed by E1 and E2 of the circular nucleic acid, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, and the complementarity between the first positively selected probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%; or

[0799] The first positive probe is at least 60% complementary to at least a portion of the link formed by the circularization of the 3' and 5' ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary, and the complementarity between the first positive probe and any portion of the uncirculated target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0800] 359. The method according to any one of claims 349-358, wherein the negative label has a polynucleotide sequence comprising any one of SEQ ID NO:7189-7202; or the negative probe has a polynucleotide sequence comprising any one of SEQ ID NO:7175-7188.

[0801] Preferably, the positive selection probe has a polynucleotide sequence comprising any one of SEQ ID NO.7203-7206 and SEQ ID NO.7222-7225.

[0802] 360. The method of claim 359, wherein the negative label and the negative probe are selected from any combination of:

[0803] (a) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7189, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7175;

[0804] (b) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7190, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7176;

[0805] (c) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7191, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7177;

[0806] (d) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7192, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7178;

[0807] (e) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7193, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7179;

[0808] (f) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7194, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7180;

[0809] (g) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7195, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7181;

[0810] (h) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7196, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7182;

[0811] (i) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7197, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7183;

[0812] (j) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7198, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7184;

[0813] (k) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7199, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7185;

[0814] (l) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7200, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7186;

[0815] (m) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7201, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7187;

[0816] (n) The polynucleotide sequence of the negative selection tag is shown in SEQ ID NO:7202, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO:7188.

[0817] Preferably, the positive selection probe includes a first positive selection probe and a second positive selection probe. The second positive selection probe is at least 60% complementary to any portion of any two sequences on the outer sides near both ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% complementary. The complementarity between the first positive selection probe and any portion of the uncirculated target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%. More preferably, the sequence of the second positive selection probe is selected from TT, GATT, or TTTC.

[0818] 361. The method according to any one of items 349-360, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II introns of the unpaired region into two fragments.

[0819] 362. The method according to claim 361, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting group II introns at the loop region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0820] The 5' and 3' intron segments are obtained by cutting group II introns in linear regions between domain 1 and domain 2, between domain 2 and domain 3, between domain 3 and domain 4, between domain 4 and domain 5, or between domain 5 and domain 6.

[0821] 363. The method according to claim 361 or 362, wherein the group II intron comprises one or more nucleotide modifications relative to its wild-type form, and the modifications are selected from one or more deletions, substitutions, and additions.

[0822] Preferably, the modification comprises one or more EBS sequences of group II introns, wherein the EBS sequences are complementary to one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0823] 364. The method according to item 363, wherein the modification is a modification of two EBS sequences (such as EBS1 and EBS3) of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0824] 365. The method according to item 363, wherein the modification is a modification of two EBS sequences (such as EBS1' and EBS3') of a group II intron, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at opposite ends of the target sequence.

[0825] 366. The method according to claim 364 or 365, wherein the modification is a modification of the EBS1 and / or δ sequence of group II introns, or a modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence for at least 60% of the nucleotides.

[0826] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to regions of corresponding length in the target sequence. Optionally, the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein 100% of the nucleotides of the EBS1 and / or δ sequence and its upstream sequence are complementary to regions of corresponding length in the target sequence.

[0827] 367. The method according to claim 366, wherein the modification includes partial or complete deletion of domain 4, such as deletion of the protein (IEP) sequence encoded by an intron in domain 4, preferably complete deletion of domain 4; or

[0828] The modification includes the omission of the open reading frame (ORF).

[0829] 368. The method according to claim 364, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence, preferably the near-scarless circRNA having a scar region of length equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0830] 369. The method of claim 365, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0831] 370. The method according to any one of claims 351-369, wherein the group II intron is a group II intron derived from a microorganism, preferably the group II intron is Cte1, and more preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0832] 371. The method according to any one of claims 352-370, wherein the 3' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO: 42-52; and / or

[0833] The 5' intron fragment has at least 95%, at least 98%, at least 99%, or 100% identity with a polynucleotide sequence selected from SEQ ID NO:75-88.

[0834] 372. The method according to any one of items 352-371, wherein the length of E1 and / or E2 is 0-20 nucleotides, preferably 0-10 nucleotides, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides;

[0835] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NO:53-63 and SEQ ID NO:7211-7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NO:64-74 and SEQ ID NO:7213.

[0836] 373. The method according to item 372, wherein E1, E2, or both are 0 nucleotides in length.

[0837] 374. The method according to any one of claims 352-373, wherein the target sequence is a non-coding sequence selected from the group consisting of: spacer sequences of SEQ ID NO:4-6, polyA sequences, poly-AC sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences, and protein binding sites.

[0838] 375. The method according to any one of claims 352-373, wherein the target sequence comprises: an internal ribosome entry site (IRES)-like polynucleotide sequence or a natural IRES sequence, and a protein-coding sequence, preferably the protein-coding sequence encoding a therapeutic product.

[0839] 376. The method according to any one of claims 349-375, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0840] 377. The method according to claim 376, wherein at least one of the modified RNA nucleotide and / or modified nucleoside is m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), Y (pseudouridine), m1A (1-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine), Am (2'-O-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxyn-valine) Acylcarbamoyl adenosine), ms2hn6A (2-methylthio-N6-hydroxyn-valinecarbamoyl adenosine), Ar(p)(2'-O-ribosyl adenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-O-methylcytidine), s2C (2-thiocytidine), ac4C (N4-acetylcytidine), (5-formylcytidine), m5Cm (5,2'-O-dimethylcytidine), ac4Cm (N4-acetyl-2'-O-methylcytidine), k2C (lysine), m! G(1-methylguanosine), m2G(N2-methylguanosine), m7G(7-methylguanosine), Gm(2'-O-methylguanosine), m22G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(huaitin), oayW(peroxyhuaitin), OH yW (hydroxyhuaitoside), OHyW* (insufficiently modified hydroxyhuaitoside), imG (huaitoside), mimG (methylhuaitoside), Q (pigmentoside), oQ (epoxypigmentoside), galQ (galactosylpigmentoside), manQ (mannosylpigmentoside), preQo (7-cyano-7-deazoguanosine), preQi (7-aminomethyl-7-deazoguanosine), G+ (archopurinoside), D (dihydrouridine), m5Um (5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), nmm5U (5-carbamoylmethyluridine), nmm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2'-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6,2'-O-dimethyladenosine), m6 2Am (N6,N6,O-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m'Gm (l,2'-O-dimethylguanosine), m'Am (l,2'-O-dimethyladenosine), rm 5U (5-taurate methyluridine), τm5s2U (5-taurate methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), or ac6A (N6-acetyladenosine).

[0841] 378. The method according to any one of claims 352-373, wherein the precursor RNA comprises unmodified RNA nucleotides and / or unmodified nucleosides; or the precursor RNA does not comprise modified RNA nucleotides and / or modified nucleosides.

[0842] 379. The method according to any one of items 352-378, further comprising a solid surface,

[0843] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

[0844] Further preferably, the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads, or silica beads, or

[0845] Further preferably, the solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion base materials, or an integral support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

[0846] 380. The method according to claim 379, wherein the anion probe is fixed on the solid surface by physical adsorption, covalent fixation, or affinity fixation, preferably covalent fixation is achieved by covalent linkage of NH2 and NHS-modified carboxyl groups.

[0847] The cation selection probe is fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation, preferably by covalent connection of NH2 and NHS modified carboxyl groups.

[0848] 381. The method according to item 379 or 380, wherein the solid surface is a magnetic bead.

[0849] Preferably, the magnetic beads are functionally modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specific groups, or combinations of two or more of the above, more preferably NHS-modified magnetic beads, or

[0850] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0851] Preferably, the magnetic beads are magnetic beads with a diameter of 0.1μm to 10μm, more preferably magnetic beads with a diameter of 0.5μm to 5μm, more preferably magnetic beads with a diameter of 0.8μm to 1.2μm, and more preferably magnetic beads with a diameter of 1μm.

[0852] 382. The method according to claim 379 or 380, wherein the solid surface is a chromatography packing material containing agarose, preferably the diameter of the chromatography packing material is 5 mm or more and the height is 20 cm or more, more preferably the diameter of the chromatography packing material is 10 mm or more and the height is 30 cm or more, and even more preferably the diameter of the chromatography packing material is 16 mm and the height is 40 cm.

[0853] Further, the agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, an agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, agarose chromatography packing material with CNBr-modified surface, agarose chromatography packing material with affinity or specificity groups, or a combination of two or more of the above, and is more preferably an NHS-modified agarose chromatography packing material.

[0854] 383. The method according to any one of claims 379-382, wherein the 3' end of the anion probe is covalently fixed to the solid surface; or the 5' end of the anion probe is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and preferably the covalent fixation is achieved through the covalent connection of NH2 and NHS-modified carboxyl groups.

[0855] The 3' end of the anode probe is covalently fixed to the solid surface; or the 5' end of the anode probe is covalently fixed to the solid surface. Preferably, the solid surface is a magnetic bead, and the covalent fixation is preferably achieved through the covalent connection of NH2 and NHS-modified carboxyl groups.

[0856] The anion probe and the cation probe are fixed on different solid surfaces.

[0857] 384. The method according to any one of claims 379-383, wherein the 3' end and / or 5' end of the anion probe is further modified with a group selected from: amino, carboxyl, NHS-carboxyl, mercapto.

[0858] The 3' and / or 5' ends of the cation-selective probe are further modified with groups selected from the following: amino, carboxyl, NHS-carboxyl, and thiol.

[0859] 385. The composition according to any one of claims 349-384, wherein the minimum free energy (MFE) value of the negative tag ranges from -2 to 2, and the change in MFE value (ΔMFE) of the precursor nucleic acid resulting from the inclusion of the tag ranges from -2 to 2.

[0860] Preferably, the anode probe includes a second anode probe, a first anode probe, and a second anode probe connected in sequence, wherein the 3' end of one second anode probe is covalently fixed to the solid surface; or the 5' end of another second anode probe is covalently fixed to the solid surface. Preferably, the solid surface is a magnetic bead, and the covalent fixation is preferably achieved through the covalent connection of NH2 and NHS modified carboxyl groups.

[0861] 386. The composition according to claim 385, wherein the MFE value of the negative label is approximately 0.

[0862] Preferably, the cation selection probe comprises a second cation selection probe, a first cation selection probe, and a second cation selection probe connected in sequence, wherein the 3' end of one second cation selection probe and / or the 5' end of the other second cation selection probe are further modified with groups selected from the following: amino, carboxyl, NHS-carboxyl, and thiol.

[0863] 387. The composition according to claim 385 or 386, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag is in the range of 0-2, preferably approximately 0.

[0864] Preferably, the positive selection probe has a polynucleotide sequence comprising any one of SEQ ID NO.7205-7206.

[0865] 388. The method according to any one of claims 349-387, wherein a sample comprising the circular nucleic acid to be purified is contacted with the negative probe or the positive probe at a pH of 4.0-9.0, preferably 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, or 8.5-9.0; or

[0866] The sample is in contact with the anion probe or the cation probe at a temperature of 0℃-60℃; or

[0867] The sample is in contact with the anion-selective probe or the cation-selective probe in a binding solution. The binding solution comprises salts, preferably further comprising buffering substances, chelating agents, and optionally stabilizers. More preferably, the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I- and SCN - Or a combination thereof, preferably, the concentration of the salt in the binding solution is 50 mM-5 M.

[0868] Further preferably, the salt is a cation of Li. + Or Na + Salts.

[0869] 389. The method according to claim 388, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl, preferably further comprising EDTA and Tris-HCl, and the pH range of the binding solution is 6.0-8.0, preferably 7.5.

[0870] Preferably, the method further includes the step of using an eluent to elute a portion of the anion probe bound to the solid surface to recover the solid surface coupled with the anion probe for reuse.

[0871] 390. The method according to any one of claims 349-389, wherein the concentration of salts in the eluent is 0M, and the salts comprise cations selected from the group consisting of: Ba 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 3- CH3CO3 - Cl - ,Br - NO3 3- ClO4 3- I - and SCN - Or a combination thereof, preferably the salt being a cation of Li. + Or Na + Salts,

[0872] Preferably, the eluent further includes buffering substances, chelating agents, and optionally stabilizers.

[0873] 391. The method according to claim 390, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.0, preferably 7.5; or

[0874] The eluent has a pH range of 2.0-4.0, preferably a pH range of 2.5-3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5-3.0.

[0875] 392. The method according to any one of items 349-391, wherein the method is scalable and may include a continuous flow process, preferably performed using a liquid chromatograph (LC) or an enzyme-linked immunosorbent assay (ELISA) reader, and more preferably the LC is a rapid liquid chromatograph (FPLC) or a high-performance liquid chromatograph (HPLC).

[0876] 393. A method for purifying circular nucleic acids from a sample, comprising the following steps:

[0877] Couple the magnetic bead to the negative selection probe;

[0878] Couple the magnetic bead with the anode probe;

[0879] The IVT reaction solution containing the circular nucleic acid obtained from in vitro transcription was incubated with magnetic beads coupled with a negative selection probe.

[0880] The magnetic beads are collected and eluted to recover the magnetic beads coupled with the anion selection probe;

[0881] Collect the unbound parts.

[0882] The unbound portion is incubated with magnetic beads coupled with anode probes.

[0883] The magnetic beads are collected and eluted to obtain the eluted circular nucleic acid, and the magnetic beads coupled with the positive probe are recovered.

[0884] The negative selection probe is at least 90% complementary to the negative selection tag, the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid containing an oligonucleotide negative selection tag, wherein the negative selection tag is removed during the self-splicing of the precursor nucleic acid, preferably the negative selection tag is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the negative selection probe, and more preferably 100% complementary;

[0885] The circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, wherein the positive probe is at least 60% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the splice portion of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive probe.

[0886] 394. The method according to claim 393, wherein the IVT reaction solution is incubated with magnetic beads coupled with an anion-selective probe in a binding solution, and the magnetic beads coupled with anion-selective probe are eluted and recovered using an elution buffer.

[0887] The IVT reaction solution was incubated with magnetic beads coupled with positive probes in the binding solution. The magnetic beads were eluted with elution buffer to obtain the eluted circular nucleic acid, and the magnetic beads coupled with positive probes were recovered.

[0888] 395. The method according to claim 394, wherein the eluent and binding solution are the eluent and binding solution involved in any one of claims 388-391, and more preferably the anion probe, anion label and anion probe involved in any one of claims 349-392.

[0889] 396. Circular nucleic acid purified by the method according to any one of items 349-395 or by using the composition containing oligonucleotide probes for purifying circular nucleic acids according to any one of items 301-339 or by using the kit according to any one of items 340-348.

[0890] 397. A composition having the circular nucleic acid of claim 396, wherein the cyclization percentage (PC) of the composition is at least 90%, at least 95%, at least 98%, or at least 99%; or

[0891] The total cyclic purity of the composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, and above 95%, preferably with an polymer content below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, and below 1.5%.

[0892] Further preferably, the size of the circular nucleic acid is 1000nt-5000nt.

[0893] 398. A composition for generating circular nucleic acids, comprising:

[0894] Linearized plasmids used to generate precursor nucleic acids;

[0895] The generated precursor nucleic acid;

[0896] The generated circular nucleic acid;

[0897] Non-circular precursor nucleic acids;

[0898] Residual impurities from in vitro transcription reaction, wherein the precursor nucleic acid and circular nucleic acid are the precursor nucleic acid and circular nucleic acid involved in any of items 301-348.

[0899] The preferred composition has a total cyclic purity ranging from 20% to 50% and a polymer content ranging from 10% to 20%.

[0900] 399. The composition according to claim 398, used as a sample by any one of claims 349-396, or purified as a sample by any one of claims 301-339 using a composition comprising an oligonucleotide negative probe for purifying circular nucleic acids, or by any one of claims 340-348 using a kit.

[0901] 400. The composition according to claim 399, wherein the purified cyclic nucleic acid composition has a total cyclic purity of ≥90.0%, ≥91%, ≥92%, ≥93%, ≥94%, or ≥95%.

[0902] Preferably, the polymer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, or below 1.5%, and more preferably, the size of the circular nucleic acid is 1000nt-5000nt. 6. Description of the attached drawings

[0904] Figure 1A-1D Self-splicing of precursor RNA with group II intron self-splicing activity is provided, along with a schematic diagram of the tags disclosed herein used for purifying circRNA products. Figure 1A This is a schematic diagram of a near-scarless system designed based on the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The self-catalytically self-splicing group II introns are split into two fragments at the D4 domain, and custom exons containing E1, E2, and the target sequence are inserted between the split introns. Arrows indicate the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3.

[0905] Figure 1B This is a schematic diagram of a near-scarless system designed based on the interaction between δ and IBS3. The self-catalytically self-splicing group II introns are split into two fragments at the D4 domain, and custom exons containing E1, E2, and the target sequence are inserted between the split introns. Arrows indicate the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and δ.

[0906] Figure 1C This is a schematic diagram of a scarless system designed based on the interaction between IBS1' and EBS1. The self-catalytically self-splicing group II introns are split into two fragments at the D4 domain, and a target sequence is inserted between the split introns. The arrows indicate the interactions between IBS1' and EBS1, and between IBS3' and EBS3.

[0907] Figure 1D This is a schematic diagram of a scarless system designed based on the interaction between δ and IBS3'. The self-catalytically self-splicing group II intron is split into two fragments at the D4 domain, and a target sequence is inserted between the split introns. The arrows indicate the interactions between IBS1' and EBS1, and between IBS3' and δ.

[0908] Figure 2 The results of an exemplary purification process are shown. As illustrated, the IVT product is a reaction product of precursor RNA generated through in vitro transcription and subjected to circularization conditions. FPLC purification as disclosed herein produces a P1 fraction containing highly purified circRNA (results from Example 2).

[0909] Figure 3A A schematic diagram of the negative selection system of this application is provided. Figure 3A This is a schematic diagram of a near-scarless system designed based on the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The self-catalytically self-splicing group II introns are split into two fragments at the D4 domain, and custom exons containing E1, E2, and the target sequence are inserted between the split introns. Arrows indicate the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The anion tags are located at both ends of the D4 domain, which is split into two fragments.

[0910] Figure 3B A schematic diagram of the positive selection system of this application is provided. Figure 3B This is a schematic diagram of a near-scarless system designed based on the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The self-catalytically self-splicing group II introns are split into two fragments at the D4 domain, and custom exons containing E1, E2, and the target sequence are inserted between the split introns. Arrows indicate the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The positive tag is located on IBS1 and IBS3. In some embodiments, a portion of the positive tag sequence is also located on the target sequence.

[0911] Figure 3C A schematic diagram of a solid surface (such as magnetic beads or agarose chromatography packing material) coupled with a negative probe is provided in this application.

[0912] Figure 3D A schematic diagram is provided of a solid surface (such as magnetic beads or agarose chromatography packing material) coupled with a first cathode probe and a second cathode probe in this application.

[0913] Figure 3E A schematic diagram of a solid surface (such as magnetic beads or agarose chromatography packing material) coupled with a cation-selective probe is provided in this application.

[0914] Figure 4 The results before and after chromatography purification are shown. (Example) Figure 4 As shown, after chromatography purification, most impurities were removed, and the purity of the circular RNA was improved.

[0915] Figure 5A and 5B The capillary electrophoresis results before and after negative selection magnetic bead purification are shown. As shown in the figure, after negative selection magnetic bead purification, introns and precursors were removed, and the purity of the circular RNA was improved.

[0916] Figure 6 The HPLC results for detecting the polymer content after purification are shown. As shown in the figure, the polymer content is extremely low after purification.

[0917] Figure 7 A and Figure 7 Figure B shows the agarose gel electrophoresis results after purification with negative and positive magnetic beads, respectively. As shown in the figure, after purification with negative and positive magnetic beads, most impurities were removed, and the purity of the circular RNA was improved.

[0918] Figure 8 The results of translating purified Gluc and Fluc are shown in the figure. As shown in the figure, G1-G7 are Gluc and F1-F12 are Fluc, all of which are purified samples. The main band obtained is circular RNA, and as can be seen from the figure, the impurity bands above and below are basically invisible.

[0919] Figure 9 This is a schematic diagram of the type II intron structure.

[0920] Figure 10 It is a schematic diagram of a structure with IBS1, IBS2, IBS3, EBS1, EBS2, EBS3, and δ (shown in bold) type II introns.

[0921] Figure 11 This demonstrates an amplified representation of circRNA production.

[0922] Figure 12 This study demonstrates the analysis of four batches of CVB3-Gluc circRNA purified from HPLC using capillary electrophoresis with an Agilent 2100 Bioanalyzer.

[0923] Figure 13 This is a schematic diagram of the CircRNA-LNP complex and the particle size of CircRNAGluc-LNP.

[0924] Figure 14 The Gaussia luciferase activity was measured in mouse serum 24 hours after injection of CircRNAGluc-LNP with different formulations.

[0925] Figure 15 Representative IVIS images of BALB / c mice administered 20 μg of CircRNAGluc-LNP via intramuscular (im) administration of two formulations are shown. Relative luminescence plots are displayed, and the luminescence scale is indicated.

[0926] Figure 16 A schematic diagram of the RNA high-throughput screening workstation of this application is shown.

[0927] Figure 17 This is an image illustrating an in vivo imaging system (IVIS) spectrum of protein expression following an IV injection of an exemplary circular RNA containing a combination of natural IRES and IRES-like sequences of the present disclosure.

[0928] Figure 18A A schematic diagram of a circular RNA is provided, which includes, in the following order: 5' tag, 3' intron fragment, translation initiation element, target sequence, 5' intron fragment, and 3' tag.

[0929] Figure 18B A schematic diagram of a circular RNA is provided, comprising, in the following order: a 3' intron fragment, a translation initiation element, a target sequence, and a 5' intron fragment. The translation initiation element and the upper portion of the target sequence serve as positive select tags.

[0930] Figure 18C A schematic diagram of a circular RNA is provided, which includes, in the following order: 5' tag, 3' intron fragment, target sequence, translation initiation element, 5' intron fragment, and 3' tag.

[0931] Figure 18D A schematic diagram of a circular RNA is provided, comprising, in the following order: a 3' intron fragment, a target sequence, and a 5' intron fragment of the translation initiation element. The translation initiation element and the upper portion of the target sequence serve as positive tags.

[0932] Figure 19A A schematic diagram of a circular RNA is provided, which includes, in the following order: 5' tag, 3' intron fragment, adapter sequence 2, translation initiation element, target sequence, adapter sequence 1, 5' intron fragment, and 3' tag.

[0933] Figure 19B A schematic diagram of a circular RNA is provided, comprising, in the following order: a 3' intron fragment, adapter sequence 2, a translation initiation element, a target sequence, adapter sequence 1, and a 5' intron fragment. The upper portions of adapter sequence 1 and adapter sequence 2 serve as positive select tags.

[0934] Figure 19C A schematic diagram of a circular RNA is provided, which includes, in the following order: 5' tag, 3' intron fragment, target sequence, adapter sequence 1, adapter sequence 2, translation initiation element, 5' intron fragment, and 3' tag.

[0935] Figure 19D A schematic diagram of a circular RNA is provided, comprising, in the following order: a 3' intron fragment, a target sequence, adapter sequence 1, adapter sequence 2, a translation initiation element, and a 5' intron fragment. The translation initiation element and the upper portion of the target sequence serve as positive tags. 7. Detailed Description of the Invention

[0937] This article provides methods and systems for purifying circular nucleic acids. Circular nucleic acids are single-stranded nucleic acids linked end-to-end. As is known in the art, circular nucleic acids can be produced in vitro using chemical means or through enzymatic activity. For example, the 5' and 3' ends of linear nucleic acids can be chemically linked by catalysis with bromide cyanide and morpholino derivatives, or linked end-to-end by the activity of nucleic acid ligases. As used herein, a "precursor nucleic acid" is a linear nucleic acid molecule that directly produces a circular nucleic acid, regardless of the cyclization method.

[0938] Circular nucleic acids may include ribonucleotides, deoxyribonucleotides, and modified nucleotides and / or analogues thereof. In some embodiments, the circular nucleic acid is circular DNA. In some embodiments, the circular nucleic acid is circular RNA.

[0939] In some embodiments, circular RNA (also known as “circRNA” or “cRNA”) can be generated by splicing. When a linear precursor undergoes splicing, a portion of the molecule is removed, resulting in circRNA with fewer total nucleotides than the linear precursor. In some embodiments, a negative selection tag may be included in the intronic portion of the linear precursor, which can then be used to negatively select circRNA from the precursor and introns. Specifically, a sample containing circular RNA, such as the product of a splicing reaction starting from a labeled linear precursor, can be mixed with a negative selection probe immobilized on a solid surface, wherein the negatively tagged precursor or any other negatively tagged impurity can bind to the negative selection probe and be removed from the solution, producing a circRNA solution substantially free of the precursor, introns, and any other tagged impurities.

[0940] In some implementations, a positive probe can be selected to be at least 60% complementary to at least a portion of the formed circular nucleic acid, but not to be less than 60% complementary to the precursor nucleic acid, i.e., there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive probe. Thus, the circular nucleic acid product can be directly bound to the IVT reaction solution by the positive probe, thereby achieving the purification of the circular nucleic acid product.

[0941] As is known in the art, RNA molecules with enzymatic activity are called ribozymes. circRNA can also be produced by ribozyme-catalyzed RNA splicing. Furthermore, some ribozymes can catalyze self-splicing independently of the spliceosome; these ribozymes are referred to as "self-splicing ribozymes," "self-splicing ribozymes," or "self-splicing introns." In some embodiments, the precursor RNA is an engineered ribozyme with in vitro self-splicing activity, which forms circular RNA after self-splicing. Such engineered ribozymes are also referred to herein as "cRNAases."

[0942] Therefore, in some embodiments, this document provides compositions comprising oligonucleotide negative probes for purifying circular nucleic acids, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid comprising an oligonucleotide negative tag, wherein the oligonucleotide negative tag is complementary to the negative probe and is removed during the self-splicing of the precursor nucleic acid. This document also provides a method for purifying circular nucleic acids from a sample, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, and wherein the precursor nucleic acid has an oligonucleotide negative tag that is removed during the self-splicing, the method comprising: (i) contacting a sample with an oligonucleotide negative probe complementary to the negative tag under conditions allowing the negative tag to bind to the probe, wherein the negative probe is immobilized on a solid surface; and (ii) collecting unbound portions of the sample.

[0943] In some embodiments, this document provides compositions comprising oligonucleotide positive probes for purifying circular nucleic acids, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, wherein the positive probe is at least 60% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, the at least portion of the circular nucleic acid spanning the splice portion of the circular nucleic acid, and the precursor nucleic acid lacking a sequence at least 60% complementary to the positive probe. The method includes: (i) contacting a sample comprising the circular nucleic acid generated by the self-splicing of the precursor nucleic acid with a positive probe at least 60% complementary to at least a portion of the circular nucleic acid, wherein the positive probe is immobilized on a solid surface, under conditions allowing the circular nucleic acid generated by the self-splicing of the precursor nucleic acid to bind to the positive probe; and (ii) collecting the portion of the positive probe bound to the solid surface, the at least portion of the circular nucleic acid spanning the splice portion of the circular nucleic acid, and the precursor nucleic acid lacking a sequence at least 60% complementary to the positive probe.

[0944] Before further describing this disclosure, it should be understood that this disclosure is not limited to the specific embodiments set forth herein, and it should also be understood that the terminology used herein is for the purpose of describing specific embodiments and is not intended to be limiting.

[0945] 7.1 Definition

[0946] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization as described herein are those well-known and commonly used in the art.

[0947] As used herein in the specification, “a” or “one” can refer to one or more. As used herein in the context of a section, when used in conjunction with the word “comprising”, the word “a” or “one” can mean one or more.

[0948] As used herein, the term “or” is used to mean “and / or” unless explicitly indicated to refer only to alternatives, or the alternatives are mutually exclusive, although this disclosure supports the definition of alternatives only and “and / or”. As used herein, “another” or “additional” may mean at least a second or more.

[0949] As used herein, the term "about" is used to indicate a value that includes variations in the apparatus, the inherent error of the method used to determine the value, or variations existing between the subjects under study. The term "about" encompasses the exact figure referenced. In some embodiments, "about" means within ±10% of a given value or range. In some embodiments, "about" means a variation of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by "about". In some embodiments, "about" means a variation of ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by "about".

[0950] As used herein, “substantially free” with respect to a particular component means that no particular component is intentionally formulated into the composition and / or exists only as a contaminant or in trace amounts. Therefore, the total amount of the particular component resulting from any unintentional contamination of the composition is well below 0.1%, preferably below 0.05%, more preferably below 0.01%. The most preferred composition is one in which the amount of the specified component cannot be detected by standard analytical methods.

[0951] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to a polymeric form of consecutive amino acids comprising at least two or more chemically or biochemically modified or derived amino acids. As used herein, the term “peptide” refers to a class of short polypeptides. The term peptide can also refer to a polymer of amino acids (naturally or non-naturally occurring) with a length of up to about 100 amino acids. For example, the length of a peptide can be: about 1 to about 10, about 10 to about 25, about 25 to about 50, about 50 to about 75, or about 75 to about 100 amino acid residues. In some embodiments, the length of the peptide can be: about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1250, about 1500, about 1750, about 2000, about 2250, about 2500, about 2750, about 3000, about 3250, about 3500, about 3750, about 4000, about 4250, about 4500, about 4750, or about 5000 amino acid residues.

[0952] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably herein and refer to polymers or oligomers of nucleotides of any length. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases (such as methylation, hydroxymethylation, or glycosylation), non-natural nucleotides, non-nucleotide components exhibiting a similar structure and / or function to natural nucleotides (i.e., “nucleotide analogs”), and / or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Nucleic acids or polynucleotides can be heterologous or homologous in composition, can be isolated from naturally occurring sources, or can be artificially or synthetically produced. Furthermore, nucleic acids can be DNA or RNA, or mixtures thereof, and can exist permanently or temporarily in single-stranded or double-stranded form (including homoduplexes, heteroduplexes, and hybrid states). Nucleic acid structures also include, for example, DNA / RNA helices, peptide nucleic acids (PNA), morpholinonucleotides (see, for example, Braasch and Corey, Biochemistry, 4(14):4503-4510 (2002) and US Patent 5,034,506), locked nucleic acids (LNA; see Wahllestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, Am. Chem. Soc., 122:8595-8602 (2000)), and / or ribozymes.

[0953] When referring to polynucleotide sequences or protein sequences, the term "identity" is used to indicate the similarity between two sequences. Sequence similarity or identity can be determined using standard techniques known in the art, including but not limited to: the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981); the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48, 443 (1970); the similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988); computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI); the best-fit sequence procedure described in Devereux et al., Nucl. Acid Res. 12, 387-395 (1984); or by inspection. Another algorithm is the BLAST algorithm, described in Altschul et al., JMol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program, which is obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which can be set to default values. The parameters are dynamic values ​​and are established by the program itself based on the composition of a particular sequence and the composition of a particular database of sequences of interest being searched; however, these values ​​can be adjusted to improve sensitivity. In addition, another useful algorithm is BLAST with gaps, as reported by Altschul et al., (1997) Nucleic Acids Res. 25, 3389-3402. Unless otherwise stated, the identity percentages in this paper were determined using an algorithm available from the Internet address (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0954] The terms “coding sequence,” “coding region,” “coding area,” and “CDS,” when referring to a polynucleotide sequence, are used interchangeably herein to refer to a portion of a DNA or RNA sequence, for example, that is or can be translated into a protein. The terms “reading frame,” “open reading frame,” and “ORF,” when used interchangeably herein, refer to a nucleotide sequence that begins with a start codon (e.g., ATG) and, in some embodiments, ends with a stop codon (e.g., TAA, TAG, or TGA). An open reading frame may contain introns and exons; therefore, all CDS are ORFs, but not all ORFs are CDSs.

[0955] As used herein, the terms “complementary” and “complementarity” refer to the relationship between two nucleic acid molecules that have the ability to form hydrogen bonds with each other through conventional Watson-Crick base pairing or other non-traditional types of pairing. Two DNA / RNA strands with complementary sequences combine to form a doublet following the Watson-Crick base pairing rule: A binds to T (U) through two hydrogen bonds; G binds to C through three hydrogen bonds. The degree of complementarity between two polynucleotide sequences can be indicated by the percentage of nucleotides in the polynucleotide sequence that can form hydrogen bonds (e.g., Watson-Crick base pairing) with the second polynucleotide sequence (e.g., approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, and 100% complementarity). Two polynucleotide sequences are “perfectly complementary” if all consecutive nucleotides in the polynucleotide sequence will form hydrogen bonds with the same number of consecutive nucleotides in the second polynucleotide sequence. The two polynucleotide sequences are considered "substantially complementary" if the complementarity between them is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) over a region of at least 8 nucleotides (e.g., 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, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more nucleotides), or if the two polynucleotide sequences hybridize under at least moderate conditions, or in some embodiments under highly stringent conditions. Exemplary moderately stringent conditions include: incubation overnight at 37°C in a solution containing 20% ​​formamide, 5% SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured cleaved salmon sperm DNA, followed by washing the filter in 1* SSC at approximately 37–50°C; or under substantially similar conditions, such as those described in Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th edition (June 15, 2012).Highly stringent conditions are used, for example: (1) low ionic strength and high temperature washing, such as 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate (SDS) at 50°C, (2) denaturing agents such as formamide are used during hybridization at 42°C, for example 50% (v / v) formamide with 0.1% bovine serum albumin (BSA) / 0.1% Ficoll / 0.1% polyvinylpyrrolidone (PVP) / 50mM sodium phosphate buffer (pH 6.5), along with 750mM sodium chloride and 75mM sodium citrate. Or (3) at 42°C, using 50% formamide, 5xSSC (0.75M sodium chloride, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt solution, sonicated salmon sperm DNA (50 pg / ml), 0.1% SDS and 10% dextran sulfate, (i) washed at 42°C in 0.2*SSC, (ii) washed at 55°C in 50% formamide, and (iii) washed at 55°C in 0.1*SSC (optionally in combination with EDTA). Further details and explanations of the strictness of the hybridization reaction are provided, for example, in Sambrook, supra, and Ausubel et al., eds., Short Protocols in Molecular Biology, 5th ed., John Wiley & Sons, Inc., Hoboken, NJ (2002).

[0956] When polynucleotide sequences are involved, the term “hybrid” or “hybrid” refers to a combination formed between and / or within sequences that are complementary.

[0957] The term "homology" refers to the percentage of similarity between nucleic acid residues of two polynucleotides or amino acid residues of two polypeptides. The correspondence between one sequence and another can be determined using techniques known in the art. For example, homology can be determined by directly comparing the sequence information of two polypeptides using readily available computer programs. As determined using the methods described above, two polynucleotide (e.g., DNA) or two polypeptide sequences are "substantially homologous" to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids match at a defined length of the molecule.

[0958] The term "scar" refers to the length of the region in the circular product that does not include the target sequence. Scarless circRNAs contain 0 nucleotides of scar sequence. Nearly scarless circRNAs contain scar sequences of equal to or less than 20 nucleotides in length.

[0959] The term "Tris" refers to tris(hydroxymethyl)aminomethane, which is widely used in the preparation of buffer solutions for biochemical and molecular biology experiments.

[0960] The term "EDTA" refers to ethylenediaminetetraacetic acid.

[0961] The term "rpm" refers to the rotational speed per minute, or revolutions per minute. The full name of the English word RPM is Revolution(s) Per Minute.

[0962] The term "Tween-20" refers to polysorbate-20.

[0963] The term "PCR" refers to Polymerase Chain Reaction, a molecular biology technique used to amplify specific DNA fragments.

[0964] The term "ATP" refers to adenosine triphosphate; the term "GTP" refers to guanosine triphosphate; the term "CTP" refers to cytidine triphosphate; and the term "UTP" refers to uridine triphosphate.

[0965] The term "DNase" refers to deoxyribonuclease, also known as DNA enzyme, which is an enzyme that can cleave the phosphodiester bonds on the DNA backbone through hydrolysis. DNA enzymes are a type of nuclease, and there are many different types of known DNA enzymes, mainly differing in their receptor specificity, chemical reaction mechanisms, and biological functions.

[0966] The term "RNase" refers to ribonuclease, a nuclease that hydrolyzes RNA into smaller molecules.

[0967] The term "CIP" or "Calf Intestinal Alkaline Phosphatase" refers to calf intestinal alkaline phosphatase, an alkaline phosphatase commonly used to remove the 5'-phosphate group from DNA, RNA, and ribose and deoxyribonucleoside triphosphates.

[0968] The term "DEPC water" refers to ultrapure water that has been treated with DEPC (diethyl pyrocarbonate) and sterilized under high temperature and pressure.

[0969] The term "PBS" refers to phosphate-buffered saline, a commonly used buffer solution in biological research. It is a sodium phosphate salt solution with water as the solvent; some formulations also include potassium chloride and potassium phosphate. The osmolality and ion concentration of this solution are similar to those in the human body.

[0970] The term "total circular purity" refers to the proportion of circular RNA in the total RNA content of the sample being tested.

[0971] The term "polymer content" refers to the proportion of polymers in the total RNA content of the sample being tested.

[0972] The term "Gaussia luciferase" or "Gluc" refers to a luciferase derived from the marine copepod Gaussiaprinceps. It has advantages such as being secretible, easy to monitor, highly sensitive, having a short half-life, requiring no ATP to react, and being highly tolerant to temperature and pH values, and can be used for real-time monitoring of living cells or organisms.

[0973] The term "Firefly luciferase" or "Fluc" refers to firefly luciferase, an enzyme that catalyzes the oxidation of firefly luciferin, which requires oxygen and ATP.

[0974] The term "Lipid nanoparticle" or "LNP" refers to lipid-based nanoparticles, representing a novel route of drug delivery.

[0975] The term "Fast protein liquid chromatography" or "FPLC" refers to rapid protein liquid chromatography, a type of liquid chromatography commonly used for the analysis or purification of protein mixtures.

[0976] The term "High performance liquid chromatography" or "HPLC" refers to high performance liquid chromatography, a chromatographic analysis technique used to separate mixtures in order to identify and quantify the proportions of individual components.

[0977] The terms "autocatalytic self-splicing," "autocatalytic self-splicing," "self-splicing," or "self-splicing" refer to self-splicing that occurs independently of the spliceosome. Some ribozymes can catalyze self-splicing independently of the spliceosome; these ribozymes are called "self-splicing ribozymes," "self-splicing ribozymes," or "self-splicing introns."

[0978] The term "intravenous injection (IV)" or "iv" refers to intravenous injection, a medical procedure in which liquid substances such as blood, medication, or nutrient solutions are injected directly into a vein.

[0979] The term "Intramuscular injection (IM)" or "im" refers to intramuscular injection, a method of injecting substances directly into the muscle, which is one of the routes of drug administration in medicine.

[0980] The term "Intraperitoneal injection (IP)" or "ip" refers to intraperitoneal injection, which is more commonly used in non-human animals compared to humans.

[0981] The term "DSPC" or "Distearoylphosphatidylcholine" refers to distearate phosphatidylcholine, a type of phosphatidylcholine and a natural component of cell membranes.

[0982] The term "PEG" stands for polyethylene glycol.

[0983] The term "DMG-PEG" refers to a synthetic lipid formed by PEGylating myristoyl diglyceride.

[0984] The term "nt" stands for "nucleotide," the number of nucleotides used to describe the length of a nucleotide.

[0985] The nomenclature of nucleotides, nucleic acids, nucleosides, and amino acids used in this article conforms to the standards of the International Union of Pure and Applied Chemistry (IUPAC) (see, for example, bioinformatics.org / smsylupac.html). Exemplary genes and peptides are described in this article with reference to GenBank numbers, GI numbers, and / or SEQ ID NOS. It should be understood that those skilled in the art can readily identify homologous sequences using reference sequence sources, including but not limited to: Uniprot (https: / / www.uniprot.org / ), GenBank (ncbi.nlm.nih.gov / genbank / ), and EMBL (embl.org / ).

[0986] The terms "domain 1" or "D1" are used herein to refer to the stem-loop structure of domain 1 of type II introns. The terms "domain 2" or "D2" are used herein to refer to the stem-loop structure of domain 2 of type II introns. The terms "domain 3" or "D3" are used herein to refer to the stem-loop structure of domain 3 of type II introns. The terms "domain 4" or "D4" are used herein to refer to the stem-loop structure of domain 4 of type II introns. The terms "domain 5" or "D5" are used herein to refer to the stem-loop structure of domain 5 of type II introns. The terms "domain 6" or "D6" are used herein to refer to the stem-loop structure of domain 6 of type II introns. The stem-loop structure is a type of RNA secondary structure that can be determined using any suitable polynucleotide folding algorithm. Some procedures are based on the calculation of the minimum Gibbs free energy. One example of such an algorithm is mFold, described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is RNAfold, an online web server developed by the Institute of Theoretical Chemistry at the University of Vienna using a centroid structure prediction algorithm (e.g., AR Gruber et al., 2008, Cell 106). (1):23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12):1151-62). Further algorithms can be found in U.S. Provisional Patent Application No. 61 / 836,080 (Attorney's File No. 44790.11.2022; Broad Reference Number BI-2013 / 004A), which is incorporated herein by reference. Type II introns mainly consist of six stem-loop structures, called domains 1-6 (D1-D6), which are arranged sequentially and contain multiple exon-binding sequences (EBSs), such as EBS1, EBS2, and EBS3. These EBS sequences interact with intron-binding sequences (IBSs) in the exon regions through complementary pairing, and splicing is triggered by the hydroxyl groups within the EBS nucleic acid sequences themselves.

[0987] As used in this paper, EBS1', EBS2', and EBS3' are used to refer to regions on the target sequence that are functionally similar to EBS1, EBS2, and EBS3, respectively.

[0988] As used herein, the term "type II intron" refers to RNA molecules encoded by type II introns that have similar secondary and tertiary structures. Type II intronic RNA molecules typically have six domains. See also Figure 9 and Figure 10Domain 4 (also known as domain IV) of the type II intron RNA contains a nucleotide sequence encoding a "type II intron-encoded protein". In some embodiments, the type II intron sequence is selected from the sequence disclosed in WO2022247943.

[0989] The term “IBS” is used in this paper to refer to the intron-binding sequence, which interacts with the exon-binding sequence (EBS) to locate the splice site.

[0990] The term “IBS1” is used in this document to refer to intron-binding sequence 1, which interacts with exon-binding sequence 1 (EBS1) to locate the splice site.

[0991] The term “IBS1” is used in this paper to refer to a region on the target sequence that is functionally similar to IBS1.

[0992] The term “IBS2” is used in this document to refer to intron-binding sequence 2, which interacts with exon-binding sequence 2 (EBS2) to locate the splice site.

[0993] The term “IBS3” is used in this document to refer to intron-binding sequence 3, which interacts with exon-binding sequence 3 (EBS3) to locate the splice site.

[0994] The term “IBS3’” is used in this paper to refer to a region on the target sequence that is functionally similar to IBS3.

[0995] The term "δ" (delta) is used in this text to refer to the region on domain 1 of the type II intron, which is a single nucleotide directly upstream of EBS1. δ pairs with IBS3, and the interaction between δ and IBS3 is called δ-IBS3 pairing. See also Figure 10 .

[0996] The term “δ” (delta”) is used in this document to refer to the region on domain 1 of the type II intron, which is a single nucleotide directly upstream of EBS1'. δ” pairs with IBS3', and the interaction between δ” and IBS3' is called δ”-IBS3' pairing. See also Figure 10 .

[0997] The terms “internal ribosome entry site,” “internal ribosome entry site sequence,” “IRES,” and “IRES sequence region” are used interchangeably herein and refer to cis-elements of viral or human cellular RNA (e.g., messenger RNA (mRNA) and / or circRNA) that bypass the classical eukaryotic cap-dependent translation initiation step. The classical cap-dependent mechanism used by the vast majority of eukaryotic mRNAs requires an m7G cap at the 5' end of the mRNA, the initiator Met-tRNAmet, more than a dozen initiation factor proteins, directional scanning, and GTP hydrolysis to place a translationally capable ribosome at the start codon. IRES typically consists of a long and highly structured 5-UTR, which mediates the binding of the translation initiation complex and catalyzes the formation of a functional ribosome. In some embodiments, sequences are selected from those disclosed in PCT / CN2023 / 096813 and sequences screened by the methods disclosed in PCT / CN2023 / 096813.

[0998] The term "IRES-like sequence" or "internal ribosome entry site-like sequence" refers to a synthetic nucleotide sequence that exhibits the function of a native IRES. In some embodiments, the IRES-like sequence can recruit ribosomal components to mediate cap-independent translation. In some embodiments, sequences are selected from those disclosed in PCT / CN2023 / 096813 and sequences screened by the methods disclosed in PCT / CN2023 / 096813.

[0999] Scope: Throughout this disclosure, all aspects of this application may be presented in a scope format. It should be understood that the scope format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of this application. Therefore, the scope description should be considered to have specifically disclosed all possible sub-scopes and the individual values ​​within those scopes. For example, a scope such as 1-6 should be considered to have specifically disclosed sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual numbers within those scopes, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the size of the scope.

[1000] 7.2 Probes and Tags

[1001] 7.2.1 Negative selection tags and negative selection probes

[1002] This document provides compositions and methods for purifying circular nucleic acids. In some embodiments, circular nucleic acids can be generated by the self-splicing of a precursor nucleic acid containing an oligonucleotide negative select tag, which is removed during the self-splicing. Therefore, an oligonucleotide negative select probe that is at least 60% complementary to the negative select tag can be used to capture precursors, introns, and any other negatively tagged impurities in a mixture of products, thereby purifying negatively tagged circular nucleic acids. A schematic diagram of the negative select system and method described herein is shown below. Figure 3A As shown. A schematic diagram of the magnetic beads coupled with the anion selection probe used in the composition discussed herein is shown below. Figure 3C As shown, a schematic diagram of the magnetic beads coupled with a first cathode probe and a second cathode probe used in the composition discussed herein is shown below. Figure 3D As shown.

[1003] In some embodiments, the negative probe and negative label may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or at least 99% complementary. In some embodiments, the negative probe and negative label may be at least 65% complementary. The negative probe and negative label may be at least 70% complementary. The negative probe and negative label may be at least 75% complementary. The negative probe and negative label may be at least 80% complementary. The negative probe and negative label may be at least 85% complementary. The negative probe and negative label may be at least 90% complementary. The negative probe and negative label may be at least 95% complementary. The negative probe and negative label may be at least 98% complementary. The negative probe and negative label may be at least 99% complementary. In some embodiments, the cathode probe and cathode label may be approximately 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% complementary. In some embodiments, the cathode probe and cathode label may be approximately 65% ​​complementary. The cathode probe and cathode label may be approximately 70% complementary. The cathode probe and cathode label may be approximately 75% complementary. The cathode probe and cathode label may be approximately 80% complementary. The cathode probe and cathode label may be approximately 85% complementary. The cathode probe and cathode label may be approximately 90% complementary. The cathode probe and cathode label may be approximately 95% complementary. The cathode probe and cathode label may be approximately 98% complementary. The cathode probe and cathode label may be approximately 99% complementary. In some embodiments, the cathode probe and cathode label may be 100% complementary.

[1004] In some embodiments, the precursor nucleic acid may have one negative tag. In some embodiments, the precursor nucleic acid may have more than one negative tag. The negative tag needs to be included in the portion removed during the self-splicing process of the precursor nucleic acid. In some embodiments, the precursor nucleic acid is a cRNA enzyme that forms a circular nucleic acid and removes its terminal portion from the circular product during self-splicing. Therefore, in some embodiments, the precursor nucleic acid may have a negative tag at its 5' end or 3' end. In some embodiments, the precursor nucleic acid may have one negative tag at its 5' end. In some embodiments, the precursor nucleic acid may have one negative tag at its 3' end. In some embodiments, where both the 5' and 3' negative tags are present, the 5' and 3' negative tags may be the same or different.

[1005] In some embodiments, the precursor nucleic acid may have two negative select tags. In some embodiments, the two negative select tags on the same precursor nucleic acid are identical. In some embodiments, the two negative select tags on the same precursor nucleic acid are different. In some embodiments, the precursor nucleic acid is a cRNA enzyme. In some embodiments, the precursor nucleic acid may have two negative select tags, one at its 5' end and the other at its 3' end. For purification of circular nucleic acids generated by self-splicing of a precursor nucleic acid with two negative select tags, one or two oligonucleotide negative select probes may be used. In some embodiments, the two negative select tags are identical, and the negative select probe may be substantially complementary to the negative select tags (i.e., at least 90% complementary). In some embodiments, the two negative select tags are different, and one negative select probe substantially complementary to at least one negative select tag (i.e., at least 90% complementary) may be used. In some embodiments, the two negative select tags are different, and two different negative select probes, each substantially complementary to one of the negative select tags (i.e., at least 90% complementary), may be used.

[1006] In some embodiments, this document provides compositions comprising a first oligonucleotide negative probe and a second oligonucleotide negative probe for purifying circular nucleic acids, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid comprising first and second oligonucleotide negative tags, wherein the first negative tag and the second negative tag are at least 60% complementary to the first negative probe and the second negative probe, respectively, and are removed during the self-splicing of the precursor nucleic acid.

[1007] In some embodiments, the first and second cathode labels may be complementary to the first and second cathode probes by at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, respectively. In some embodiments, the first and second cathode labels may be at least 65% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be at least 70% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be at least 75% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be at least 80% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be at least 85% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be at least 90% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be at least 95% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be at least 98% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be at least 99% complementary to the first and second cathode probes, respectively. In some embodiments, the cathode probes and cathode labels may be about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% complementary. In some embodiments, the first and second cathode labels may be about 65% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be about 70% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be about 75% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be about 80% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be about 85% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be about 90% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be about 95% complementary to the first and second cathode probes, respectively. The first and second cathode labels may be about 98% complementary to the first and second cathode probes, respectively. The first and second cathode tags are approximately 99% complementary to the first and second cathode probes, respectively. In some embodiments, the first and second cathode tags are 100% complementary to the first and second cathode probes, respectively.

[1008] While all substantially complementary negative probe and negative tag pairs can be used to remove negatively tagged precursors, intron fragments, and / or other impurities from negatively tagged circular nucleic acids, it is important that the inclusion of the negative tag in the precursor nucleic acid has minimal impact on the secondary structure of the precursor nucleic acid, so as not to negatively affect the self-splicing activity of the precursor nucleic acid. Oligonucleotide negative tags found to be usable in the compositions and methods disclosed herein should meet the following criteria: (1) they have a minimum free energy (MFE) value in the range of -2 to 2, and (2) the change in the MFE value (ΔMFE) of the precursor nucleic acid resulting from the inclusion of the negative tag is in the range of -2 to 2. That is, the difference between the MFE value of the negatively tagged precursor nucleic acid and the MFE value of the negatively tagged precursor nucleic acid is in the range of -2 to 2.

[1009] In some implementations, the oligonucleotide negative tag has an MFE value ranging from -1.9 to 1.9, -1.8 to 1.8, -1.7 to 1.7, -1.6 to 1.6, -1.5 to 1.5, -1.4 to 1.4, -1.3 to 1.3, -1.2 to 1.2, -1.1 to 1.1, -1.0 to 1.0, -0.9 to 0.9, -0.8 to 0.8, -0.7 to 0.7, -0.6 to 0.6, -0.5 to 0.5, -0.4 to 0.4, -0.3 to 0.3, -0.2 to 0.2, or -0.1 to 0.1.

[1010] In some implementations, the oligonucleotide negative tag has an MFE value ranging from -1.9 to 0, -1.8 to 0, -1.7 to 0, -1.6 to 0, -1.5 to 0, -1.4 to 0, -1.3 to 0, -1.2 to 0, -1.1 to 0, -1.0 to 0, -0.9 to 0, -0.8 to 0, -0.7 to 0, -0.6 to 0, -0.5 to 0, -0.4 to 0, -0.3 to 0, -0.2 to 0, or -0.1 to 0. In some implementations, the oligonucleotide negative tag has an MFE value ranging from -2.0 to -1.9, -1.9 to -1.8, -1.8 to -1.7, -1.7 to -1.6, -1.6 to -1.5, -1.5 to -1.4, -1.4 to -1.3, -1.3 to -1.2, -1.2 to -1.1, -1.1 to -1.0, -1.0 to -0.9, -0.9 to -0.8, -0.8 to -0.7, -0.7 to -0.6, -0.6 to -0.5, -0.5 to -0.4, -0.4 to -0.3, -0.3 to -0.2, -0.2 to -0.1, or -0.1 to 0.

[1011] In some implementations, the oligonucleotide negative tag has an MFE value ranging from 0 to 1.9, 0 to 1.8, 0 to 1.7, 0 to 1.6, 0 to 1.5, 0 to 1.4, 0 to 1.3, 0 to 1.2, 0 to 1.1, 0 to 1.0, 0 to 0.9, 0 to 0.8, 0 to 0.7, 0 to 0.6, 0 to 0.5, 0 to 0.4, 0 to 0.3, 0 to 0.2, or 0 to 0.1. In some implementations, the oligonucleotide negative tag has an MFE value ranging from 1.9 to 2.0, 1.8 to 1.9, 1.7 to 1.8, 1.6 to 1.7, 1.5 to 1.6, 1.4 to 1.5, 1.3 to 1.4, 1.2 to 1.3, 1.1 to 1.2, 1.0 to 1.1, 0.9 to 1.0, 0.8 to 0.9, 0.7 to 0.8, 0.6 to 0.7, 0.5 to 0.6, 0.4 to 0.5, 0.3 to 0.4, 0.2 to 0.3, 0.1 to 0.2, or 0 to 0.1.

[1012] In some embodiments, the oligonucleotide negative label has an MFE value ranging from -1.8 to 0. In some embodiments, the oligonucleotide negative label has an MFE value ranging from -1.5 to 0. In some embodiments, the oligonucleotide negative label has an MFE value ranging from -1.2 to 0. In some embodiments, the oligonucleotide negative label has an MFE value ranging from -1.0 to 0. In some embodiments, the oligonucleotide negative label has an MFE value ranging from -0.8 to 0. In some embodiments, the oligonucleotide negative label has an MFE value ranging from -0.5 to 0. In some embodiments, the oligonucleotide negative label has an MFE value ranging from -0.2 to 0. In some embodiments, the oligonucleotide negative label has an MFE value ranging from -0.1 to 0. In some embodiments, the oligonucleotide negative label has an MFE value ranging from 0 to 1.8. In some embodiments, the oligonucleotide negative label has an MFE value ranging from 0 to 1.5. In some embodiments, the oligonucleotide negative label has an MFE value ranging from 0 to 1.2. In some embodiments, the oligonucleotide negative label has an MFE value ranging from 0 to 1.0. In some embodiments, the oligonucleotide negative label has an MFE value ranging from 0 to 0.8. In some embodiments, the oligonucleotide negative label has an MFE value ranging from 0 to 0.5. In some embodiments, the oligonucleotide negative label has an MFE value ranging from 0 to 0.2. In some embodiments, the oligonucleotide negative label has an MFE value ranging from 0 to 0.1.

[1013] In some implementations, the MFE value of the oligonucleotide negative tag is about -2.0, about -1.9, about -1.8, about -1.7, about -1.6, about -1.5, about -1.4, about -1.3, about -1.2, about -1.1, about -1.0, about -0.9, about -0.8, about -0.7, about -0.6, about -0.5, about -0.4, about -0.3, about -0.2, about -0.1, about 0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0. Oligonucleotide negative select tags can have an MFE value of approximately -1.8. Oligonucleotide negative select tags can have an MFE value of approximately -1.5. Oligonucleotide negative select tags can have an MFE value of approximately -1.2. Oligonucleotide negative select tags can have an MFE value of approximately -1.0. Oligonucleotide negative select tags can have an MFE value of approximately -0.8. Oligonucleotide negative select tags can have an MFE value of approximately -0.5. Oligonucleotide negative select tags can have an MFE value of approximately -0.2. Oligonucleotide negative select tags can have an MFE value of approximately -0.1. Oligonucleotide negative select tags can have an MFE value of approximately 1.8. Oligonucleotide negative select tags can have an MFE value of approximately 1.5. Oligonucleotide negative select tags can have an MFE value of approximately 1.2. Oligonucleotide negative select tags can have an MFE value of approximately 1.0. Oligonucleotide negative select tags can have an MFE value of approximately 0.8. Oligonucleotide negative select tags can have an MFE value of approximately 0.5. The oligonucleotide negative tag may have an MFE value of about 0.2. The oligonucleotide negative tag may have an MFE value of about 0.1. In a preferred embodiment, the oligonucleotide negative tag has an MFE value of about 0.

[1014] In some embodiments, the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag ranges from -2 to 2. In a preferred embodiment, the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag ranges from 0 to 2. In some implementations, the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag ranges from -1.9 to 1.9, -1.8 to 1.8, -1.7 to 1.7, -1.6 to 1.6, -1.5 to 1.5, -1.4 to 1.4, -1.3 to 1.3, -1.2 to 1.2, -1.1 to 1.1, -1.0 to 1.0, -0.9 to 0.9, -0.8 to 0.8, -0.7 to 0.7, -0.6 to 0.6, -0.5 to 0.5, -0.4 to 0.4, -0.3 to 0.3, -0.2 to 0.2, or -0.1 to 0.1.

[1015] In some implementations, the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag ranges from -1.9 to 0, -1.8 to 0, -1.7 to 0, -1.6 to 0, -1.5 to 0, -1.4 to 0, -1.3 to 0, -1.2 to 0, -1.1 to 0, -1.0 to 0, -0.9 to 0, -0.8 to 0, -0.7 to 0, -0.6 to 0, -0.5 to 0, -0.4 to 0, -0.3 to 0, -0.2 to 0, or -0.1 to 0. In some implementations, the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative label ranges from -2.0 to -1.9, -1.9 to -1.8, -1.8 to -1.7, -1.7 to -1.6, -1.6 to -1.5, -1.5 to -1.4, -1.4 to -1.3, -1.3 to -1.2, -1.2 to -1.1, -1.1 to -1.0, -1.0 to -0.9, -0.9 to -0.8, -0.8 to -0.7, -0.7 to -0.6, -0.6 to -0.5, -0.5 to -0.4, -0.4 to -0.3, -0.3 to -0.2, -0.2 to -0.1, or -0.1 to 0.

[1016] In some implementations, the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag ranges from 0 to 1.9, 0 to 1.8, 0 to 1.7, 0 to 1.6, 0 to 1.5, 0 to 1.4, 0 to 1.3, 0 to 1.2, 0 to 1.1, 0 to 1.0, 0 to 0.9, 0 to 0.8, 0 to 0.7, 0 to 0.6, 0 to 0.5, 0 to 0.4, 0 to 0.3, 0 to 0.2, or 0 to 0.1. In some implementations, the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative tag ranges from 1.9 to 2.0, 1.8 to 1.9, 1.7 to 1.8, 1.6 to 1.7, 1.5 to 1.6, 1.4 to 1.5, 1.3 to 1.4, 1.2 to 1.3, 1.1 to 1.2, 1.0 to 1.1, 0.9 to 1.0, 0.8 to 0.9, 0.7 to 0.8, 0.6 to 0.7, 0.5 to 0.6, 0.4 to 0.5, 0.3 to 0.4, 0.2 to 0.3, 0.1 to 0.2, or 0 to 0.1.

[1017] In some embodiments, the ΔMFE value ranges from -1.8 to 0. In some embodiments, the ΔMFE value ranges from -1.5 to 0. In some embodiments, the ΔMFE value ranges from -1.2 to 0. In some embodiments, the ΔMFE value ranges from -1.0 to 0. In some embodiments, the ΔMFE value ranges from -0.8 to 0. In some embodiments, the ΔMFE value ranges from -0.5 to 0. In some embodiments, the ΔMFE value ranges from -0.2 to 0. In some embodiments, the ΔMFE value ranges from -0.1 to 0. In some embodiments, the ΔMFE value ranges from 0 to 1.8. In some embodiments, the ΔMFE value ranges from 0 to 1.5. In some embodiments, the ΔMFE value ranges from 0 to 1.2. In some embodiments, the ΔMFE value ranges from 0 to 1.0. In some embodiments, the ΔMFE value ranges from 0 to 0.8. In some embodiments, the ΔMFE value ranges from 0 to 0.5. In some implementations, the ΔMFE value ranges from 0 to 0.2. In some implementations, the ΔMFE value ranges from 0 to 0.1.

[1018] In some implementations, the ΔMFE value of the precursor nucleic acid resulting from the inclusion of a negative label is: approximately -2.0, approximately -1.9, approximately -1.8, approximately -1.7, approximately -1.6, approximately -1.5, approximately -1.4, approximately -1.3, approximately -1.2, approximately -1.1, approximately -1.0, approximately -0.9, approximately -0.8, approximately -0.7, approximately -0.6, approximately -0.5, approximately -0.4, approximately -0.3, approximately -0.2, approximately -0.1, approximately 0, approximately 0.1, approximately 0, approximately 0.2, approximately 0.3, approximately 0.4, appr...

Claims

1. A composition comprising an oligonucleotide positivity probe for purifying circular nucleic acids, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid, wherein, The positive selector probe is 100% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, and at least a portion of the circular nucleic acid spans the splice region of the circular nucleic acid. The precursor nucleic acid does not contain any sequence 100% complementary to the positive selector probe. The polynucleotide sequence of the positive selector probe is shown in SEQ ID NO. 7204. The circular nucleic acid mentioned above is circular RNA (circRNA). The precursor nucleic acid mentioned therein is an RNA with group II intron self-splicing activity.

2. The composition according to claim 1, further comprising a solid surface.

3. The composition according to claim 2, wherein the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

4. The composition according to claim 2, wherein the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads or silica beads, or the solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion base materials or integral support.

5. The composition according to claim 4, wherein the polymer filler is selected from polystyrene-divinylbenzene or polymethacrylate.

6. The composition according to claim 2, wherein the anode probe is fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation.

7. The composition according to claim 6, wherein the covalent fixation is achieved by covalent linkage of NH2 and NHS-modified carboxyl groups.

8. The composition according to claim 4, wherein the solid surface is a magnetic bead.

9. The composition according to claim 8, wherein the magnetic beads are functionally modified magnetic beads.

10. The composition according to claim 9, wherein the magnetic beads are amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, or a combination of two or more of the above.

11. The composition according to claim 10, wherein the magnetic beads are NHS-modified magnetic beads.

12. The composition according to claim 8, wherein the magnetic bead is a magnetic bead with a diameter of 0.1µm to 10µm.

13. The composition according to claim 12, wherein the magnetic bead is a magnetic bead with a diameter of 0.5µm to 5µm.

14. The composition according to claim 13, wherein the magnetic bead is a magnetic bead with a diameter of 0.8µm to 1.2µm.

15. The composition according to claim 14, wherein the magnetic bead is a magnetic bead with a diameter of 1µm.

16. The composition according to claim 4, wherein the solid surface is an agarose-containing resin, and the agarose-containing resin is an agarose chromatography packing material.

17. The composition according to claim 16, wherein the diameter of the chromatography packing is 5 mm or more, and the height of the packing is 20 cm or more, or The agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, or agarose chromatography packing material with CNBr-modified surface, or a combination of two or more of the above.

18. The composition according to claim 17, wherein the diameter of the chromatography packing is 10 mm or more and the height of the packing is 30 cm or more.

19. The composition according to claim 18, wherein the diameter of the chromatography packing is 16 mm or more and the height of the packing is 40 cm or more.

20. The composition according to any one of claims 1-19, wherein the 3' end of the anode probe is covalently fixed to the solid surface; or the 5' end of the anode probe is covalently fixed to the solid surface.

21. The composition of claim 20, wherein the solid surface is a magnetic bead.

22. The composition of claim 20, wherein the covalent fixation is achieved by covalent linkage of NH2 and NHS-modified carboxyl groups.

23. The composition of claim 20, wherein the 3' end and / or 5' end of the anodic probe is further modified with a group selected from the group consisting of amino, carboxyl, NHS-carboxyl, and thiol.

24. A positive selection kit for purifying circular nucleic acids, comprising: The composition comprising an oligonucleotide positivity probe for purifying circular nucleic acids according to any one of claims 1-23, the binding solution, and the elution solution.

25. The kit of claim 24, wherein the binding solution comprises a salt, the salt comprising a cation selected from the group consisting of Ba. 2+ Ca 2+ Mg 2+ Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 2- CH3COO - Cl - ,Br - NO3 - ClO4 - I - and SCN - Or a combination thereof.

26. The kit according to claim 25, wherein the concentration of the salt in the binding solution is 50 mM-5 M.

27. The kit according to claim 25, wherein the salt is a cation, specifically Li. + Or Na + Salts.

28. The kit according to claim 24, wherein the pH range of the binding solution is 4.0-9.

0.

29. The kit according to claim 28, wherein the pH range of the binding solution is 5.0-9.

0.

30. The kit according to claim 29, wherein the pH range of the binding solution is 5.5-8.

5.

31. The kit according to claim 30, wherein the pH range of the binding solution is 6.0-8.

0.

32. The kit according to claim 31, wherein the pH range of the binding solution is 6.5-7.

5.

33. The kit according to claim 32, wherein the pH range of the binding solution is 6.5-7.

0.

34. The kit according to claim 25, wherein the binding solution further comprises a buffer substance and a chelating agent.

35. The kit of claim 34, wherein the binding solution further comprises a stabilizer.

36. The kit according to claim 27, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl.

37. The kit of claim 34, wherein the binding solution further comprises EDTA and Tris-HCl.

38. The kit according to claim 37, wherein the pH range of the binding solution is 6.0-8.

0.

39. The kit according to claim 38, wherein the pH of the binding solution is 7.

5.

40. The kit of claim 24, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.

0.

41. The kit according to claim 40, wherein the pH of the elution buffer is 7.

5.

42. A method for purifying circular nucleic acids from a sample, wherein the circular nucleic acids are generated by the self-splicing of precursor nucleic acids, the method comprising: (i) Under conditions that allow the circular nucleic acid generated by the self-splicing of the precursor nucleic acid to bind to the positive probe, a sample comprising the circular nucleic acid generated by the self-splicing of the precursor nucleic acid and a positive probe that is 100% complementary to at least a portion of the circular nucleic acid are brought into contact, wherein the positive probe is fixed on a solid surface. and (ii) Collect the portion of the anode probe that has bonded to the solid surface. At least a portion of the circular nucleic acid spans the splice region of the circular nucleic acid, and the precursor nucleic acid does not contain a sequence that is 100% complementary to the positive probe. The polynucleotide sequence of the positive probe is shown in SEQ ID NO. 7204. The circular nucleic acid mentioned above is circular RNA (circRNA). The precursor nucleic acid mentioned therein is an RNA with group II intron self-splicing activity.

43. The method of claim 42, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked together: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II introns; (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

44. The method of claim 43, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 3' intron fragment; (b) exon fragment 2 (E2); (c) a adapter sequence; (d) a target sequence; (e) an adapter sequence; (f) exon fragment 1 (E1); and (g) a 5' intron fragment; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II introns; (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

45. The method of claim 43, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a target sequence; (e) exon fragment 1 (E1); (f) a 5' intron fragment; and (g) a 3' homologous arm; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II introns; (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

46. ​​The method of claim 42, wherein the precursor RNA comprises, from 5' to 3', the following elements operatively linked: (a) a 5' homologous arm; (b) a 3' intron fragment; (c) exon fragment 2 (E2); (d) a adapter sequence; (e) a target sequence; (f) an adapter sequence; (g) exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II introns; (2) E1 is the 5' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; (3) The E2 is the 3' adjacent exon fragment of the group II intron, with a length ≥ 0 nucleotides; and (4) The target sequence is not present, or it is a protein-coding sequence, a non-coding sequence, or a combination of both.

47. The method according to any one of claims 42-46, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II introns of the unpaired region into two fragments.

48. The method of claim 47, wherein the 5' intron fragment and the 3' intron fragment are obtained by cleaving group II introns at the ring region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or The 5' and 3' intron segments are obtained by cutting group II introns in linear regions between domain 1 and domain 2, between domain 2 and domain 3, between domain 3 and domain 4, between domain 4 and domain 5, or between domain 5 and domain 6.

49. The method of claim 47, wherein the group II intron comprises one or more nucleotide modifications relative to its wild-type form, and the modifications are selected from one or more deletions, substitutions, and additions.

50. The method of claim 49, wherein the modification comprises modification of one or more EBS sequences of group II introns, wherein the EBS sequences are complementary to one or more regions of corresponding length in the target sequence at 100% nucleotide positions.

51. The method of claim 49, wherein the modification is a modification of two EBS sequences of a group II intron, the EBS sequences being EBS1 and EBS3, wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at 100% nucleotide positions.

52. The method of claim 51, wherein the two regions are located at opposite ends of the target sequence.

53. The method of claim 49, wherein the modification is a modification of two EBS sequences of a group II intron, the EBS sequences being EBS1' and EBS3', wherein the EBS sequences are complementary to two regions of corresponding length in the target sequence at 100% nucleotide positions.

54. The method of claim 53, wherein the two regions are located at opposite ends of the target sequence.

55. The method of claim 49, wherein the modification is a modification of the EBS1 and / or δ sequence of a group II intron, or a modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence on 100% of the nucleotides.

56. The method of claim 55, wherein the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream are complementary to a region of corresponding length in the target sequence on 100% of the nucleotides.

57. The method of claim 55, wherein the EBS1 and / or δ sequence is complementary to a region of corresponding length in the target sequence on 100% of the nucleotides, and the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence on 100% of the nucleotides.

58. The method of claim 55, wherein the modification includes the partial or complete absence of domain 4.

59. The method of claim 58, wherein all of the structural domains 4 are missing.

60. The method of claim 58, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence.

61. The method of claim 60, wherein the near-scarless circRNA has a scar region of length equal to or less than 20 nucleotides.

62. The method of claim 61, wherein the near-scarless circRNA has a scar region of length equal to or less than 19 nucleotides.

63. The method of claim 62, wherein the near-scarless circRNA has a scar region of length equal to or less than 18 nucleotides.

64. The method of claim 63, wherein the near-scarless circRNA has a scar region of length equal to or less than 17 nucleotides.

65. The method of claim 64, wherein the near-scarless circRNA has a scar region of length equal to or less than 16 nucleotides.

66. The method of claim 65, wherein the near-scarless circRNA has a scar region of length equal to or less than 15 nucleotides.

67. The method of claim 66, wherein the near-scarless circRNA has a scar region of length equal to or less than 14 nucleotides.

68. The method of claim 67, wherein the near-scarless circRNA has a scar region of length equal to or less than 13 nucleotides.

69. The method of claim 68, wherein the near-scarless circRNA has a scar region of length equal to or less than 12 nucleotides.

70. The method of claim 69, wherein the near-scarless circRNA has a scar region of length equal to or less than 11 nucleotides.

71. The method of claim 70, wherein the near-scarless circRNA has a scar region of length equal to or less than 10 nucleotides.

72. The method of claim 71, wherein the near-scarless circRNA has a scar region of length equal to or less than 9 nucleotides.

73. The method of claim 72, wherein the near-scarless circRNA has a scar region of length equal to or less than 8 nucleotides.

74. The method of claim 73, wherein the near-scarless circRNA has a scar region of length equal to or less than 7 nucleotides.

75. The method of claim 74, wherein the near-scarless circRNA has a scar region of length equal to or less than 6 nucleotides.

76. The method of claim 75, wherein the near-scarless circRNA has a scar region of length equal to or less than 5 nucleotides.

77. The method of claim 76, wherein the near-scarless circRNA has a scar region of length equal to or less than 4 nucleotides.

78. The method of claim 77, wherein the near-scarless circRNA has a scar region of length equal to or less than 3 nucleotides.

79. The method of claim 78, wherein the near-scarless circRNA has a scar region of length equal to or less than 2 nucleotides.

80. The method of claim 79, wherein the near-scarless circRNA has a scar region of length equal to or less than 1 nucleotide.

81. The method of claim 53, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

82. The method according to any one of claims 43-81, wherein the group II introns are group II introns derived from microorganisms.

83. The method of claim 82, wherein the group II intron is Cte1.

84. The method of claim 82, wherein the group II introns are polynucleotide sequences selected from SEQ ID NO:33-41.

85. The method according to any one of claims 43-81, wherein the 3' intron fragment is a polynucleotide sequence selected from SEQ ID NO: 42-52; and / or The 5' intron fragment is a polynucleotide sequence selected from SEQ ID NO:75-88.

86. The method according to any one of claims 43-81, wherein the length of E1 and / or E2 is 0-20 nucleotides.

87. The method of claim 86, wherein the length of E1 and / or E2 is 0-10 nucleotides.

88. The method according to claim 86, wherein E2 is a polynucleotide sequence selected from SEQ ID NO:53-63 and SEQ ID NO:7211-7212; and / or E1 is a polynucleotide sequence selected from SEQ ID NO:64-74 and SEQ ID NO:7213.

89. The method of claim 86, wherein E1, E2, or both are 0 nucleotides in length.

90. The method according to any one of claims 43-81, wherein the target sequence is a non-coding sequence selected from the group consisting of: spacer sequences of SEQ ID NO:4-6, polyA sequences, poly-AC sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), small RNA binding sites, translation regulatory sequences, and protein binding sites.

91. The method according to any one of claims 43-81, wherein the target sequence comprises: an internal ribosome entry site (IRES)-like polynucleotide sequence or a natural IRES sequence, and a protein-coding sequence, wherein the protein-coding sequence encodes a therapeutic product.

92. The method according to any one of claims 43-81, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

93. The method according to claim 92, wherein at least one of the modified RNA nucleotide and / or modified nucleoside is m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), Ψ (pseudouridine), m1A (1-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine); m2A (2-methyladenosine), Am (2'-O-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine), ms2i6A (2-methylthio-N6-isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl)adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine), g6A (N6-glycylcarbamoyladenosine), t6A (N6-threonylcarbamoyladenosine), ms2t6A (2-methylthio-N6-threonylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonylcarbamoyladenosine), hn6A (N6-hydroxyn-valinecarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy (n-Valylcarbamoyladenosine), Ar(p)(2'-O-ribosyladenosine (phosphate)), I(inosine), m1I(1-methylinosine), m1hn(1,2'-O-dimethylinosine), m3C(3-methylcytidine), Cm(2'-O-methylcytidine), s2C(2-thiocytidine), ac4C(N4-acetylcytidine), (5-formylcytidine), m5Cm(5,2'-O-dimethylcytidine), ac4Cm(N4-acetyl-2'-O-methylcytidine), k2C(2-lysylcytidine), m1G(1-methylguanosine), m2G(N2-methylguanosine), m7G(7-methylguanosine), Gm(2'-O-methylguanosine), m2 2G(N2,N2-dimethylguanosine), m2Gm(N2,2'-O-dimethylguanosine), m2aGm(N2,N2,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW(huaitoside), oayW(peroxyhuaitoside), OHyW(hydroxyhuaitoside), OHyW*(undermodified hydroxyhuaitoside), imG(huaitoside), mimG(methylhuaitoside), Q(huaitoside), oQ(epoxyhuaitoside), galQ(galactosyl-huaitoside), manQ(mannosyl-huaitoside), preQo(7-cyano-7-deazoguanosine), preQi(7-aminomethyl-7-deazoguanosine), G+(archopurinol), D(dihydrouridine), m5Um(5,2'-O-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-O-methyluridine), acp3U (3-(3-amino-3-carboxypropyl)uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), nmm5U (5-carbamoylmethyluridine), nmm5Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm5U (5-carboxymethylaminomethyluridine), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6,N6-dimethyladenosine), Im (2'-O-methylinosine), m4C (N4-methylcytidine), m4Cm (N4,2'-O-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6,2'-O-dimethyladenosine), m6 2Am (N6,N6,O-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-O-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-O-methylcytidine), m'Gm (l,2'-O-dimethylguanosine), m'Am (l,2'-O-dimethyladenosine), rm 5U (5-taurate methyluridine), τm5s2U (5-taurate methyl-2-thiouridine), imG-14 (4-demethylwoyoside), imG2 (isowoyoside), or ac6A (N6-acetyladenosine).

94. The method according to any one of claims 43-81, wherein the precursor RNA comprises unmodified RNA nucleotides and / or unmodified nucleosides; or the precursor RNA does not comprise modified RNA nucleotides and / or modified nucleosides.

95. The method of claim 42, wherein the solid surface is a bead, resin, tube, nanoparticle, chip, plate, or paper.

96. The method according to claim 95, wherein the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads, or silica beads, or The solid surface is a resin containing agarose, carbohydrate-based materials, polymer fillers, silica, glass particles, infusion-based materials, or an integral support.

97. The method of claim 96, wherein the polymer filler is selected from polystyrene-divinylbenzene or polymethacrylate.

98. The method of claim 42, wherein the anode probe is fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation.

99. The method of claim 98, wherein the covalent fixation is achieved by covalently linking NH2 and NHS-modified carboxyl groups.

100. The method of claim 96, wherein the solid surface is a magnetic bead.

101. The method according to claim 100, wherein the magnetic bead is a functionally modified magnetic bead.

102. The method according to claim 101, wherein the magnetic beads are amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleamide-modified magnetic beads, CNBr-modified magnetic beads, or a combination of two or more of the above.

103. The method according to claim 102, wherein the magnetic bead is an NHS-modified magnetic bead.

104. The method according to claim 96, wherein the magnetic bead is a magnetic bead with a diameter of 0.1µm to 10µm.

105. The method according to claim 104, wherein the magnetic bead is a magnetic bead with a diameter of 0.5µm to 5µm.

106. The method according to claim 105, wherein the magnetic bead is a magnetic bead with a diameter of 0.8µm to 1.2µm.

107. The method according to claim 106, wherein the magnetic bead is a magnetic bead with a diameter of 1µm.

108. The method according to claim 96, wherein the solid surface is a resin containing agarose, and the resin containing agarose is an agarose chromatography packing material.

109. The method according to claim 108, wherein the diameter of the chromatography packing is 5 mm or more, and the height of the packing is 20 cm or more, or The agarose chromatography packing material is an agarose chromatography packing material with amino-modified surface, agarose chromatography packing material with carboxyl-modified surface, agarose chromatography packing material with NHS-modified surface, agarose chromatography packing material with maleamide-modified surface, agarose chromatography packing material with CNBr-modified surface, or a combination of two or more of the above.

110. The method according to claim 109, wherein the diameter of the chromatography packing is 10 mm or more and the height of the packing is 30 cm or more.

111. The method according to claim 110, wherein the diameter of the chromatography packing is 16 mm and the height of the packing is 40 cm.

112. The method of claim 42, wherein the 3' end of the anode probe is covalently fixed to the solid surface; or the 5' end of the anode probe is covalently fixed to the solid surface.

113. The method according to claim 112, wherein the solid surface is a magnetic bead.

114. The method of claim 112, wherein the covalent fixation is achieved by covalently linking NH2 and NHS-modified carboxyl groups.

115. The method of claim 112, wherein the 3' end and / or 5' end of the anode probe is further modified with a group selected from: amino, carboxyl, NHS-carboxyl, mercapto.

116. The method of claim 42, wherein a sample of the circular nucleic acid to be purified is contacted with the cation-selective probe at a pH range of 4.0-9.

0.

117. The method of claim 116, wherein the pH range is 5.0-9.0; or The sample is in contact with the anode probe at a temperature of 0℃-60℃; or The sample is in contact with the cation-selected probe in a binding solution comprising salts, a buffer, a chelating agent, and optionally a stabilizer. Further, the salts comprise cations selected from the group consisting of: Ba... 2+ Ca 2+ Mg 2 + Mn 2+ Fe 2+ Cu 2+ Zn 2+ Cd 2+ Pb 2+ Li + Cs + Na + K + 、Rb + and NH4 + and / or anions selected from the following group: PO4 3- SO4 2- CH3COO - Cl - ,Br - NO3 - ClO4 - I - and SCN - Or a combination thereof.

118. The method of claim 117, wherein the pH range is 5.5-8.

5.

119. The method of claim 118, wherein the pH range is 6.0-8.

0.

120. The method of claim 119, wherein the pH range is 6.5-7.

5.

121. The method of claim 120, wherein the pH range is 7.0-7.

5.

122. The method according to claim 117, wherein the concentration of the salt in the binding solution is 50 mM-5 M.

123. The method according to claim 117, wherein the salt is a cation, specifically Li. + Or Na + Salts.

124. The method according to claim 117, wherein the binding liquid comprises 0.1M-0.5M LiCl or NaCl.

125. The method of claim 117, wherein the binding solution further comprises EDTA and Tris-HCl.

126. The method of claim 117, wherein the pH range of the binding solution is 6.0-8.

0.

127. The method of claim 126, wherein the pH of the binding solution is 7.

5.

128. The method of claim 42, wherein the method further comprises the step of using an eluent to elute a portion of the anode probe bound to the solid surface to recover the solid surface coupled with the anode probe for reuse.

129. The method of claim 128, wherein the eluent comprises EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.

0.

130. The method of claim 129, wherein the pH of the eluent is 7.

5.

131. The method of claim 42, wherein the method is scalable and may include a continuous flow process.

132. The method of claim 131, wherein the continuous flow process is performed using a liquid chromatograph (LC) or an enzyme-linked immunosorbent assay (ELISA) reader.

133. The method according to claim 132, wherein the LC is a rapid liquid chromatograph (FPLC) or a high-performance liquid chromatograph (HPLC).

134. A method for purifying circular nucleic acids from a sample, comprising the following steps: Couple the magnetic bead with the anode probe; The IVT reaction solution containing the circular nucleic acid obtained by in vitro transcription was incubated with magnetic beads coupled with a positively selected probe. The magnetic beads are collected and eluted to obtain the eluted circular nucleic acid, and the magnetic beads coupled with the positive probe are recovered. The circular nucleic acid is generated through the self-splicing of the precursor nucleic acid. The positive selector probe is 100% complementary to at least a portion of the circular nucleic acid generated by the self-splicing of the precursor nucleic acid, and at least a portion of the circular nucleic acid spans the splice portion of the circular nucleic acid. The precursor nucleic acid does not contain any sequence 100% complementary to the positive selector probe. The polynucleotide sequence of the positive selector probe is shown in SEQ ID NO. 7204. The circular nucleic acid mentioned above is circular RNA (circRNA). The precursor nucleic acid mentioned therein is an RNA with group II intron self-splicing activity.

135. The method according to claim 134, wherein the IVT reaction solution is incubated with magnetic beads coupled with a positively selected probe in a binding solution, the magnetic beads are eluted with an elution buffer to obtain the eluted circular nucleic acid, and the magnetic beads coupled with the positively selected probe are recovered, wherein the elution buffer and the binding buffer are the elution buffer or binding buffer according to any one of claims 117-130, the polynucleotide sequence of the positively selected probe is shown in SEQ ID NO. 7204, wherein the circular nucleic acid is a circular RNA (circRNA), and wherein the precursor nucleic acid is RNA with group II intron self-splicing activity.

Images