Negative selection method and system for purifying cyclic nucleic acids

Abstract

Disclosed herein are compositions comprising an oligonucleotide cathodic probe for purifying a cyclic nucleic acid wherein the cyclic nucleic acid is produced by self-splicing of a precursor nucleic acid comprising an oligonucleotide cathodic tag wherein the cathodic tag is at least 90% complementary to the cathodic probe, wherein the negative tag is removed during the self-splicing of the precursor nucleic acid, preferably the negative tag is complementary to the negative probe by 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%, further preferably 100%. The invention also relates to a related purification method using the negative selection probe composition.

Description

[0001] This is a divisional application of the case with an application date of January 10, 2024, an invention title of "Negative Selection Methods and Systems for Purifying Circular Nucleic Acids", and an application number of 2024100398987.

[0002] 1. Related Applications

[0003] This application has no related priority applications.

[0004] 2. Incorporation of Sequence Listing by Reference

[0005] The content of the electronic sequence listing TPG03609-sequencing list.xml (file size: 6.17 MB; created on January 10, 2024) is incorporated herein by reference in its entirety. Technical Field

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

[0007] Circular RNAs (circRNAs) are a class of RNA molecules formed by head-to-tail connection and have been shown to have multiple 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.

[0008] Purity is a key factor in RNA therapy because contaminants often cause immunogenicity. Although there are available methods for purifying RNA, there is still a lack of effective methods and systems for purifying RNA, especially circRNAs. The compositions, methods, and systems provided herein meet this need and provide related advantages. Summary of the Invention

[0010] 1. A composition for purifying circular nucleic acids, comprising an oligonucleotide negative selection probe, wherein the circular nucleic acid is generated by self-splicing of a precursor nucleic acid comprising an oligonucleotide negative selection tag, wherein the negative selection tag is at least 90% complementary to the negative selection probe, and 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.

[0011] 2. The composition according to item 1, wherein the precursor nucleic acid comprises the negative selection tag at its 5' end; and / or the precursor nucleic acid comprises the negative selection tag at its 3' end.

[0012] Under the condition that the negative selection tags at the 5' end and the 3' end are both present, the negative selection tags at the 5' end and the 3' end may be the same or different.

[0013] 3. The composition according to item 1 or 2, wherein the circular nucleic acid is circular RNA (circRNA), and preferably the precursor nucleic acid is an RNA having group II intron self-splicing activity.

[0014] 4. The composition according to item 3, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 3' intron fragment; (b) an exon fragment 2 (E2); (c) a target sequence; (d) an exon fragment 1 (E1); and (e) a 5' intron fragment, wherein:

[0015] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a group II intron, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0016] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides;

[0017] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and

[0018] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0019] 5. The composition according to item 3, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0020] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a Group II intron, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the Group II intron;

[0021] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥ 0 nucleotides;

[0022] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥ 0 nucleotides; and

[0023] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0024] 6. The composition according to item 3, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0025] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a Group II intron, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the Group II intron;

[0026] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥ 0 nucleotides;

[0027] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥ 0 nucleotides; and

[0028] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0029] 7. The composition according to item 3, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0030] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II introns;

[0031] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0032] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides; and

[0033] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0034] 8. The composition according to any one of items 1-7, wherein the lengths of the negative selection tag and the negative selection probe are 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides; preferably, the lengths of the negative selection tag and the negative selection probe are 20-30 nucleotides.

[0035] 9. The composition according to any one of items 1-8, wherein the negative selection probe is an RNA probe; or the negative selection probe is a DNA probe.

[0036] 10. The composition according to any one of items 1-9, wherein the negative selection tag has a polynucleotide sequence comprising SEQ ID NO: n, and the negative selection probe has a polynucleotide sequence comprising SEQ ID NO: n + 1100, where n is an integer from 135 to 1234; or wherein the negative selection tag has a polynucleotide sequence comprising SEQ ID NO: m, and the negative selection probe has a polynucleotide sequence comprising SEQ ID NO: m + 1210, where m is an integer from 2335 to 3544 or 4755 to 5964.

[0037] 11. The composition according to any one of items 1-9, the negative selection tag has a polynucleotide sequence comprising any one of the polynucleotide sequences shown in SEQ ID NOs: 7189-7202; or the negative selection probe has a polynucleotide sequence comprising any one of the polynucleotide sequences shown in SEQ ID NOs: 7175-7188.

[0038] 12. The composition according to item 11, wherein the negative selection tag and the negative selection probe of the composition are selected from any one of the following combinations:

[0039] (a) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7189, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7175;

[0040] The polynucleotide sequence of the negative selection tag described in (b) is as shown in SEQ ID NO:7190, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7176;

[0041] The polynucleotide sequence of the negative selection tag described in (c) is as shown in SEQ ID NO:7191, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7177;

[0042] The polynucleotide sequence of the negative selection tag described in (d) is as shown in SEQ ID NO:7192, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7178;

[0043] The polynucleotide sequence of the negative selection tag described in (e) is as shown in SEQ ID NO:7193, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7179;

[0044] The polynucleotide sequence of the negative selection tag described in (f) is as shown in SEQ ID NO:7194, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7180;

[0045] The polynucleotide sequence of the negative selection tag described in (g) is as shown in SEQ ID NO:7195, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7181;

[0046] The polynucleotide sequence of the negative selection tag described in (h) is as shown in SEQ ID NO:7196, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7182;

[0047] The polynucleotide sequence of the negative selection tag described in (i) is as shown in SEQ ID NO:7197, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7183;

[0048] The polynucleotide sequence of the negative selection tag described in (j) is as shown in SEQ ID NO:7198, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7184;

[0049] The polynucleotide sequence of the negative selection tag described in (k) is as shown in SEQ ID NO:7199, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7185;

[0050] The polynucleotide sequence of the negative selection tag described in (l) is as shown in SEQ ID NO:7200, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7186;

[0051] (m) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7201, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7187;

[0052] (n) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7202, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7188.

[0053] 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 a Group II intron in an unpaired region into two fragments.

[0054] 14. The composition according to item 13, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the Group II intron at the loop region of the stem - loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0055] the 5' intron fragment and the 3' intron fragment are obtained by cutting the Group II intron at the linear region 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.

[0056] 15. The composition according to item 13 or 14, wherein the Group II intron contains one or more nucleotide modifications relative to its wild - type form, and the modifications are selected from one or more of deletions, substitutions, and additions,

[0057] preferably the modifications include modifications of one or more EBS sequences of the Group II intron, wherein the EBS sequences are complementary paired with one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0058] 16. The composition according to item 15, wherein the modifications are modifications of two EBS sequences (such as EBS1 and EBS3) of the Group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably the two regions are located at both ends of the target sequence respectively.

[0059] 17. The composition according to item 15, wherein the modifications are modifications of two EBS sequences (such as EBS1' and EBS3') of the Group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably the two regions are located at both ends of the target sequence respectively.

[0060] 18. The composition according to item 15 or 16, wherein the modification is a modification of the EBS1 and / or δ sequence of group II intron, or a modification of the EBS1' and / or δ'' sequence, wherein the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at 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 are complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides,

[0061] Preferably, the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at 100% 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 are complementary paired with a region of corresponding length in the target sequence at 100% of the nucleotides.

[0062] 19. The composition according to item 14, wherein the modification includes the deletion of part or all of domain 4, such as the deletion of the intron-encoded protein (IEP) sequence in domain 4, preferably the entire deletion of domain 4; or

[0063] The modification includes the deletion of an open reading frame (ORF).

[0064] 20. The composition according to item 16, wherein the precursor RNA is capable of forming a near scarless circRNA of the target sequence, preferably the near scarless circRNA has a scar region with a 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.

[0065] 21. The composition according to item 17, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0066] 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 further preferably the group II intron contains a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0067] 23. The composition according to any one of items 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

[0068] 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 NOs: 75 - 88.

[0069] 24. The composition according to any one of items 4 - 23, 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;

[0070] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NOs: 53 - 63 and SEQ ID NOs: 7211 - 7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NOs: 64 - 74 and SEQ ID NO: 7213.

[0071] 25. The composition according to item 24, wherein the length of E1, E2 or both is 0 nucleotides.

[0072] 26. The composition according to any one of items 4 - 25, wherein the target sequence is a non - coding sequence selected from the group consisting of: the spacer sequences of SEQ ID NOs: 4 - 6, polyA sequence, poly - A - C sequence, polyC sequence, polyU sequence, IRES, ribosome - binding site, aptamer sequence, RNA scaffold, riboswitch, ribozymes other than self - splicing ribozymes, antisense oligonucleotide (ASO), scaffold, small RNA - binding site, translation regulatory sequence and protein - binding site.

[0073] 27. The composition according to any one of items 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 encodes a therapeutic product.

[0074] 28. The composition according to any one of items 1 - 27, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0075] 29. The composition according to item 28, wherein at least one of the modified RNA nucleotides and / or modified nucleosides 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-hydroxy-norvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine), 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 (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 (wybutosine), oayW (oxywybutosine), OHyW (hydroxywybutosine), OHyW* (under-modified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archaeosine), 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 (methyl ester of uridine 5-oxyacetic acid), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (methyl ester of 5-(carboxyhydroxymethyl)uridine), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (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 (1,2'-O-dimethylguanosine), m'Am (1,2'-O-dimethyladenosine), rm 5U (5-tauromethyluridine), τm5s2U (5-tauromethyl-2-thiouridine), imG-14 (4-demethylwyeoside), imG2 (isowyeoside), or ac6A (N6-acetyladenosine).

[0076] 30. The composition according to any one of items 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.

[0077] 31. The composition according to any one of items 1 - 30 further comprises a solid surface,

[0078] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate or paper,

[0079] More preferably, the solid surface is a magnetic bead, agarose bead, streptavidin - coated bead, colloidal bead, glass bead, polymer bead, methacrylate bead, polystyrene bead, latex bead or silica bead, or

[0080] More preferably, the solid surface is a resin containing agarose, carbohydrate - based material, polymer filler, silica, glass particles, perfusion - based material or monolithic support, and the polymer filler is optionally selected from polystyrene - divinylbenzene or polymethacrylate.

[0081] 32. The composition according to item 31, wherein the negative selection probe is immobilized on the solid surface by physical adsorption, covalent immobilization or affinity immobilization, and preferably the covalent immobilization is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups.

[0082] 33. The composition according to item 31 or 32, wherein the solid surface is a magnetic bead,

[0083] Preferably, the magnetic bead is a functional - group - modified magnetic bead, more preferably an amino - modified magnetic bead, carboxyl - modified magnetic bead, NHS - modified magnetic bead, maleimide - modified magnetic bead, CNBr - modified magnetic bead, magnetic bead modified with an affinity or specific group, or a combination of two or more of the above, and more preferably an NHS - modified magnetic bead, or

[0084] Preferably, the magnetic bead is a hydrophilic magnetic bead; or

[0085] Preferably, the magnetic bead has a diameter in the range of 0.1 μm to 10 μm, more preferably 0.5 μm to 5 μm, more preferably 0.8 μm to 1.2 μm, and more preferably 1 μm.

[0086] 34. The composition according to item 31 or 32, wherein the solid surface is an agarose chromatography packing material,

[0087] Preferably, the diameter of the chromatography packing material is more than 5 mm and the packing height is more than 20 cm. More preferably, the diameter of the chromatography packing material is more than 10 mm and the packing height is more than 30 cm. More preferably, the diameter of the chromatography packing material is 16 mm and the packing height is 40 cm.

[0088] Furthermore, the agarose chromatography packing material is an agarose chromatography packing material with amino modification on the surface, a carboxyl-modified agarose chromatography packing material, an NHS-modified agarose chromatography packing material, a maleimide-modified agarose chromatography packing material, a CNBr-modified agarose chromatography packing material, an agarose chromatography packing material with affinity or specific group modification, or a combination of two or more of the above. Further preferably, it is an NHS-modified agarose chromatography packing material.

[0089] 35. The composition according to any one of items 31-34, wherein the 3'-end of the negative selection probe is covalently fixed to the solid surface; or the 5'-end of the negative selection 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 the carboxyl group modified by NHS.

[0090] 36. The composition according to any one of items 31-35, wherein the 3'-end and / or 5'-end of the negative selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto.

[0091] 37. The composition according to any one of items 1-36, wherein the minimum free energy (MFE) value of the negative selection tag ranges from -2 to 2, and the change in the MFE value (ΔMFE) of the precursor nucleic acid caused by including the tag ranges from -2 to 2.

[0092] 38. The composition according to item 37, wherein the MFE value of the negative selection tag is about 0.

[0093] 39. The composition according to item 37 or 38, wherein the range of the ΔMFE value of the precursor nucleic acid caused by including the negative selection tag is 0-2, and preferably the ΔMFE value of the precursor nucleic acid caused by including the negative selection tag is about 0.

[0094] 40. A negative selection kit for purifying circular nucleic acids, which comprises: the composition for purifying circular nucleic acids containing an oligonucleotide negative selection probe according to any one of items 1-39, a binding solution, and an elution solution.

[0095] 41. The kit according to item 40, wherein the binding solution comprises salts, and the salts comprise cations selected from the following group: Ba 2+ 、Ca 2+ 、Mg 2+ 、Mn 2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、Cd 2+ 、Pb 2+ 、Li + 、Cs + 、Na+ , K + , Rb + and NH 4 + , and / or anions selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the concentration of the salts in the binding solution is 50 mM - 5 M,

[0096] More preferably, the salts have a cation of Li + or Na + .

[0097] 42. The kit according to item 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;

[0098] Preferably, the binding solution further comprises a buffering substance, a chelating agent, and optionally a stabilizing agent.

[0099] 43. The kit according to item 41 or 42, wherein the binding solution comprises 0.1 M - 0.5 M 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.

[0100] 44. The kit according to any one of items 40 - 43, wherein the concentration of the salts in the eluent is 0 M, and the salts include 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 NH 4 + , and / or an anion selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the salts are salts with cations being Li + or Na + .

[0101] Preferably, the eluent further comprises a buffering substance, a chelating agent, and optionally a stabilizer.

[0102] 45. The kit according to item 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

[0103] the pH range of the eluent is 2.0 - 4.0, preferably the pH range is 2.5 - 3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5 - 3.0.

[0104] 46. A negative selection kit for purifying circular nucleic acids, comprising: magnetic beads coupled with a negative selection probe, an eluent, and a binding solution, wherein the negative selection probe is at least 90% complementary to a negative selection tag, and the circular nucleic acid is generated by 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.

[0105] 47. The kit according to item 46, wherein the negative selection probe and the negative selection tag are the negative selection probe and the negative selection tag involved in any one of items 1 - 39, or

[0106] the eluent and the binding solution are the eluent and the binding solution involved in any one of items 40 - 45.

[0107] 48. The kit according to item 46 or 47, wherein the magnetic beads immobilized with the negative selection probes can be reused.

[0108] 49. A method for purifying circular nucleic acids from a sample, wherein the circular nucleic acids are generated by self-splicing of a precursor nucleic acid, and wherein the precursor nucleic acid has an oligonucleotide negative selection tag that is removed during the self-splicing process, the method comprising:

[0109] (i) contacting the sample and an oligonucleotide negative selection probe that is at least 90% complementary to the negative selection tag under conditions that permit binding of the negative selection tag to the negative selection probe, wherein the negative selection probe is immobilized on a solid surface; and

[0110] (ii) collecting the unbound portion of the sample,

[0111] 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.

[0112] 50. The method according to item 49, wherein the precursor nucleic acid comprises the negative selection tag at its 5' end; or the precursor nucleic acid comprises the negative selection tag at its 3' end,

[0113] Under the condition that the negative selection tag at the 5' end and the negative selection tag at the 3' end are both present, the negative selection tag at the 5' end and the negative selection tag at the 3' end may be the same or different.

[0114] 51. The method according to item 49 or 50, wherein the circular nucleic acid is circular RNA (circRNA), and preferably the precursor nucleic acid is an RNA having group II intron self-splicing activity.

[0115] 52. The method according to item 51, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 3' intron fragment; (b) an exon fragment 2 (E2); (c) a target sequence; (d) an exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0116] (1) both the 5' intron fragment and the 3' intron fragment are fragments of a group II intron, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0117] (2) the E1 is a 5'-adjacent exon fragment of the group II intron, and its length ≥ 0 nucleotides;

[0118] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length ≥ 0 nucleotides; and

[0119] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0120] 53. The method according to item 51, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 3'-intron fragment; (b) an exon fragment 2 (E2); (c) a linker sequence; (d) a target sequence; (e) a linker sequence; (f) an exon fragment 1 (E1); and (g) a 5'-intron fragment; wherein:

[0121] (1) Both the 5'-intron fragment and the 3'-intron fragment are fragments of the Group II intron, and the 5'-intron fragment is located on the 5' side of the 3'-intron fragment in the Group II intron;

[0122] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length ≥ 0 nucleotides;

[0123] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length ≥ 0 nucleotides; and

[0124] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0125] 54. The method according to item 51, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 5'-homologous arm; (b) a 3'-intron fragment; (c) an exon fragment 2 (E2); (d) a target sequence; (e) an exon fragment 1 (E1); (f) a 5'-intron fragment; and (g) a 3'-homologous arm; wherein:

[0126] (1) Both the 5'-intron fragment and the 3'-intron fragment are fragments of the Group II intron, and the 5'-intron fragment is located on the 5' side of the 3'-intron fragment in the Group II intron;

[0127] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length ≥ 0 nucleotides;

[0128] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length ≥ 0 nucleotides; and

[0129] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0130] 55. The method according to item 51, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 5' homologous arm; (b) a 3' intron fragment; (c) an exon fragment 2 (E2); (d) a linker sequence; (e) a target sequence; (f) a linker sequence; (g) an exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0131] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a group II intron, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0132] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0133] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides; and

[0134] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0135] 56. The method according to any one of items 49-55, wherein the length of the negative selection tag and the negative selection probe is 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides; preferably, the length of the negative selection tag and the negative selection probe is 20-30 nucleotides.

[0136] 57. The method according to any one of items 49-56, wherein the negative selection probe is an RNA probe; or the negative selection probe is a DNA probe.

[0137] 58. The method according to any one of items 49-57, wherein the negative selection tag has a polynucleotide sequence comprising SEQ ID NO:n, and the negative selection probe has a polynucleotide sequence comprising SEQ ID NO:n+1100, where n is an integer from 135 to 1234; or wherein the negative selection tag has a polynucleotide sequence comprising SEQ ID NO:m, and the negative selection probe has a polynucleotide sequence comprising SEQ ID NO:m+1210, where m is an integer from 2335 to 3544 or 4755 to 5964.

[0138] 59. The method according to any one of items 49-58, wherein the negative selection tag has a polynucleotide sequence comprising any one selected from SEQ ID NO:7189-7202; or the negative selection probe has a polynucleotide sequence comprising any one selected from SEQ ID NO:7175-7188.

[0139] 60. The method according to item 59, wherein the negative selection tag and the negative selection probe are selected from any of the following combinations:

[0140] (a) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7189, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7175;

[0141] (b) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7190, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7176;

[0142] (c) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7191, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7177;

[0143] (d) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7192, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7178;

[0144] (e) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7193, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7179;

[0145] (f) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7194, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7180;

[0146] (g) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7195, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7181;

[0147] (h) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7196, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7182;

[0148] (i) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7197, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7183;

[0149] (j) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7198, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7184;

[0150] (k) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7199, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7185;

[0151] (l) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7200, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7186;

[0152] (m) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7201, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7187;

[0153] (n) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7202, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7188.

[0154] 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 a group II intron of an unpaired region into two fragments.

[0155] 62. The method according to item 61, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron at the loop region of the stem - loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0156] the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron at the linear region 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.

[0157] 63. The method according to item 61 or 62, wherein the group II intron contains one or more nucleotide modifications relative to its wild - type form, and the modifications are selected from one or more of deletion, substitution, and addition,

[0158] preferably, the modifications include modifications of one or more EBS sequences of the group II intron, wherein the EBS sequences are complementary paired with one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0159] 64. The method according to item 63, wherein the modification is the modification of two EBS sequences (such as EBS1 and EBS3) of group II introns, and the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are respectively located at both ends of the target sequence.

[0160] 65. The method according to item 63, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of group II introns, and the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are respectively located at both ends of the target sequence.

[0161] 66. The method according to item 64 or 65, wherein the modification is the modification of EBS1 and / or δ sequence of group II introns, or the modification of EBS1' and / or δ” sequence, and the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides. Optionally, the modification is the modification of EBS1 and / or δ sequence and its upstream sequence, and the EBS1 and / or δ sequence and its upstream are complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides.

[0162] Preferably, the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at 100% of the nucleotides. Optionally, the modification is the modification of EBS1 and / or δ sequence and its upstream sequence, and the EBS1 and / or δ sequence and its upstream are complementary paired with a region of corresponding length in the target sequence at 100% of the nucleotides.

[0163] 67. The method according to item 66, wherein the modification includes the partial or complete deletion of domain 4, such as the deletion of the intron-encoded protein (IEP) sequence in domain 4, preferably the complete deletion of domain 4; or

[0164] The modification includes the deletion of the open reading frame (ORF).

[0165] 68. The method according to item 64, wherein the precursor RNA can form a near scarless circRNA of the target sequence. Preferably, the near scarless circRNA has a scar region with a 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.

[0166] 69. The method according to item 65, wherein the precursor RNA can form a scarless circRNA of the target sequence.

[0167] 70. The method according to any one of items 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 NOs: 33 - 41.

[0168] 71. The method according to any one of items 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 NOs: 42 - 52; and / or

[0169] 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 NOs: 75 - 88.

[0170] 72. The method according to any one of items 52 - 71, wherein the length of E1 and / or E2 is 0 - 20 nucleotides, preferably the length is 0 - 10 nucleotides, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides;

[0171] preferably E2 comprises a polynucleotide sequence selected from SEQ ID NOs: 53 - 63 and SEQ ID NOs: 7211 - 7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NOs: 64 - 74 and SEQ ID NO: 7213.

[0172] 73. The method according to item 72, wherein the length of E1, E2 or both is 0 nucleotides.

[0173] 74. The method according to any one of items 52 - 73, wherein the target sequence is a non - coding sequence selected from the group consisting of: spacer sequences of SEQ ID NOs: 4 - 6, polyA sequence, poly - A - C sequence, polyC sequence, polyU sequence, IRES, ribosome - binding site, aptamer sequence, RNA scaffold, riboswitch, ribozyme other than self - splicing ribozyme, antisense oligonucleotide (ASO), scaffold, small RNA - binding site, translation regulatory sequence and protein - binding site.

[0174] 75. The method according to any one of items 52 - 73, 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 encodes a therapeutic product.

[0175] 76. The method according to any one of items 49 - 75, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0176] 77. The method according to item 76, wherein at least one of the modified RNA nucleotides and / or modified nucleosides 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-hydroxy-norvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine), 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 (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 (wybutosine), oayW (oxywybutosine), OHyW (hydroxywybutosine), OHyW* (under-modified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archaeosine), 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 (methyl ester of uridine 5-oxyacetic acid), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (methyl ester of 5-(carboxyhydroxymethyl)uridine), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (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 (1,2'-O-dimethylguanosine), m'Am (1,2'-O-dimethyladenosine), rm 5U (5-taurinomethyluridine), τm5s2U (5-taurinomethyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine).

[0177] 78. The method according to any one of items 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.

[0178] 79. The method according to any one of items 52 - 78 further comprises a solid surface,

[0179] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate or paper,

[0180] More preferably, the solid surface is a magnetic bead, agarose bead, streptavidin - coated bead, colloidal bead, glass bead, polymer bead, methacrylate bead, polystyrene bead, latex bead or silica bead, or

[0181] More preferably, the solid surface is a resin containing agarose, carbohydrate - based material, polymer filler, silica, glass particles, perfusion - based material or monolithic support, and the polymer filler is optionally selected from polystyrene - divinylbenzene or polymethacrylate.

[0182] 80. The method according to item 79, wherein the negative selection probe is immobilized on the solid surface by physical adsorption, covalent fixation or affinity fixation, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups.

[0183] 81. The method according to item 79 or 80, wherein the solid surface is a magnetic bead,

[0184] Preferably, the magnetic bead is a functional - group - modified magnetic bead, more preferably an amino - modified magnetic bead, carboxyl - modified magnetic bead, NHS - modified magnetic bead, maleimide - modified magnetic bead, CNBr - modified magnetic bead, magnetic bead modified with an affinity or specific group, or a combination of two or more of the above, and more preferably an NHS - modified magnetic bead, or

[0185] Preferably, the magnetic bead is a hydrophilic magnetic bead; or

[0186] Preferably, the magnetic bead has a diameter in the range of 0.1 μm to 10 μm, more preferably 0.5 μm to 5 μm, more preferably 0.8 μm to 1.2 μm, and most preferably 1 μm.

[0187] 82. The method according to item 79 or 80, wherein the solid surface is a chromatography packing containing agarose, and preferably the chromatography packing has a diameter of more than 5 mm and a packing height of more than 20 cm, more preferably a diameter of more than 10 mm and a packing height of more than 30 cm, and most preferably a diameter of 16 mm and a packing height of 40 cm,

[0188] Furthermore, the agarose chromatography packing material is an agarose chromatography packing material with an amino modification on the surface, a carboxyl modification on the surface, an NHS modification on the surface, a maleimide modification on the surface, a CNBr modification on the surface, an agarose chromatography packing material with an affinity or specific group modification, or a combination of two or more of the above. Further preferably, it is an agarose chromatography packing material with an NHS modification.

[0189] 83. The method according to any one of items 79 - 82, wherein the 3'-end of the negative selection probe is covalently fixed to the solid surface; or the 5'-end of the negative selection probe is covalently fixed to the solid surface. Preferably, the solid surface is a magnetic bead. Preferably, the covalent fixation is achieved through the covalent connection of NH2 and the carboxyl group modified by NHS.

[0190] 84. The method according to any one of items 79 - 83, wherein the 3'-end and / or 5'-end of the negative selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto.

[0191] 85. The composition according to any one of items 49 - 84, wherein the minimum free energy (MFE) value of the negative selection tag ranges from -2 to 2, and the change in the MFE value (ΔMFE) of the precursor nucleic acid caused by including the tag ranges from -2 to 2.

[0192] 86. The composition according to item 85, wherein the MFE value of the negative selection tag is about 0.

[0193] 87. The composition according to item 85 or 86, wherein the range of the ΔMFE value of the precursor nucleic acid caused by including the negative selection tag is 0 - 2. Preferably, the ΔMFE value of the precursor nucleic acid caused by including the negative selection tag is about 0.

[0194] 88. The method according to any one of items 49 - 87, wherein the sample containing the circular nucleic acid to be purified is contacted with the negative selection 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

[0195] the sample is contacted with the negative selection probe at a temperature of 0°C - 60°C; or

[0196] the sample is contacted with the negative selection probe in a binding solution, and the binding solution includes salts. Preferably, the binding solution further includes a buffering substance, a chelating agent, and optionally includes a stabilizer. Further preferably, the salts include cations selected from the following group: Ba 2+ 、Ca 2+ 、Mg2+ , Mn 2+ , Fe 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Pb 2+ , Li + , Cs + , Na + , K + , Rb + and NH 4 + , and / or anions selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the concentration of the salts in the binding solution is 50 mM - 5 M,

[0197] Further preferably, the salts have a cation of Li + or Na + .

[0198] 89. The method according to item 88, wherein the binding solution comprises 0.1 M - 0.5 M 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,

[0199] Preferably, the method further comprises the step of eluting a part of the anion selection probe bound to the solid surface with an eluent to recover the solid surface conjugated with the anion selection probe for reuse.

[0200] 90. The method according to any one of items 49 - 89, wherein the concentration of the 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 NH 4 + , and / or anions selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the salts are salts with cations being Li + or Na + of the salts,

[0201] Preferably, the eluent further comprises a buffering substance, a chelating agent, and optionally a stabilizer.

[0202] 91. The method according to item 90, wherein the eluent comprises EDTA and Tris-HCl, the pH range of the eluent is 6.0 - 8.0, preferably 7.5; or

[0203] the pH range of the eluent is 2.0 - 4.0, preferably the pH range is 2.5 - 3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5 - 3.0.

[0204] 92. The method according to any one of items 49 - 91, wherein the method is scalable and may include a continuous flow process, preferably carried out using a liquid chromatograph (LC) or an enzyme-linked immunosorbent assay (ELISA) reader, and further preferably the LC is a fast protein liquid chromatograph (FPLC) or a high performance liquid chromatograph (HPLC).

[0205] 93. A method for purifying circular nucleic acids from a sample, comprising the following steps:

[0206] Coupling magnetic beads with a negative selection probe;

[0207] Incubating the in vitro transcription (IVT) reaction solution containing the circular nucleic acid with the magnetic beads coupled with the negative selection probe;

[0208] Collecting the magnetic beads and eluting to recover the magnetic beads coupled with the negative selection probe;

[0209] Collecting the unbound fraction,

[0210] Wherein the negative selection probe is at least 90% complementary to the negative selection tag, and the circular nucleic acid is generated by 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.

[0211] 94. The method according to item 93, wherein the IVT reaction solution is incubated with magnetic beads coupled with a negative selection probe in a binding solution, and the magnetic beads are eluted with an eluent to recover the magnetic beads coupled with the negative selection probe.

[0212] 95. The method according to item 94, wherein the eluent and the binding solution are the eluent and the binding solution involved in any one of items 88-91, and more preferably, the negative selection probe and the negative selection tag are the negative selection probe and the negative selection tag involved in any one of items 49-92.

[0213] 96. The method according to any one of items 49-95, or the circular nucleic acid purified by using the composition containing an oligonucleotide negative selection probe for purifying circular nucleic acid according to any one of items 1-39, or the kit according to any one of items 40-48.

[0214] 97. A composition having the circular nucleic acid according to item 96, wherein the cyclization percentage (PC) of the composition is at least 90%, at least 95%, at least 98%, or at least 99%; or

[0215] The total ring purity of the composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, above 95%. Preferably, the multimer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%.

[0216] More preferably, the size of the circular nucleic acid is 1000 nt - 5000 nt.

[0217] 98. A composition for generating a circular nucleic acid, comprising:

[0218] A linearized plasmid for generating a precursor nucleic acid;

[0219] The generated precursor nucleic acid;

[0220] The generated circular nucleic acid;

[0221] The uncircularized precursor nucleic acid;

[0222] Residual impurities in the in vitro transcription reaction, wherein the precursor nucleic acid and the circular nucleic acid are the precursor nucleic acid and the circular nucleic acid involved in any one of Items 1-48,

[0223] Preferably, the total ring purity of the composition ranges from 20% to 50%, and the content of the multimer ranges from 10% to 20%.

[0224] 99. The composition according to Item 98, which is used as a sample or as a sample for the method described in any one of Items 49-96 and is purified by the composition containing an oligonucleotide negative selection probe for purifying circular nucleic acid described in any one of Items 1-39 or by using the kit described in any one of Items 40-48.

[0225] 100. The composition according to Item 99, wherein the total ring purity of the purified circular nucleic acid composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, above 95%,

[0226] Preferably, the content of the multimer is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%,

[0227] More preferably, the size of the circular nucleic acid is 1000 nt - 5000 nt.

[0228] 101. A composition for purifying circular nucleic acid, which contains a first oligonucleotide negative selection probe and a second oligonucleotide negative selection probe, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid containing a first oligonucleotide negative selection tag and a second oligonucleotide negative selection tag, wherein the first and second negative selection tags are at least 90% complementary to the first and second negative selection probes respectively and are removed during the self-splicing of the precursor nucleic acid. Preferably, the first and second negative selection 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 selection probes respectively. More preferably, they are 100% complementary.

[0229] 102. The composition according to Item 101, wherein the precursor nucleic acid contains the first negative selection tag at its 5' end and the second negative selection tag at its 3' end, and the first negative selection tag and the second negative selection tag can be the same or different.

[0230] 103. The composition according to Item 101 or 102, wherein the circular nucleic acid is circular RNA (circRNA). Preferably, the precursor nucleic acid is an RNA with group II intron self-splicing activity.

[0231] 104. The composition according to item 103, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0232] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a group II intron, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0233] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides;

[0234] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and

[0235] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both,

[0236] Further preferably, the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection 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 selection tag.

[0237] 105. The composition according to item 103, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0238] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a group II intron, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0239] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides;

[0240] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and

[0241] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both,

[0242] More preferably, the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection tag; (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); (g) a 5' intron fragment; and a second negative selection tag.

[0243] 106. The composition according to item 103, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0244] (1) Both the 5' intron fragment and the 3' intron fragment are 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;

[0245] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0246] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides; and

[0247] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both,

[0248] More preferably, the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection 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 selection tag.

[0249] 107. The composition according to item 103, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0250] (1) Both the 5' intron fragment and the 3' intron fragment are 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;

[0251] (2) E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0252] (3) E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0253] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of the two.

[0254] Further preferably, the precursor RNA contains the following operably linked elements from 5' to 3': a first negative selection tag; (a) a 5' homologous arm; (b) a 3' intron fragment; (c) an exon fragment 2 (E2); (d) a linker sequence; (e) a target sequence; (f) a linker sequence; (g) an exon fragment 1 (E1); (h) a 5' intron fragment; (i) a 3' homologous arm; and a second negative selection tag.

[0255] 108. The composition according to any one of items 101-107, wherein the lengths of the first negative selection tag, the second negative selection tag, the first negative selection probe, and the second negative selection probe are 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides respectively; preferably, the lengths of the first negative selection tag, the second negative selection tag, the first negative selection probe, and the second negative selection probe are 20-30 nucleotides respectively.

[0256] 109. The composition according to any one of items 101-108, wherein the first negative selection probe and the second negative selection probe are RNA probes; or the first negative selection probe and the second negative selection probe are DNA probes.

[0257] 110. The composition according to any one of items 101-109, wherein

[0258] when the first negative selection tag and the negative selection probe pair are the same as the second negative selection tag and the negative selection probe pair, the first negative selection tag and the second negative selection tag have a polynucleotide sequence containing SEQ ID NO: n, and the first negative selection probe and the second negative selection probe have a polynucleotide sequence containing SEQ ID NO: n + 1100, where n is an integer from 135 to 1234; or

[0259] When the first negative selection tag and negative selection probe pair are different from the second negative selection tag and negative selection probe pair, the first negative selection tag has a polynucleotide sequence comprising SEQ ID NO: n; the first negative selection probe has a polynucleotide sequence comprising SEQ ID NO: n + 1210; the second negative selection tag has a polynucleotide sequence comprising SEQ ID NO: n + 2420; the second negative selection probe has a polynucleotide sequence comprising SEQ ID NO: n + 3630; where n is an integer from 2335 to 3544.

[0260] 111. The composition according to any one of items 101 - 109, wherein the first negative selection tag and negative selection probe pair are the same as the second negative selection tag and negative selection probe pair, and the first negative selection tag and the second negative selection tag have a polynucleotide sequence comprising any one shown in SEQ ID NOs: 7189 - 7202; or the first negative selection probe and the second negative selection probe have a polynucleotide sequence comprising any one shown in SEQ ID NOs: 7175 - 7188.

[0261] 112. The composition according to item 111, wherein the first negative selection tag and the first negative selection probe of the composition are selected from any of the following combinations, and the second negative selection tag and the second negative selection probe of the composition are selected from any of the following combinations

[0262] (a) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7189, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7175;

[0263] (b) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7190, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7176;

[0264] (c) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7191, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7177;

[0265] (d) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7192, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7178;

[0266] (e) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7193, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7179;

[0267] (f) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7194, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7180;

[0268] (g) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7195, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7181;

[0269] (h) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7196, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7182;

[0270] (i) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7197, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7183;

[0271] (j) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7198, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7184;

[0272] (k) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7199, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7185;

[0273] (l) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7200, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7186;

[0274] (m) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7201, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7187;

[0275] (n) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7202, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7188.

[0276] 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 a Group II intron of an unpaired region into two fragments.

[0277] 114. The composition according to item 113, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the Group II intron at the loop region of the stem - loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0278] the 5' intron fragment and the 3' intron fragment are obtained by cutting the Group II intron at the linear region 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.

[0279] 115. The composition according to item 113 or 114, wherein the Group II intron contains one or more nucleotide modifications relative to its wild - type form, and the modifications are selected from one or more of deletion, substitution, and addition,

[0280] preferably the modification comprises modification of one or more EBS sequences of the Group II intron, wherein the EBS sequences are complementary paired with one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0281] 116. The composition according to item 115, wherein the modification is the modification of two EBS sequences (such as EBS1 and EBS3) of the Group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably the two regions are located at both ends of the target sequence respectively.

[0282] 117. The composition according to item 115, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of the Group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably the two regions are located at both ends of the target sequence respectively.

[0283] 118. The composition according to item 115 or 116, 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 paired with a region of corresponding length in the target sequence at 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 are complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides,

[0284] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary paired with a region 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 are complementary paired with a region of corresponding length in the target sequence.

[0285] 119. The composition according to item 114, wherein the modification includes the deletion of part or all of domain 4, such as the deletion of the intron-encoded protein (IEP) sequence in domain 4, preferably the entire deletion of domain 4; or

[0286] The modification includes the deletion of an open reading frame (ORF).

[0287] 120. The composition according to item 116, wherein the precursor RNA is capable of forming a near scarless circRNA of the target sequence, preferably the near scarless circRNA has a scar region with a 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.

[0288] 121. The composition according to item 117, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0289] 122. The composition according to any one of items 103 - 121, wherein the Group II intron is a Group II intron derived from a microorganism, preferably the Group II intron is Cte1, and further preferably the Group II intron contains a polynucleotide sequence selected from SEQ ID NO: 33 - 41.

[0290] 123. The composition according to any one of items 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

[0291] 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 NOs: 75 - 88.

[0292] 124. The composition according to any one of items 104 - 123, 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;

[0293] Preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NOs: 53 - 63 and SEQ ID NOs: 7211 - 7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NOs: 64 - 74 and SEQ ID NO: 7213.

[0294] 125. The composition according to item 124, wherein the length of E1, E2 or both is 0 nucleotides.

[0295] 126. The composition according to any one of items 104 - 125, wherein the target sequence is a non-coding sequence selected from the group consisting of: the spacer sequences of SEQ ID NOs: 4 - 6, polyA sequence, poly-A-C sequence, polyC sequence, polyU sequence, IRES, ribosome binding site, aptamer sequence, RNA scaffold, riboswitch, ribozyme other than self-splicing ribozyme, antisense oligonucleotide (ASO), scaffold, small RNA binding site, translation regulatory sequence and protein binding site.

[0296] 127. The composition according to any one of items 106 - 15, 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 encodes a therapeutic product.

[0297] 128. The composition according to any one of items 101 - 127, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0298] 129. The composition according to item 128, wherein at least one of the modified RNA nucleotides and / or modified nucleosides 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-hydroxy-norvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine), 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 (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 (wybutosine), oayW (oxywybutosine), OHyW (hydroxywybutosine), OHyW* (under-modified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archaeosine), 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 (methyl ester of uridine 5-oxyacetic acid), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (methyl ester of 5-(carboxyhydroxymethyl)uridine), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (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 (1,2'-O-dimethylguanosine), m'Am (1,2'-O-dimethyladenosine), rm 5U (5-tauromethyluridine), τm5s2U (5-tauromethyl-2-thiouridine), imG-14 (4-demethylwyeoside), imG2 (isowyeoside), or ac6A (N6-acetyladenosine).

[0299] 130. The composition according to any one of items 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.

[0300] 131. The composition according to any one of items 101 - 130 further comprises a solid surface,

[0301] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate or paper,

[0302] More preferably, the solid surface is a magnetic bead, agarose bead, streptavidin - coated bead, colloidal bead, glass bead, polymer bead, methacrylate bead, polystyrene bead, latex bead or silica bead, or

[0303] More preferably, the solid surface is a resin containing agarose, carbohydrate - based material, polymer filler, silica, glass particles, perfusion - based material or monolithic support, and the polymer filler is optionally selected from polystyrene - divinylbenzene or polymethacrylate.

[0304] 132. The composition according to item 131, wherein the first negative selection probe and the second negative selection probe are immobilized on the solid surface by physical adsorption, covalent fixation or affinity fixation, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups.

[0305] 133. The composition according to item 131 or 132, wherein the solid surface is a magnetic bead,

[0306] Preferably, the magnetic bead is a magnetic bead modified with a functional group, more preferably an amino - modified magnetic bead, a carboxyl - modified magnetic bead, an NHS - modified magnetic bead, a maleimide - modified magnetic bead, a CNBr - modified magnetic bead, a magnetic bead modified with an affinity or specific group, or a combination of two or more of the above, and more preferably an NHS - modified magnetic bead, or

[0307] Preferably, the magnetic bead is a hydrophilic magnetic bead; or

[0308] Preferably, the magnetic bead has a diameter in the range of 0.1 μm to 10 μm, more preferably 0.5 μm to 5 μm, more preferably 0.8 μm to 1.2 μm, and most preferably 1 μm.

[0309] 134. The composition according to item 131 or 132, wherein the solid surface is an agarose chromatography packing material,

[0310] Preferably, the diameter of the chromatography packing material is more than 5 mm and the packing height is more than 20 cm. More preferably, the diameter of the chromatography packing material is more than 10 mm and the packing height is more than 30 cm. More preferably, the diameter of the chromatography packing material is 16 mm and the packing height is 40 cm.

[0311] Furthermore, the agarose chromatography packing material is an agarose chromatography packing material with amino modification on the surface, a carboxyl-modified agarose chromatography packing material, an NHS-modified agarose chromatography packing material, a maleimide-modified agarose chromatography packing material, a CNBr-modified agarose chromatography packing material, an agarose chromatography packing material modified with an affinity or specific group, or a combination of two or more of the above, and is further preferably an NHS-modified agarose chromatography packing material.

[0312] 135. The composition according to any one of items 131-134, wherein the 3'-ends of the first negative selection probe and the second negative selection probe are covalently fixed to the solid surface; or the 5'-ends of the first negative selection probe and the second negative selection 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 connection of NH2 and a carboxyl group modified with NHS.

[0313] 136. The composition according to any one of items 131-135, wherein the 3'-ends and / or 5'-ends of the first negative selection probe and the second negative selection probe are further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto.

[0314] 137. The composition according to any one of items 101-136, wherein the minimum free energy (MFE) value of the first negative selection label and the second negative selection label ranges from -2 to 2, and the change in the MFE value (ΔMFE) of the precursor nucleic acid caused by including the label ranges from -2 to 2.

[0315] 138. The composition according to item 137, wherein the MFE values of the first negative selection label and the second negative selection label are about 0.

[0316] 139. The composition according to item 137 or 138, wherein the range of the ΔMFE value of the precursor nucleic acid caused by including the first negative selection label and the second negative selection label is 0-2, and preferably the ΔMFE value of the precursor nucleic acid caused by including the first negative selection label and the second negative selection label is about 0.

[0317] 140. A negative selection kit for purifying circular nucleic acids, comprising: the composition for purifying circular nucleic acids containing oligonucleotide negative selection probes according to any one of items 101-139, a binding solution, and an elution solution.

[0318] 141. The kit according to item 140, wherein the binding solution includes salts, and the salts include cations selected from the following groups: Ba 2+ 、Ca 2+ 、Mg 2+ 、Mn 2+ 、Fe 2+ 、Cu 2+, Zn 2+ , Cd 2+ , Pb 2+ , Li + , Cs + , Na + , K + , Rb + and NH 4 + , and / or anions selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the concentration of the salts in the binding solution is 50 mM - 5 M,

[0319] more preferably the salts are those with cations Li + or Na + .

[0320] 142. The kit according to item 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;

[0321] Preferably, the binding solution further comprises a buffering substance, a chelating agent, and optionally a stabilizing agent.

[0322] 143. The kit according to item 141 or 142, wherein the binding solution comprises 0.1 M - 0.5 M 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.

[0323] 144. The kit according to any one of items 140 - 143, wherein the concentration of the 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 NH 4 + , and / or anions selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the salts are salts with cations being Li + or Na + .

[0324] Preferably, the eluent further comprises a buffering substance, a chelating agent, and optionally a stabilizer.

[0325] 145. The kit according to item 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

[0326] the pH range of the eluent is 2.0 - 4.0, preferably the pH range is 2.5 - 3.0, and preferably the eluent is glycine - HCl with a pH range of 2.5 - 3.0.

[0327] 146. A negative selection kit for purifying circular nucleic acids, comprising: magnetic beads conjugated with negative selection probes, an eluent, and a binding solution, wherein the negative selection probes are at least 90% complementary to the negative selection tags, and the circular nucleic acids are generated by self - splicing of a precursor nucleic acid comprising 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 probes, and more preferably 100% complementary.

[0328] 147. The kit according to item 146, wherein the negative selection probes and negative selection tags are the negative selection probes and negative selection tags involved in any one of items 101 - 139, or

[0329] The eluent and the binding solution are the eluent and the binding solution involved in any one of items 140-145.

[0330] 148. The kit according to item 146 or 147, wherein the magnetic beads immobilized with the negative selection probes can be reused.

[0331] 149. A method for purifying circular nucleic acids from a sample, wherein the circular nucleic acids are generated by self-splicing of precursor nucleic acids, and wherein the precursor nucleic acids have an oligonucleotide first negative selection tag and an oligonucleotide second negative selection tag, and the first negative selection tag and the second negative selection tag are removed during the self-splicing process. The method includes:

[0332] (i) contacting the sample with the first negative selection probe and the second negative selection probe that are at least 90% complementary to the first negative selection tag and the second negative selection tag, respectively, under conditions that allow the first negative selection tag to bind to the first negative selection probe and the second negative selection tag to bind to the second negative selection probe, wherein the first negative selection probe and the second negative selection probe are immobilized on a solid surface; and

[0333] (ii) collecting the unbound portion of the sample,

[0334] Preferably, the first and second negative selection 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 selection probes, respectively, and more preferably 100% complementary.

[0335] 150. The method according to item 149, wherein the precursor nucleic acid contains the first negative selection tag at its 5' end and the second negative selection tag at its 3' end, and the first negative selection tag and the second negative selection tag may be the same or different.

[0336] 151. The method according to item 149 or 150, wherein the circular nucleic acid is circular RNA (circRNA), and preferably the precursor nucleic acid is an RNA having group II intron self-splicing activity.

[0337] 152. The method according to item 151, wherein the precursor RNA contains the following operably linked elements from 5' to 3': (a) a 3' intron fragment; (b) an exon fragment 2 (E2); (c) a target sequence; (d) an exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0338] (1) both the 5' intron fragment and the 3' intron fragment are fragments of group II introns, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0339] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0340] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0341] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both,

[0342] More preferably, the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection tag; (a) a 3'-intron fragment; (b) an exon fragment 2 (E2); (c) a target sequence; (d) an exon fragment 1 (E1); (e) a 5'-intron fragment; and a second negative selection tag.

[0343] 153. The method according to item 151, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 3'-intron fragment; (b) an exon fragment 2 (E2); (c) a linker sequence; (d) a target sequence; (e) a linker sequence; (f) an exon fragment 1 (E1); and (g) a 5'-intron fragment; wherein:

[0344] (1) Both the 5'-intron fragment and the 3'-intron fragment are fragments of the Group II intron, and the 5'-intron fragment is located on the 5'-side of the 3'-intron fragment in the Group II intron;

[0345] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0346] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0347] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both,

[0348] More preferably, the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection tag; (a) a 3'-intron fragment; (b) an exon fragment 2 (E2); (c) a linker sequence; (d) a target sequence; (e) a linker sequence; (f) an exon fragment 1 (E1); (g) a 5'-intron fragment; and a second negative selection tag.

[0349] 154. The method according to item 151, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0350] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a group II intron, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0351] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0352] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides; and

[0353] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both,

[0354] Further preferably, the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection 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 selection tag.

[0355] 155. The method according to item 151, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0356] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a group II intron, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0357] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0358] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides; and

[0359] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both,

[0360] More preferably, the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection tag; (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; (i) a 3' homologous arm; and a second negative selection tag.

[0361] 156. The method according to any one of items 149-155, wherein the lengths of the first negative selection tag, the second negative selection tag, the first negative selection probe, and the second negative selection probe are 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides, respectively; preferably, the lengths of the first negative selection tag, the second negative selection tag, the first negative selection probe, and the second negative selection probe are 20-30 nucleotides, respectively.

[0362] 157. The method according to any one of items 149-156, wherein the first negative selection probe and the second negative selection probe are RNA probes; or the first negative selection probe and the second negative selection probe are DNA probes.

[0363] 158. The method according to any one of items 149-157, wherein,

[0364] when the first negative selection tag and the negative selection probe pair are the same as the second negative selection tag and the negative selection probe pair, the first negative selection tag and the second negative selection tag have a polynucleotide sequence comprising SEQ ID NO: n, and the first negative selection probe and the second negative selection probe have a polynucleotide sequence comprising SEQ ID NO: n + 1100, where n is an integer from 135 to 1234; or

[0365] when the first negative selection tag and the negative selection probe pair are different from the second negative selection tag and the negative selection probe pair, the first negative selection tag has a polynucleotide sequence comprising SEQ ID NO: n; the first negative selection probe has a polynucleotide sequence comprising SEQ ID NO: n + 1210; the second negative selection tag has a polynucleotide sequence comprising SEQ ID NO: n + 2420; the second negative selection probe has a polynucleotide sequence comprising SEQ ID NO: n + 3630; where n is an integer from 2335 to 3544.

[0366] 159. The method according to any one of items 49 - 58, wherein the first negative selection tag and the negative selection probe pair are the same as the second negative selection tag and the negative selection probe pair, and the first negative selection tag and the second negative selection tag have a polynucleotide sequence comprising any one shown in SEQ ID NO: 7189 - 7202; or the first negative selection probe and the second negative selection probe have a polynucleotide sequence comprising any one shown in SEQ ID NO: 7175 - 7188.

[0367] 160. The method according to item 159, wherein the first negative selection tag and the first negative selection probe of the composition are selected from any one of the following combinations, and the second negative selection tag and the second negative selection probe of the composition are selected from any one of the following combinations

[0368] (a) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7189, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7175;

[0369] (b) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7190, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7176;

[0370] (c) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7191, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7177;

[0371] (d) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7192, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7178;

[0372] (e) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7193, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7179;

[0373] (f) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7194, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7180;

[0374] (g) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7195, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7181;

[0375] (h) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7196, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7182;

[0376] (i) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7197, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7183;

[0377] (j) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7198, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7184;

[0378] (k) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7199, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7185;

[0379] (l) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7200, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7186;

[0380] (m) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7201, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7187;

[0381] (n) The polynucleotide sequence of the first or second negative selection tag is as shown in SEQ ID NO: 7202, and the polynucleotide sequence of the first or second negative selection probe is as shown in SEQ ID NO: 7188.

[0382] 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 a group II intron in an unpaired region into two fragments.

[0383] 162. The method according to item 161, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron at the loop region of the stem - loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0384] The 5' intron fragment and the 3' intron fragment are obtained by cleaving a Group II intron at a linear region 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.

[0385] 163. The method according to item 161 or 162, wherein the Group II intron contains a modification of one or more nucleotides relative to its wild-type form, and the modification is selected from one or more of deletion, substitution, and addition.

[0386] Preferably, the modification comprises a modification of one or more EBS sequences of the Group II intron, wherein the EBS sequences are complementary paired with one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0387] 164. The method according to item 163, wherein the modification is a modification of two EBS sequences (such as EBS1 and EBS3) of the Group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at both ends of the target sequence respectively.

[0388] 165. The method according to item 163, wherein the modification is a modification of two EBS sequences (such as EBS1' and EBS3') of the Group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are located at both ends of the target sequence respectively.

[0389] 166. The method according to item 164 or 165, wherein the modification is a modification of the EBS1 and / or δ sequence of the Group II intron, or a modification of the EBS1' and / or δ'' sequence, wherein the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at 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 are complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides.

[0390] Preferably, the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at 100% 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 are complementary paired with a region of corresponding length in the target sequence at 100% of the nucleotides.

[0391] 167. The method according to item 166, wherein the modification includes the deletion of part or all of domain 4, such as the deletion of the intron-encoded protein (IEP) sequence in domain 4, preferably the complete deletion of domain 4; or

[0392] the modification includes the deletion of an open reading frame (ORF).

[0393] 168. The method according to item 164, wherein the precursor RNA is capable of forming a near scarless circRNA of the target sequence, preferably the near scarless circRNA has a scar region with a 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.

[0394] 169. The method according to item 165, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0395] 170. The method according to any one of items 151-169, wherein the group II intron is a group II intron derived from a microorganism, preferably the group II intron is Cte1, and further preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0396] 171. The method according to any one of items 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

[0397] 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.

[0398] 172. The method according to any one of items 152-171, wherein the length of E1 and / or E2 is 0-20 nucleotides, preferably the length is 0-10 nucleotides, such as the length is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides;

[0399] 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.

[0400] 173. The method according to item 172, wherein the length of E1, E2 or both is 0 nucleotides.

[0401] 174. The method according to any one of items 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 sequence, poly-A-C sequence, polyC sequence, polyU sequence, IRES, ribosome binding site, aptamer sequence, RNA scaffold, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotide (ASO), scaffold, small RNA binding site, translation regulatory sequence, and protein binding site.

[0402] 175. The method according to any one of items 152 - 173, 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 encodes a therapeutic product.

[0403] 176. The method according to any one of items 149 - 175, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0404] 177. The method according to item 176, wherein at least one of the modified RNA nucleotides and / or modified nucleosides 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'-0-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-hydroxy-norvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine), 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 (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 (wybutosine), oayW (oxywybutosine), OHyW (hydroxywybutosine), OHyW* (under-modified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archaeosine), 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 (methyl ester of uridine 5-oxyacetic acid), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (methyl ester of 5-(carboxyhydroxymethyl)uridine), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (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 (1,2'-O-dimethylguanosine), m'Am (1,2'-O-dimethyladenosine), rm 5U (5-taurinomethyluridine), τm5s2U (5-taurinomethyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine).

[0405] 178. The method according to any one of items 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.

[0406] The method according to any one of items 152 - 178 further comprises a solid surface,

[0407] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate or paper,

[0408] More preferably, the solid surface is a magnetic bead, agarose bead, streptavidin - coated bead, colloidal bead, glass bead, polymer bead, methacrylate bead, polystyrene bead, latex bead or silica bead, or

[0409] More preferably, the solid surface is a resin containing agarose, carbohydrate - based material, polymer filler, silica, glass particles, perfusion - based material or monolithic support, and the polymer filler is optionally selected from polystyrene - divinylbenzene or polymethacrylate.

[0410] 180. The method according to item 179, wherein the first negative selection probe and the second negative selection probe are immobilized on the solid surface by physical adsorption, covalent fixation or affinity fixation, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups.

[0411] 181. The method according to item 179 or 180, wherein the solid surface is a magnetic bead,

[0412] Preferably, the magnetic bead is a functional - group - modified magnetic bead, more preferably an amino - modified magnetic bead, carboxyl - modified magnetic bead, NHS - modified magnetic bead, maleimide - modified magnetic bead, CNBr - modified magnetic bead, magnetic bead modified with an affinity or specific group, or a combination of two or more of the above, and more preferably an NHS - modified magnetic bead, or

[0413] Preferably, the magnetic bead is a hydrophilic magnetic bead; or

[0414] Preferably, the magnetic bead has a diameter in the range of 0.1 μm to 10 μm, more preferably 0.5 μm to 5 μm, more preferably 0.8 μm to 1.2 μm, and most preferably 1 μm.

[0415] 182. The method according to item 179 or 180, wherein the solid surface is a chromatography packing containing agarose, preferably the chromatography packing has a diameter of more than 5 mm and a packing height of more than 20 cm, more preferably a diameter of more than 10 mm and a packing height of more than 30 cm, and most preferably a diameter of 16 mm and a packing height of 40 cm,

[0416] Furthermore, the agarose chromatography packing material is an agarose chromatography packing material with an amino modification on the surface, a carboxyl modification on the surface, an NHS modification on the surface, a maleimide modification on the surface, a CNBr modification on the surface, an agarose chromatography packing material with an affinity or specific group modification, or a combination of two or more of the above, and is further preferably an agarose chromatography packing material with an NHS modification.

[0417] 183. The method according to any one of items 179 - 182, wherein the 3' ends of the first negative selection probe and the second negative selection probe are covalently fixed to the solid surface; or the 5' ends of the first negative selection probe and the second negative selection probe are 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 the carboxyl group modified by NHS.

[0418] 184. The method according to any one of items 179 - 183, wherein the 3' ends and / or 5' ends of the first negative selection probe and the second negative selection probe are further modified with a group selected from the following: amino, carboxyl, NHS - carboxyl, mercapto.

[0419] 185. The method according to any one of items 149 - 184, wherein the minimum free energy (MFE) value of the first negative selection label and the second negative selection label ranges from -2 to 2, and the change in the MFE value (ΔMFE) of the precursor nucleic acid caused by including the label ranges from -2 to 2.

[0420] 186. The method according to item 185, wherein the MFE value of the first negative selection label and the second negative selection label is about 0.

[0421] 187. The method according to item 185 or 186, wherein the range of the ΔMFE value of the precursor nucleic acid caused by including the first negative selection label and the second negative selection label is 0 - 2, and preferably the ΔMFE value of the precursor nucleic acid caused by including the first negative selection label and the second negative selection label is about 0.

[0422] 188. The method according to any one of items 149 - 187, wherein the sample containing the circular nucleic acid to be purified is contacted with the first negative selection probe and the second negative selection 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

[0423] the sample is contacted with the first negative selection probe and the second negative selection probe at a temperature of 0°C - 60°C; or

[0424] The sample is contacted with the first negative selection probe and the second negative selection probe in a binding solution, wherein the binding solution includes salts, preferably the binding solution also includes a buffer substance, a chelating agent, and optionally a stabilizer, and further preferably the salt includes a cation selected from the following group: Ba 2+ , Ca 2+ Mg 2+ , Mn 2+ , Fe 2+ , Cu 2+ 、Zn 2+ 、Cd 2+ , Pb 2+ , Li + , Cs + 、Na + , K + , Rb + and NH 4 + , and / or an anion selected from the group consisting of: PO 4 3- 、SO 4 3- , CH 3 CO 3 - , Cl - Br - 、NO 3 3- , ClO 4 3- ,I - and SCN - or a combination thereof, preferably the concentration of the salt in the binding solution is 50mM-5M,

[0425] It is further preferred that the salt is a cation of Li + Or Na + of salts.

[0426] 189. The method according to item 188, 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,

[0427] Preferably, the method further comprises the step of eluting the first negative selection probe and the second negative selection probe bound to the solid surface with an elution solution to recover the solid surface coupled with the first negative selection probe and the second negative selection probe for reuse.

[0428] 190. A method according to any one of items 149-189, wherein the concentration of the salt in the eluent is 0M, and the salt includes a cation selected from the group consisting of: Ba 2+ , Ca2+ , Mg 2+ , Mn 2+ , Fe 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Pb 2+ , Li + , Cs + , Na + , K + , Rb + and NH 4 + , and / or anions selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the salts are salts with cations being Li + or Na + .

[0429] Preferably, the eluent further comprises a buffering substance, a chelating agent, and optionally a stabilizer.

[0430] 191. The method according to item 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

[0431] the pH range of the eluent is 2.0 - 4.0, preferably the pH range is 2.5 - 3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5 - 3.0.

[0432] 192. The method according to any one of items 149 - 191, wherein the method is scalable and may include a continuous flow process, preferably carried out using a liquid chromatograph (LC) or a microplate reader, and further preferably the LC is a fast protein liquid chromatograph (FPLC) or a high performance liquid chromatograph (HPLC).

[0433] 193. A method for purifying circular nucleic acids from a sample, comprising the following steps:

[0434] Coupling magnetic beads with a first negative selection probe and a second negative selection probe;

[0435] Incubate the in vitro transcription (IVT) reaction solution containing the circular nucleic acid with magnetic beads conjugated with a first negative selection probe and a second negative selection probe;

[0436] Collect the magnetic beads and elute them to recover the magnetic beads conjugated with the first negative selection probe and the second negative selection probe;

[0437] Collect the unbound portion,

[0438] wherein the first and second negative selection tags are at least 90% complementary to the first and second negative selection probes respectively, and the circular nucleic acid is generated by self-splicing of a precursor nucleic acid containing a first oligonucleotide negative selection tag and a second oligonucleotide negative selection tag, and wherein the negative selection tags are removed during the self-splicing of the precursor nucleic acid. Preferably, the first and second negative selection 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 selection probes respectively, and more preferably 100% complementary.

[0439] 194. The method according to item 193, wherein the incubation of the IVT reaction solution with the magnetic beads conjugated with the first negative selection probe and the second negative selection probe is carried out in a binding solution, and the magnetic beads conjugated with the first negative selection probe and the second negative selection probe are eluted and recovered using an eluent.

[0440] 195. The method according to item 194, wherein the eluent and the binding solution are the eluent and the binding solution involved in any one of items 188 - 191. More preferably, the negative selection probe and the negative selection tag are the first negative selection probe and the second negative selection probe, and the first negative selection tag and the second negative selection tag involved in any one of items 149 - 195.

[0441] 196. The method according to any one of items 149 - 195, or the circular nucleic acid purified by using the composition containing a first oligonucleotide negative selection probe and a second oligonucleotide negative selection probe for purifying circular nucleic acid according to any one of items 101 - 139, or the kit according to any one of items 140 - 148.

[0442] 197. A composition having the circular nucleic acid according to item 196, wherein the cyclization percentage (PC) of the composition is at least 90%, at least 95%, at least 98%, or at least 99%; or

[0443] the total ring purity of the composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, above 95%. Preferably, the content of multimers is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%.

[0444] More preferably, the size of the circular nucleic acid is 1000 nt - 5000 nt.

[0445] 198. A composition for generating circular nucleic acid, comprising:

[0446] A linearized plasmid for generating precursor nucleic acid;

[0447] The generated precursor nucleic acid;

[0448] The generated circular nucleic acid;

[0449] The uncircularized precursor nucleic acid;

[0450] Impurities remaining from in vitro transcription reaction, wherein the precursor nucleic acid and the circular nucleic acid are the precursor nucleic acid and the circular nucleic acid involved in any one of items 101 - 148,

[0451] Preferably, the total ring purity range of the composition is 20 - 50%, and the multimer content range is 10 - 20%.

[0452] 199. The composition according to item 198, which is used as a sample for the method described in any one of items 149 - 196 or purified as a sample via the composition comprising a first oligonucleotide negative selection probe and a second oligonucleotide negative selection probe for purifying circular nucleic acid described in any one of items 101 - 139 or using the kit described in any one of items 140 - 148.

[0453] 200. The composition according to item 199, wherein the total ring purity of the purified circular nucleic acid composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, above 95%,

[0454] Preferably, the multimer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%,

[0455] More preferably, the size of the circular nucleic acid is 1000 nt - 5000 nt.

[0456] 201. A composition comprising an oligonucleotide positive selection probe for purifying circular nucleic acid, wherein the circular nucleic acid is generated by self - splicing of a precursor nucleic acid, wherein the positive selection probe is at least 60% complementary to at least a part of the circular nucleic acid generated by self - splicing of the precursor nucleic acid, at least a part of the circular nucleic acid spans the splicing part of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0457] 202. The composition according to item 201, wherein the positive selection 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 part of the circular nucleic acid generated by self-splicing of the precursor nucleic acid, and more preferably 100% complementary, and there is no sequence on the precursor nucleic acid 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 selection probe, and more preferably 100% complementary.

[0458] 203. The composition according to item 201 or 202, wherein the circular nucleic acid is circular RNA (circRNA), and preferably the precursor nucleic acid is an RNA having group II intron self-splicing activity.

[0459] 204. The composition according to item 203, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0460] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of group II introns, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0461] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length ≥ 0 nucleotides;

[0462] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length ≥ 0 nucleotides; and

[0463] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0464] 205. The composition according to item 203, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0465] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of group II introns, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0466] (2) E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0467] (3) E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0468] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0469] 206. The composition according to item 203, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0470] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of the Group II intron, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the Group II intron;

[0471] (2) E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0472] (3) E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0473] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0474] 207. The composition according to item 203, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0475] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of the Group II intron, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the Group II intron;

[0476] (2) E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0477] (3) E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0478] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0479] 208. The composition according to any one of items 201-207, wherein 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.

[0480] 209. The composition according to any one of items 201-208, wherein the positive selection probe is an RNA probe or a DNA probe.

[0481] 210. The composition according to any one of items 204-209, wherein the positive selection probe comprises a first positive selection probe and optionally a second positive selection probe,

[0482] The first positive selection probe is at least 60% complementary to the linker fragment part 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% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%; or

[0483] The first positive selection probe is at least 60% complementary to at least a part of the linker part formed by circularizing the 3'-end and 5'-end of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0484] 211. The composition according to any one of items 201-210, wherein the positive selection probe has a polynucleotide sequence shown in any one of SEQ ID NO. 7203-7206 and SEQ ID NO. 7222-7225.

[0485] 212. The composition according to item 210, wherein the positive selection probe comprises a first positive selection probe and a second positive selection probe, and the second positive selection probe is at least 60% complementary, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, to at least a part of any of the two side sequences outside both ends of the target sequence near the two ends, and the complementarity between the first positive selection probe and any part of the non-cyclized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%. Further preferably, the sequence of the second positive selection probe is selected from TT, GATT, TTTC.

[0486] 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 a group II intron of an unpaired region into two fragments.

[0487] 214. The composition according to item 213, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron at the loop region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0488] the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron at the linear region 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.

[0489] 215. The composition according to item 213 or 214, wherein the group II intron contains one or more nucleotide modifications relative to its wild-type form, and the modifications are selected from one or more of deletion, substitution, and addition,

[0490] preferably the modification comprises modification of one or more EBS sequences of the group II intron, wherein the EBS sequences are complementary paired with one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0491] 216. The composition according to item 215, wherein the modification is modification of two EBS sequences (such as EBS1 and EBS3) of the group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably the two regions are respectively located at both ends of the target sequence.

[0492] 217. The composition according to item 215, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of group II introns, and the EBS sequences are complementary paired with two regions of corresponding lengths in the target sequence at at least 60% of the nucleotide positions; preferably, these two regions are respectively located at both ends of the target sequence.

[0493] 218. The composition according to item 215 or 216, wherein the modification is the modification of EBS1 and / or δ sequence of group II introns, or the modification of EBS1' and / or δ” sequence, and the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides. Optionally, the modification is the modification of EBS1 and / or δ sequence and its upstream sequence, and the EBS1 and / or δ sequence and its upstream are complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides.

[0494] Preferably, the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at 100% of the nucleotides. Optionally, the modification is the modification of EBS1 and / or δ sequence and its upstream sequence, and the EBS1 and / or δ sequence and its upstream are complementary paired with a region of corresponding length in the target sequence at 100% of the nucleotides.

[0495] 219. The composition according to item 214, wherein the modification includes the deletion of part or all of domain 4, such as the deletion of the intron-encoded protein (IEP) sequence in domain 4, preferably the complete deletion of domain 4; or

[0496] The modification includes the deletion of the open reading frame (ORF).

[0497] 220. The composition according to item 216, wherein the precursor RNA can form a near scarless circRNA of the target sequence. Preferably, the near scarless circRNA has a scar region with a 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.

[0498] 221. The composition according to item 217, wherein the precursor RNA can form a scarless circRNA of the target sequence.

[0499] 222. The composition according to any one of items 203 - 221, wherein the group II intron is a group II intron derived from a microorganism. Preferably, the group II intron is Cte1. More preferably, the group II intron contains a polynucleotide sequence selected from SEQ ID NO: 33 - 41.

[0500] 223. The composition according to any one of items 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 NOs: 42 - 52; and / or

[0501] 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 NOs: 75 - 88.

[0502] 224. The composition according to any one of items 204 - 223, wherein the length of E1 and / or E2 is 0 - 20 nucleotides, preferably the length is 0 - 10 nucleotides, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides;

[0503] preferably E2 comprises a polynucleotide sequence selected from SEQ ID NOs: 53 - 63 and SEQ ID NOs: 7211 - 7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NOs: 64 - 74 and SEQ ID NO: 7213.

[0504] 225. The composition according to item 224, wherein the length of E1, E2 or both is 0 nucleotides.

[0505] 226. The composition according to any one of items 204 - 225, wherein the target sequence is a non - coding sequence selected from the group consisting of: the spacer sequences of SEQ ID NOs: 4 - 6, polyA sequence, poly - A - C sequence, polyC sequence, polyU sequence, IRES, ribosome binding site, aptamer sequence, RNA scaffold, riboswitch, ribozymes other than self - splicing ribozymes, antisense oligonucleotide (ASO), scaffold, small RNA binding site, translation regulatory sequence and protein binding site.

[0506] 227. The composition according to any one of items 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 encodes a therapeutic product.

[0507] 228. The composition according to any one of items 201 - 227, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0508] 229. The composition according to item 228, wherein at least one of the modified RNA nucleotides and / or modified nucleosides 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-hydroxy-norvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine), 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 (lysine), m1G (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 (wybutosine), oayW (oxywybutosine), OHyW (hydroxywybutosine), OHyW* (under-modified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archaeosine), 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 (methyl ester of uridine 5-oxyacetic acid), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (methyl ester of 5-(carboxyhydroxymethyl)uridine), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (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 (1,2'-O-dimethylguanosine), m'Am (1,2'-O-dimethyladenosine), rm 5U (5-tauromethyluridine), τm5s2U (5-tauromethyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine).

[0509] 230. The composition according to any one of items 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.

[0510] The composition according to any one of items 201 - 230, further comprising a solid surface,

[0511] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate or paper,

[0512] More preferably, the solid surface is a magnetic bead, agarose bead, streptavidin - coated bead, colloidal bead, glass bead, polymer bead, methacrylate bead, polystyrene bead, latex bead or silica bead, or

[0513] More preferably, the solid surface is a resin containing agarose, carbohydrate - based material, polymer filler, silica, glass particles, perfusion - based material or monolithic support, and the polymer filler is optionally selected from polystyrene - divinylbenzene or polymethacrylate.

[0514] 232. The composition according to item 231, wherein the positive selection probe is immobilized on the solid surface by physical adsorption, covalent fixation or affinity fixation, and preferably the covalent fixation is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups.

[0515] 233. The composition according to item 231 or 232, wherein the solid surface is a magnetic bead,

[0516] Preferably, the magnetic bead is a magnetic bead modified with a functional group, more preferably an amino - modified magnetic bead, a carboxyl - modified magnetic bead, an NHS - modified magnetic bead, a maleimide - modified magnetic bead, a CNBr - modified magnetic bead, a magnetic bead modified with an affinity or specific group, or a combination of two or more of the above, and more preferably an NHS - modified magnetic bead, or

[0517] Preferably, the magnetic bead is a hydrophilic magnetic bead; or

[0518] Preferably, the magnetic bead has a diameter in the range of 0.1 μm to 10 μm, more preferably 0.5 μm to 5 μm, more preferably 0.8 μm to 1.2 μm, and most preferably 1 μm.

[0519] 234. The composition according to item 231 or 232, wherein the solid surface is an agarose chromatography packing material,

[0520] Preferably, the chromatography packing material has a diameter of more than 5 mm and a packing height of more than 20 cm. More preferably, the chromatography packing material has a diameter of more than 10 mm and a packing height of more than 30 cm. More preferably, the chromatography packing material has a diameter of 16 mm and a packing height of 40 cm

[0521] Furthermore, the agarose chromatography packing material is an agarose chromatography packing material with amino modification on the surface, a carboxyl-modified agarose chromatography packing material, an NHS-modified agarose chromatography packing material, a maleimide-modified agarose chromatography packing material, a CNBr-modified agarose chromatography packing material, an agarose chromatography packing material modified with an affinity or specific group, or a combination of two or more of the above, and is further preferably an NHS-modified agarose chromatography packing material.

[0522] 235. The composition according to any one of items 231-234, wherein the 3' end of the positive selection probe is covalently fixed to the solid surface; or the 5' end of the positive selection 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 the carboxyl group modified by NHS.

[0523] 236. The composition according to any one of items 231-235, wherein the 3' end and / or 5' end of the positive selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto.

[0524] 237. The composition according to item 235, wherein the positive selection probe includes a second positive selection probe, a first positive selection probe, and a second positive selection probe connected in sequence, wherein the 3' end of one of the second positive selection probes is covalently fixed to the solid surface; or the 5' end of the other second positive selection 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 the carboxyl group modified by NHS.

[0525] 238. The composition according to item 236, wherein the positive selection probe includes a second positive selection probe, a first positive selection probe, and a second positive selection probe connected in sequence, wherein the 3' end of one of the second positive selection probes and / or the 5' end of the other second positive selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto.

[0526] 239. The composition according to item 237 or 238, wherein the positive selection probe has a polynucleotide sequence shown in any one of SEQ ID NO.7205-7206.

[0527] 240. A positive selection kit for purifying circular nucleic acids, comprising: the composition for purifying circular nucleic acids containing oligonucleotide positive selection probes according to any one of items 201-239, a binding solution, and an elution solution.

[0528] 241. The kit according to item 240, wherein the binding solution includes salts, and the salts include cations selected from the following group: Ba 2+ 、Ca 2+ 、Mg2+ , Mn 2+ , Fe 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Pb 2+ , Li + , Cs + , Na + , K + , Rb + and NH 4 + , and / or anions selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the concentration of the salts in the binding solution is 50 mM - 5 M,

[0529] More preferably, the salts have a cation of Li + or Na + .

[0530] 242. The kit according to item 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;

[0531] Preferably, the binding solution further comprises a buffering substance, a chelating agent, and optionally a stabilizing agent.

[0532] 243. The kit according to item 241 or 242, wherein the binding solution comprises 0.1 M - 0.5 M 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.

[0533] 244. The kit according to any one of items 240 - 243, wherein the concentration of the salts in the eluent is 0 M, and the salts comprise cations selected from the group consisting of: Ba 2+ , Ca 2+ , Mg 2+ , Mn2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、Cd 2+ 、Pb 2+ 、Li + 、Cs + 、Na + 、K + 、Rb + and NH 4 + ,and / or an anion selected from the group consisting of: PO 4 3- 、SO 4 3- 、CH 3 CO 3 - 、Cl - 、Br - 、NO 3 3- 、ClO 4 3- 、I - and SCN - or a combination thereof, preferably the salt has a cation of Li + or Na + of the salt,

[0534] Preferably, the eluent further comprises a buffering substance, a chelating agent, and optionally a stabilizer.

[0535] 245. The kit according to item 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

[0536] the pH range of the eluent is 2.0 - 4.0, preferably the pH range is 2.5 - 3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5 - 3.0.

[0537] 246. A positive selection kit for purifying circular nucleic acids, comprising: magnetic beads conjugated with a positive selection probe, an eluent, and a binding solution, wherein the circular nucleic acids are generated by self-splicing of precursor nucleic acids, the positive selection probe is at least 60% complementary to at least a part of the circular nucleic acids generated by self-splicing of the precursor nucleic acids, at least a part of the circular nucleic acids spans the splicing part of the circular nucleic acids, and there is no sequence on the precursor nucleic acids that is at least 60% complementary to the positive selection probe.

[0538] 247. The kit according to item 246, wherein the positive selection probe is the positive selection probe involved in any one of items 201-239, or the eluent and the binding solution are the eluent and the binding solution involved in any one of items 240-245.

[0539] 248. The kit according to item 246 or 247, wherein the magnetic beads immobilized with the positive selection probe can be reused.

[0540] 249. A method for purifying circular nucleic acid from a sample, wherein the circular nucleic acid is generated by self-splicing of a precursor nucleic acid, and the method includes:

[0541] (i) Under conditions allowing the circular nucleic acid generated by self-splicing of the precursor nucleic acid to bind to the positive selection probe, contacting a sample including the circular nucleic acid generated by self-splicing of the precursor nucleic acid with a positive selection probe that is at least 60% complementary to at least a part of the circular nucleic acid, wherein the positive selection probe is immobilized on a solid surface; and

[0542] (ii) Collecting the part bound to the positive selection probe on the solid surface,

[0543] At least a part of the circular nucleic acid spans the splicing part of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0544] 250. The method according to item 249, wherein the positive selection 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 part of the circular nucleic acid generated by self-splicing of the precursor nucleic acid, and further preferably 100% complementary, and there is no sequence on the precursor nucleic acid 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 selection probe, and further preferably 100% complementary.

[0545] 251. The method according to item 249 or 250, wherein the circular nucleic acid is circular RNA (circRNA), and preferably the precursor nucleic acid is an RNA having group II intron self-splicing activity.

[0546] 252. The method according to item 251, wherein the precursor RNA contains the following operably linked elements from 5' to 3': (a) a 3' intron fragment; (b) an exon fragment 2 (E2); (c) a target sequence; (d) an exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0547] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, where the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II introns;

[0548] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0549] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides; and

[0550] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0551] 253. The method according to item 251, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0552] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, where the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II introns;

[0553] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0554] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides; and

[0555] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0556] 254. The method according to item 251, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0557] (1) The 5' intron fragment and the 3' intron fragment are both fragments of group II introns, where the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II introns;

[0558] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0559] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0560] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0561] 255. The method according to item 251, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 5' homologous arm; (b) a 3' intron fragment; (c) an exon fragment 2 (E2); (d) a linker sequence; (e) a target sequence; (f) a linker sequence; (g) an exon fragment 1 (E1); (h) a 5' intron fragment; and (i) a 3' homologous arm; wherein:

[0562] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of the Group II intron, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the Group II intron;

[0563] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0564] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0565] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0566] 256. The method according to any one of items 249-255, wherein 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.

[0567] 257. The method according to any one of items 249-256, wherein the positive selection probe is an RNA probe or a DNA probe.

[0568] 258. The method according to any one of items 251-257, wherein the positive selection probe comprises a first positive selection probe and optionally a second positive selection probe,

[0569] The first positive selection probe is at least 60% complementary, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary to the linked fragment portion formed by E1 and E2 of the circular nucleic acid, and the complementarity between the first positive selection probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%; or

[0570] The first positive selection probe is at least 60% complementary to at least a part of the linking part formed by cyclization of the 3'-end and 5'-end of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the non-cyclized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0571] 259. The method according to any one of items 249-258, wherein the positive selection probe has a polynucleotide sequence shown in any one of SEQ ID NO.7203-7206 and SEQ ID NO.7222-7225.

[0572] 260. The method according to item 259, wherein 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 at least a part of any of the two side sequences near the two 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% complementary, or 100% complementary. The complementarity between the first positive selection probe and any part of the non-cyclized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%. Further preferably, the sequence of the second positive selection probe is selected from TT, GATT, TTTC.

[0573] 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 a group II intron in the unpaired region into two fragments.

[0574] 262. The method according to item 261, wherein the 5'-intron fragment and the 3'-intron fragment are obtained by cutting the group II intron at the loop region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0575] The 5'-intron fragment and the 3'-intron fragment are obtained by cutting the group II intron at the linear region 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.

[0576] 263. The method according to item 261 or 262, wherein the group II intron contains one or more nucleotide modifications relative to its wild-type form, and the modifications are selected from one or more of deletion, substitution, and addition.

[0577] Preferably, the modification comprises modifying one or more EBS sequences of a Group II intron, 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.

[0578] 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 both ends of the target sequence respectively.

[0579] 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 both ends of the target sequence respectively.

[0580] 266. The method according to item 264 or 265, 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 at 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 are complementary to a region of corresponding length in the target sequence at at least 60% of the nucleotides,

[0581] Preferably, the EBS1 and / or δ sequence is 100% complementary to a region of corresponding length in the target sequence at 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 are 100% complementary to a region of corresponding length in the target sequence at the nucleotides.

[0582] 267. The method according to item 266, wherein the modification comprises a partial or complete deletion of domain 4, such as a deletion of the intron-encoded protein (IEP) sequence in domain 4, preferably a complete deletion of domain 4; or

[0583] The modification comprises a deletion of the open reading frame (ORF).

[0584] 268. The method according to item 264, wherein the precursor RNA is capable of forming a near scarless circRNA of the target sequence, preferably the near scarless circRNA has a scar region with a 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.

[0585] 269. The method according to item 265, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0586] 270. The method according to any one of items 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.

[0587] 271. The method according to any one of items 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

[0588] 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.

[0589] 272. The method according to any one of items 252-271, wherein the length of E1 and / or E2 is 0-20 nucleotides, preferably the length is 0-10 nucleotides, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides;

[0590] 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.

[0591] 273. The method according to item 272, wherein the length of E1, E2 or both is 0 nucleotides.

[0592] 274. The method according to any one of items 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-A-C sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitches, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences, and protein binding sites.

[0593] 275. The method according to any one of items 252-273, 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 encodes a therapeutic product.

[0594] 276. The method according to any one of items 249-275, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0595] 277. The method according to item 276, wherein at least one of the modified RNA nucleotides and / or modified nucleosides 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-hydroxy-norvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine), 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 (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 (wybutosine), oayW (oxywybutosine), OHyW (hydroxywybutosine), OHyW* (undermodified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archaeosine), 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 (methyl ester of uridine 5-oxyacetic acid), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (methyl ester of 5-(carboxyhydroxymethyl)uridine), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (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 (1,2'-O-dimethylguanosine), m'Am (1,2'-O-dimethyladenosine), rm 5U (5-tauromethyluridine), τm5s2U (5-tauromethyl-2-thiouridine), imG-14 (4-demethylwyeoside), imG2 (isowyeoside), or ac6A (N6-acetyladenosine).

[0596] 278. The method according to any one of items 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.

[0597] The method according to any one of items 252 - 278 further comprises a solid surface,

[0598] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate or paper,

[0599] More preferably, the solid surface is a magnetic bead, agarose bead, streptavidin - coated bead, colloidal bead, glass bead, polymer bead, methacrylate bead, polystyrene bead, latex bead or silica bead, or

[0600] More preferably, the solid surface is a resin containing agarose, carbohydrate - based material, polymer filler, silica, glass particles, perfusion - based material or monolithic support, and the polymer filler is optionally selected from polystyrene - divinylbenzene or polymethacrylate.

[0601] 280. The method according to item 279, wherein the positive selection probe is immobilized on the solid surface by physical adsorption, covalent immobilization or affinity immobilization, and preferably the covalent immobilization is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups.

[0602] 281. The method according to item 279 or 280, wherein the solid surface is a magnetic bead,

[0603] Preferably, the magnetic bead is a functional - group - modified magnetic bead, more preferably an amino - modified magnetic bead, carboxyl - modified magnetic bead, NHS - modified magnetic bead, maleimide - modified magnetic bead, CNBr - modified magnetic bead, magnetic bead modified with an affinity or specific group, or a combination of two or more of the above, and more preferably an NHS - modified magnetic bead, or

[0604] Preferably, the magnetic bead is a hydrophilic magnetic bead; or

[0605] Preferably, the magnetic bead has a diameter in the range of 0.1 μm to 10 μm, more preferably 0.5 μm to 5 μm, more preferably 0.8 μm to 1.2 μm, and most preferably 1 μm.

[0606] 282. The method according to item 279 or 280, wherein the solid surface is a chromatography packing containing agarose, preferably the chromatography packing has a diameter of more than 5 mm and a packing height of more than 20 cm, more preferably the chromatography packing has a diameter of more than 10 mm and a packing height of more than 30 cm, and most preferably the chromatography packing has a diameter of 16 mm and a packing height of 40 cm,

[0607] Further, the agarose chromatography packing material is an agarose chromatography packing material with amino modification on the surface, a carboxyl-modified agarose chromatography packing material, an NHS-modified agarose chromatography packing material, a maleamide-modified agarose chromatography packing material, a CNBr-modified agarose chromatography packing material, an agarose chromatography packing material with affinity or specific group modification, or a combination of two or more of the above, and is further preferably an NHS-modified agarose chromatography packing material.

[0608] 283. The method according to any one of items 279-282, wherein the 3' end of the positive selection probe is covalently fixed to the solid surface; or the 5' end of the positive selection 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.

[0609] 284. The method according to any one of items 279-283, wherein the 3' end and / or 5' end of the positive selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto.

[0610] 285. The method according to item 283, wherein the positive selection probe comprises a second positive selection probe, a first positive selection probe and a second positive selection probe connected in sequence, wherein the 3' end of one of the second positive selection probes is covalently fixed to the solid surface; or the 5' end of the other second positive selection 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.

[0611] 286. The method according to item 284, wherein the positive selection probe comprises a second positive selection probe, a first positive selection probe and a second positive selection probe connected in sequence, wherein the 3' end of one of the second positive selection probes and / or the 5' end of the other second positive selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto.

[0612] 287. The method according to item 285 or 286, wherein the positive selection probe has a polynucleotide sequence comprising any one of SEQ ID NOs. 7205-7206.

[0613] 288. The method according to any one of items 249-287, wherein the sample containing the circular nucleic acid to be purified is contacted with the positive selection 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

[0614] The sample is contacted with the positive selection probe at a temperature of 0°C to 60°C; or

[0615] The sample is contacted with the positive selection probe in a binding solution, the binding solution comprising salts, preferably the binding solution further comprises a buffering substance, a chelating agent, optionally a stabilizer, and further 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 NH 4 + ,and / or anions selected from the group consisting of: PO 4 3- 、SO 4 3- 、CH 3 CO 3 - 、Cl - 、Br - 、NO 3 3- 、ClO 4 3- 、I - and SCN - or a combination thereof, preferably the concentration of the salts in the binding solution is 50 mM to 5 M,

[0616] Further preferably, the salts are salts with cations being Li + or Na + .

[0617] 289. According to the method of item 288, wherein the binding solution comprises 0.1 M to 0.5 M LiCl or NaCl, preferably the binding solution further comprises EDTA and Tris-HCl, and the pH range of the binding solution is 6.0 to 8.0, preferably 7.5,

[0618] Preferably, the method further comprises a step of eluting a part of the positive selection probe bound to the solid surface with an eluent to recover the solid surface conjugated with the positive selection probe for reuse.

[0619] The method according to any one of items 249-289, wherein the concentration of salts in the eluent is 0 M, and the salts include 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 NH 4 + , and / or anions selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the salts are salts with cations being Li + or Na + of the salts,

[0620] Preferably, the eluent further includes a buffering substance, a chelating agent, and optionally includes a stabilizer.

[0621] 291. The method according to item 290, wherein the eluent includes EDTA and Tris-HCl, and the pH range of the eluent is 6.0-8.0, preferably 7.5; or

[0622] the pH range of the eluent is 2.0-4.0, preferably the pH range is 2.5-3.0, and preferably the eluent is glycine-HCl with a pH range of 2.5-3.0.

[0623] 292. The method according to any one of items 249-291, wherein the method is scalable and may include a continuous flow process, preferably carried out using a liquid chromatograph (LC) or a microplate reader, and further preferably the LC is a fast protein liquid chromatograph (FPLC) or a high performance liquid chromatograph (HPLC).

[0624] 293. A method for purifying circular nucleic acids from a sample, comprising the following steps:

[0625] Coupling magnetic beads with a positive selection probe;

[0626] Incubating an in vitro transcription (IVT) reaction solution containing the circular nucleic acid obtained by in vitro transcription with the magnetic beads coupled with the positive selection probe;

[0627] Collecting the magnetic beads for elution to obtain the eluted circular nucleic acid, and recovering the magnetic beads coupled with the positive selection probe;

[0628] Wherein the circular nucleic acid is generated by self-splicing of a precursor nucleic acid, wherein the positive selection probe is at least 60% complementary to at least a part of the circular nucleic acid generated by self-splicing of the precursor nucleic acid, at least a part of the circular nucleic acid spans the splicing part of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0629] 294. The method according to item 293, wherein the incubation of the IVT reaction solution with the magnetic beads coupled with the positive selection probe is carried out in a binding solution, and the magnetic beads are eluted with an elution solution to obtain the eluted circular nucleic acid, and the magnetic beads coupled with the positive selection probe are recovered.

[0630] 295. The method according to item 294, wherein the elution solution and the binding solution are the elution solution and the binding solution involved in any one of items 288-291, and further preferably the positive selection probe is the positive selection probe involved in any one of items 249-292.

[0631] 296. The method according to any one of items 249-295, or the circular nucleic acid purified by using the composition containing an oligonucleotide positive selection probe for purifying circular nucleic acids according to any one of items 201-239, or by using the kit according to any one of items 240-248.

[0632] 297. A composition having the circular nucleic acid according to item 296, wherein the cyclization percentage (PC) of the composition is at least 90%, at least 95%, at least 98% or at least 99%; or

[0633] The total ring purity of the composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, above 95%, and preferably the multimer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%,

[0634] Further preferably, the size of the circular nucleic acid is 1000 nt - 5000 nt.

[0635] 298. A composition for generating circular nucleic acids, comprising:

[0636] A linearized plasmid for generating precursor nucleic acids;

[0637] The generated precursor nucleic acids;

[0638] The generated circular nucleic acids;

[0639] The uncircularized precursor nucleic acids;

[0640] Impurities remaining from in vitro transcription reactions, wherein the precursor nucleic acids and circular nucleic acids are the precursor nucleic acids and circular nucleic acids described in any one of items 201 - 248,

[0641] Preferably, the total ring purity of the composition ranges from 20% to 50%, and the multimer content ranges from 10% to 20%.

[0642] 299. The composition according to item 298, which is used as a sample or purified via a composition containing an oligonucleotide positive selection probe for purifying circular nucleic acids described in any one of items 201 - 239 or using a kit described in any one of items 240 - 248 for the method described in any one of items 249 - 296.

[0643] 300. The composition according to item 299, wherein the total ring purity of the purified circular nucleic acid composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, above 95%,

[0644] Preferably, the multimer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%,

[0645] More preferably, the size of the circular nucleic acid is 1000 nt - 5000 nt.

[0646] 301. A composition containing an oligonucleotide probe for purifying circular nucleic acids, wherein the probe includes a negative selection probe and a positive selection probe,

[0647] The circular nucleic acid is generated by self - splicing of a precursor nucleic acid containing an oligonucleotide negative selection tag, wherein the negative selection tag is at least 90% complementary to the negative selection probe, and the negative selection tag is removed during the self - splicing process 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, and

[0648] The positive selection probe is at least 60% complementary to at least a part of the circular nucleic acid generated by self-splicing of the precursor nucleic acid, at least a part of the circular nucleic acid spans the splicing part of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0649] 302. The composition according to item 301, wherein the precursor nucleic acid contains the negative selection tag at its 5' end; and / or the precursor nucleic acid contains the negative selection tag at its 3' end.

[0650] Under the condition that both the negative selection tag at the 5' end and the negative selection tag at the 3' end are present, the negative selection tag at the 5' end and the negative selection tag at the 3' end can be the same or different.

[0651] Preferably, the positive selection 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 part of the circular nucleic acid generated by self-splicing of the precursor nucleic acid, more preferably 100% complementary, and there is no sequence on the precursor nucleic acid 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 selection probe, more preferably 100% complementary.

[0652] 303. The composition according to item 301 or 302, wherein the circular nucleic acid is circular RNA (circRNA), preferably the precursor nucleic acid is an RNA with group II intron self-splicing activity.

[0653] 304. The composition according to item 303, wherein the precursor RNA contains the following operably linked elements from 5' to 3': (a) a 3' intron fragment; (b) an exon fragment 2 (E2); (c) a target sequence; (d) an exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0654] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of group II introns, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0655] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length ≥ 0 nucleotides;

[0656] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length ≥ 0 nucleotides; and

[0657] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0658] 305. The composition according to item 303, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0659] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a Group II intron, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the Group II intron;

[0660] (2) The E1 is the 5'-adjacent exon fragment of the Group II intron, and its length is ≥ 0 nucleotides;

[0661] (3) The E2 is the 3'-adjacent exon fragment of the Group II intron, and its length is ≥ 0 nucleotides; and

[0662] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0663] 306. The composition according to item 303, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0664] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of a Group II intron, wherein the 5' intron fragment is located on the 5' side of the 3' intron fragment in the Group II intron;

[0665] (2) The E1 is the 5'-adjacent exon fragment of the Group II intron, and its length is ≥ 0 nucleotides;

[0666] (3) The E2 is the 3'-adjacent exon fragment of the Group II intron, and its length is ≥ 0 nucleotides; and

[0667] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0668] 307. The composition according to item 303, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0669] (1) Both the 5' intron fragment and the 3' intron fragment are 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;

[0670] (2) The E1 is the 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0671] (3) The E2 is the 3'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides; and

[0672] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0673] 308. The composition according to any one of items 301-307, wherein the length of the negative selection tag and the negative selection probe is 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides; preferably, the length of the negative selection tag and the negative selection probe is 20-30 nucleotides;

[0674] 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.

[0675] 309. The composition according to any one of items 301-308, wherein the negative selection probe is an RNA probe; or the negative selection probe is a DNA probe; the positive selection probe is an RNA probe or a DNA probe.

[0676] 310. The composition according to any one of items 301-309, wherein the negative selection tag has a polynucleotide sequence comprising SEQ ID NO: n, and the negative selection probe has a polynucleotide sequence comprising SEQ ID NO: n + 1100, where n is an integer from 135 to 1234; or wherein the negative selection tag has a polynucleotide sequence comprising SEQ ID NO: m, and the negative selection probe has a polynucleotide sequence comprising SEQ ID NO: m + 1210, where m is an integer from 2335 to 3544 or 4755 to 5964,

[0677] Preferably, the positive selection probe includes a first positive selection probe and an optional second positive selection probe.

[0678] The first positive selection probe is at least 60% complementary to the linked fragment portion 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% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%; or

[0679] The first positive selection probe is at least 60% complementary to at least a part of the linked portion formed by cyclization of the 3'-end and 5'-end of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0680] 311. The composition according to any one of items 301-309, wherein the negative selection tag has a polynucleotide sequence selected from any one of SEQ ID NOs: 7189-7202; or the negative selection probe has a polynucleotide sequence selected from any one of SEQ ID NOs: 7175-7188.

[0681] Preferably, the positive selection probe has a polynucleotide sequence selected from any one of SEQ ID NOs: 7203-7206 and SEQ ID NOs: 7222-7225.

[0682] 312. The composition according to item 311, wherein the negative selection tag and the negative selection probe of the composition are selected from any one of the following combinations:

[0683] (a) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7189, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7175;

[0684] (b) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7190, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7176;

[0685] (c) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7191, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7177;

[0686] (d) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7192, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7178;

[0687] (e) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7193, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7179;

[0688] (f) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7194, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7180;

[0689] (g) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7195, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7181;

[0690] (h) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7196, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7182;

[0691] (i) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7197, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7183;

[0692] (j) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7198, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7184;

[0693] (k) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7199, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7185;

[0694] (l) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7200, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7186;

[0695] (m) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7201, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7187;

[0696] (n) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO:7202, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO:7188,

[0697] 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 at least a part of any of the two side sequences on the outer sides of 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% complementary, or 100% complementary. The complementarity between the first positive selection probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%. Further preferably, the sequence of the second positive selection probe is selected from TT, GATT, TTTC.

[0698] 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 a group II intron of an unpaired region into two fragments.

[0699] 314. The composition according to item 313, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron at the loop region of the stem - loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0700] The 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron at the linear region 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.

[0701] 315. The composition according to item 313 or 314, wherein the group II intron contains one or more nucleotide modifications relative to its wild - type form, and the modifications are selected from one or more of deletions, substitutions, and additions.

[0702] Preferably, the modification includes the modification of one or more EBS sequences of the group II intron, wherein the EBS sequences are complementary paired with one or more regions of corresponding lengths in the target sequence at at least 60% of the nucleotide positions.

[0703] 316. The composition according to item 315, wherein the modification is the modification of two EBS sequences (such as EBS1 and EBS3) of the group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding lengths in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are respectively located at both ends of the target sequence.

[0704] 317. The composition according to item 315, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of group II introns, and the EBS sequences are complementary paired with two regions of corresponding lengths in the target sequence at at least 60% of the nucleotide positions; preferably, the two regions are respectively located at both ends of the target sequence.

[0705] 318. The composition according to item 315 or 316, wherein the modification is the modification of EBS1 and / or δ sequence of group II introns, or the modification of EBS1' and / or δ” sequence, and the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides. Optionally, the modification is the modification of EBS1 and / or δ sequence and its upstream sequence, and the EBS1 and / or δ sequence and its upstream are complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides.

[0706] Preferably, the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at 100% of the nucleotides. Optionally, the modification is the modification of EBS1 and / or δ sequence and its upstream sequence, and the EBS1 and / or δ sequence and its upstream are complementary paired with a region of corresponding length in the target sequence at 100% of the nucleotides.

[0707] 319. The composition according to item 314, wherein the modification includes the deletion of part or all of domain 4, such as the deletion of the intron-encoded protein (IEP) sequence in domain 4, preferably the complete deletion of domain 4; or

[0708] The modification includes the deletion of the open reading frame (ORF).

[0709] 320. The composition according to item 316, wherein the precursor RNA can form a near scarless circRNA of the target sequence. Preferably, the near scarless circRNA has a scar region with a 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.

[0710] 321. The composition according to item 317, wherein the precursor RNA can form a scarless circRNA of the target sequence.

[0711] 322. The composition according to any one of items 303 - 321, wherein the group II intron is a group II intron derived from a microorganism. Preferably, the group II intron is Cte1. More preferably, the group II intron contains a polynucleotide sequence selected from SEQ ID NO: 33 - 41.

[0712] 323. The composition according to any one of items 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 NOs: 42 - 52; and / or

[0713] 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 NOs: 75 - 88.

[0714] 324. The composition according to any one of items 304 - 323, 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;

[0715] preferably, E2 comprises a polynucleotide sequence selected from SEQ ID NOs: 53 - 63 and SEQ ID NOs: 7211 - 7212; and / or E1 comprises a polynucleotide sequence selected from SEQ ID NOs: 64 - 74 and SEQ ID NO: 7213.

[0716] 325. The composition according to item 324, wherein the length of E1, E2 or both is 0 nucleotides.

[0717] 326. The composition according to any one of items 304 - 325, wherein the target sequence is a non - coding sequence selected from the group consisting of: spacer sequences of SEQ ID NOs: 4 - 6, polyA sequences, poly - A - C sequences, polyC sequences, polyU sequences, IRES, ribosome binding sites, aptamer sequences, RNA scaffolds, riboswitches, ribozymes other than self - splicing ribozymes, antisense oligonucleotides (ASO), scaffolds, small RNA binding sites, translation regulatory sequences and protein binding sites.

[0718] 327. The composition according to any one of items 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 encodes a therapeutic product.

[0719] 328. The composition according to any one of items 301 - 327, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0720] 329. The composition according to item 328, wherein at least one of the modified RNA nucleotides and / or modified nucleosides 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-hydroxy-norvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine), 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 (lysine), m1G (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 (wybutosine), oayW (oxywybutosine), OHyW (hydroxywybutosine), OHyW* (undermodified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archaeosine), 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 (methyl ester of uridine 5-oxyacetic acid), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (methyl ester of 5-(carboxyhydroxymethyl)uridine), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (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 (1,2'-O-dimethylguanosine), m'Am (1,2'-O-dimethyladenosine), rm 5U (5-tauromethyluridine), τm5s2U (5-tauromethyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine).

[0721] 330. The composition according to any one of items 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.

[0722] The composition according to any one of items 301 - 330 further comprises a solid surface,

[0723] preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate or paper,

[0724] more preferably, the solid surface is a magnetic bead, agarose bead, streptavidin - coated bead, colloidal bead, glass bead, polymer bead, methacrylate bead, polystyrene bead, latex bead or silica bead, or

[0725] more preferably, the solid surface is a resin containing agarose, carbohydrate - based material, polymer filler, silica, glass particles, perfusion - based material or monolithic support, and the polymer filler is optionally selected from polystyrene - divinylbenzene or polymethacrylate.

[0726] 332. The composition according to item 331, wherein the negative selection probe is immobilized on the solid surface by physical adsorption, covalent immobilization or affinity immobilization, and preferably the covalent immobilization is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups,

[0727] the positive selection probe is immobilized on the solid surface by physical adsorption, covalent immobilization or affinity immobilization, and preferably the covalent immobilization is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups.

[0728] 333. The composition according to item 331 or 332, wherein the solid surface is a magnetic bead,

[0729] preferably, the magnetic bead is a functional - group - modified magnetic bead, more preferably an amino - modified magnetic bead, carboxyl - modified magnetic bead, NHS - modified magnetic bead, maleimide - modified magnetic bead, CNBr - modified magnetic bead, magnetic bead modified with an affinity or specific group, or a combination of two or more of the above, and more preferably an NHS - modified magnetic bead, or

[0730] preferably, the magnetic bead is a hydrophilic magnetic bead; or

[0731] preferably, the magnetic bead has a diameter in the range of 0.1 μm to 10 μm, more preferably 0.5 μm to 5 μm, more preferably 0.8 μm to 1.2 μm, and further preferably 1 μm.

[0732] 334. The composition according to item 331 or 332, wherein the solid surface is an agarose chromatography packing material,

[0733] Preferably, the diameter of the chromatography packing material is above 5 mm, and the height of the packing material is above 20 cm. Further preferably, the diameter of the chromatography packing material is above 10 mm, and the height of the packing material is above 30 cm. Further preferably, the diameter of the chromatography packing material is 16 mm, and the height of the packing material is 40 cm

[0734] Furthermore, the agarose chromatography packing material is an agarose chromatography packing material with an amino modification on the surface, a carboxyl modification on the surface, an NHS modification on the surface, a maleimide modification on the surface, a CNBr modification on the surface, an agarose chromatography packing material with an affinity or specific group modification, or a combination of two or more of the above. Further preferably, it is an agarose chromatography packing material with an NHS modification

[0735] 335. The composition according to any one of items 331-334, wherein the 3'-end of the negative selection probe is covalently fixed to the solid surface; or the 5'-end of the negative selection probe is covalently fixed to the solid surface. Preferably, the solid surface is a magnetic bead. Preferably, the covalent fixation is achieved through the covalent connection of NH2 and a carboxyl group modified by NHS

[0736] The 3'-end of the positive selection probe is covalently fixed to the solid surface; or the 5'-end of the positive selection probe is covalently fixed to the solid surface. Preferably, the solid surface is a magnetic bead. Preferably, the covalent fixation is achieved through the covalent connection of NH2 and a carboxyl group modified by NHS

[0737] The negative selection probe and the positive selection probe are fixed on different solid surfaces

[0738] 336. The composition according to any one of items 331-335, wherein the 3'-end and / or 5'-end of the negative selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto

[0739] The 3'-end and / or 5'-end of the positive selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto

[0740] 337. The composition according to any one of items 301-336, wherein the minimum free energy (MFE) value of the negative selection tag ranges from -2 to 2, and the change in the MFE value (ΔMFE) of the precursor nucleic acid caused by including the tag ranges from -2 to 2

[0741] Preferably, the positive selection probe comprises a second positive selection probe, a first positive selection probe, and a second positive selection probe connected in sequence, wherein the 3'-end of one of the second positive selection probes is covalently fixed to the solid surface; or the 5'-end of the other second positive selection 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 a carboxyl group modified with NH2 and NHS.

[0742] 338. The composition according to item 337, wherein the MFE value of the negative selection tag is about 0.

[0743] Preferably, the positive selection probe comprises a second positive selection probe, a first positive selection probe, and a second positive selection probe connected in sequence, wherein the 3'-end of one of the second positive selection probes and / or the 5'-end of the other second positive selection probe is further modified with a group selected from the following: amino group, carboxyl group, NHS-carboxyl group, mercapto group.

[0744] 339. The composition according to item 337 or 338, wherein the range of the ΔMFE value of the precursor nucleic acid caused by the inclusion of the negative selection tag is 0-2, and preferably the ΔMFE value of the precursor nucleic acid caused by the inclusion of the negative selection tag is about 0.

[0745] Preferably, the positive selection probe has a polynucleotide sequence comprising any one of SEQ ID NOs. 7205-7206.

[0746] 340. A negative selection kit for purifying circular nucleic acids, comprising: the composition comprising oligonucleotide probes for purifying circular nucleic acids according to any one of items 301-339, a binding solution, and an elution solution.

[0747] 341. The kit according to item 340, wherein the binding solution comprises salts, and the salts comprise cations selected from the following group: Ba 2+ , Ca 2+ , Mg 2+ , Mn 2+ , Fe 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Pb 2+ , Li + , Cs + , Na + , K + , Rb + and NH 4 + , and / or anions selected from the following group: PO 4 3- , SO 4 3- , CH3 CO 3 - , Cl - Br - 、NO 3 3- , ClO 4 3- ,I - and SCN - or a combination thereof, preferably the concentration of the salt in the binding solution is 50mM-5M,

[0748] It is further preferred that the salt is a cation of Li + Or Na + of salts.

[0749] 342. A kit according to item 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;

[0750] Preferably, the binding solution further comprises a buffer substance, a chelating agent, and optionally a stabilizer.

[0751] 343. A kit according to item 341 or 342, wherein the binding solution comprises 0.1M-0.5M LiCl or NaCl, preferably the binding solution also comprises EDTA and Tris-HCl, and the pH range of the binding solution is 6.0-8.0, preferably 7.5.

[0752] 344. A kit according to any one of items 340-343, wherein the concentration of the salt in the eluent is 0M, and the salt includes 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 NH 4 + , and / or an anion selected from the group consisting of: PO 4 3- 、SO 4 3- , CH 3 CO3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof, preferably the salts are salts with cations being Li + or Na + .

[0753] Preferably, the eluent further comprises a buffering substance, a chelating agent, and optionally a stabilizer.

[0754] 345. The kit according to item 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

[0755] the pH range of the eluent is 2.0 - 4.0, preferably the pH range is 2.5 - 3.0, and preferably the eluent is glycine - HCl with a pH range of 2.5 - 3.0.

[0756] 346. A kit for purifying circular nucleic acids, comprising: magnetic beads coupled with a negative selection probe, magnetic beads coupled with a positive selection probe, an eluent, and a binding solution,

[0757] the negative selection probe is at least 90% complementary to the negative selection tag, and the circular nucleic acid is generated by self - splicing of a precursor nucleic acid containing an oligonucleotide negative selection tag, wherein the negative selection tag is removed during the self - splicing process 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 further preferably 100% complementary,

[0758] the positive selection probe is at least 60% complementary to at least a part of the circular nucleic acid generated by self - splicing of the precursor nucleic acid, at least a part of the circular nucleic acid spans the splicing part of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0759] 347. The kit according to item 346, wherein the negative selection probe and the negative selection tag are the negative selection probe, negative selection tag, and positive selection probe involved in any one of items 301 - 339, or

[0760] the eluent and the binding solution are the eluent and the binding solution involved in any one of items 340 - 345.

[0761] 348. The kit according to item 346 or 347, wherein the magnetic beads immobilized with the negative selection probes can be reused, and the magnetic beads immobilized with the positive selection probes can be reused.

[0762] 349. A method for purifying circular nucleic acids from a sample, wherein the circular nucleic acids are generated by self-splicing of a precursor nucleic acid, and the precursor nucleic acid has an oligonucleotide negative selection tag that is removed during the self-splicing process. The method includes:

[0763] (i) contacting the sample and an oligonucleotide negative selection probe that is at least 60% complementary to the negative selection tag under conditions that allow the negative selection tag to bind to the negative selection probe, wherein the negative selection probe is immobilized on a solid surface;

[0764] (ii) collecting the unbound portion of the sample;

[0765] (iii) contacting the sample comprising the circular nucleic acids generated 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 under conditions that allow the circular nucleic acids generated by self-splicing of the precursor nucleic acid to bind to the positive selection probe, wherein the positive selection probe is immobilized on a solid surface; and

[0766] (iv) collecting the portion that binds to the positive selection probe on the solid surface,

[0767] at least a portion of the circular nucleic acids spans the splicing portion of the circular nucleic acids, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe,

[0768] 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.

[0769] 350. The method according to item 349, wherein the precursor nucleic acid comprises the negative selection tag at its 5' end; or the precursor nucleic acid comprises the negative selection tag at its 3' end,

[0770] Under the condition that both the 5'-end negative selection tag and the 3'-end negative selection tag are present, the 5'-end negative selection tag and the 3'-end negative selection tag can be the same or different,

[0771] Preferably, the positive selection 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 part of the circular nucleic acid generated by self-splicing of the precursor nucleic acid, more preferably 100% complementary, and there is no sequence on the precursor nucleic acid 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 selection probe, more preferably 100% complementary.

[0772] 351. The method according to item 349 or 350, wherein the circular nucleic acid is circular RNA (circRNA), preferably the precursor nucleic acid is an RNA having group II intron self-splicing activity.

[0773] 352. The method according to item 351, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 3' intron fragment; (b) an exon fragment 2 (E2); (c) a target sequence; (d) an exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0774] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of group II introns, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0775] (2) The E1 is a 5'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides;

[0776] (3) The E2 is a 3'-adjacent exon fragment of the group II intron, and its length is ≥0 nucleotides; and

[0777] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0778] 353. The method according to item 351, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 3' intron fragment; (b) an exon fragment 2 (E2); (c) a linker sequence; (d) a target sequence; (e) a linker sequence; (f) an exon fragment 1 (E1); and (g) a 5' intron fragment; wherein:

[0779] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of group II introns, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the group II intron;

[0780] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0781] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0782] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0783] 354. The method according to item 351, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0784] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of the Group II intron, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the Group II intron;

[0785] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0786] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0787] (4) The target sequence is absent, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0788] 355. The method according to item 351, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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:

[0789] (1) Both the 5' intron fragment and the 3' intron fragment are fragments of the Group II intron, and the 5' intron fragment is located on the 5' side of the 3' intron fragment in the Group II intron;

[0790] (2) The E1 is a 5'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides;

[0791] (3) The E2 is a 3'-adjacent exon fragment of the Group II intron, and its length is ≥0 nucleotides; and

[0792] (4) The target sequence does not exist, or is a protein-coding sequence, a non-coding sequence, or a combination of both.

[0793] 356. The method according to any one of items 349 - 355, wherein the lengths of the negative selection tag and the negative selection probe are 10 - 50, 10 - 40, 10 - 30, 20 - 50, 20 - 40, or 20 - 30 nucleotides; preferably, the lengths of the negative selection tag and the negative selection probe are 20 - 30 nucleotides.

[0794] 357. The method according to any one of items 349 - 356, wherein the negative selection probe is an RNA probe; or the negative selection probe is a DNA probe, and the positive selection probe is an RNA probe or a DNA probe.

[0795] 358. The method according to any one of items 349 - 357, wherein the negative selection tag has a polynucleotide sequence comprising SEQ ID NO: n, and the negative selection probe has a polynucleotide sequence comprising SEQ ID NO: n + 1100, where n is an integer from 135 - 1234; or wherein the negative selection tag has a polynucleotide sequence comprising SEQ ID NO: m, and the negative selection probe has a polynucleotide sequence comprising SEQ ID NO: m + 1210, where m is an integer from 2335 - 3544 or 4755 - 5964,

[0796] Preferably, the positive selection probe includes a first positive selection probe and optionally a second positive selection probe,

[0797] The first positive selection probe is at least 60% complementary to the linked fragment portion 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% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%; or

[0798] The first positive selection probe is at least 60% complementary to at least a part of the linked portion formed by circularizing the 3'-end and 5'-end of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0799] 359. The method according to any one of items 349 - 358, wherein the negative selection tag has a polynucleotide sequence comprising any one shown in SEQ ID NO: 7189 - 7202; or the negative selection probe has a polynucleotide sequence comprising any one shown in SEQ ID NO: 7175 - 7188,

[0800] Preferably, the positive selection probe has a polynucleotide sequence comprising any one shown in SEQ ID NO. 7203 - 7206 and SEQ ID NO. 7222 - 7225.

[0801] 360. The method according to item 359, wherein the negative selection tag and the negative selection probe are selected from any one of the following combinations:

[0802] (a) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7189, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7175;

[0803] (b) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7190, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7176;

[0804] (c) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7191, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7177;

[0805] (d) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7192, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7178;

[0806] (e) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7193, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7179;

[0807] (f) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7194, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7180;

[0808] (g) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7195, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7181;

[0809] (h) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7196, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7182;

[0810] (i) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7197, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7183;

[0811] (j) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7198, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7184;

[0812] (k) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7199, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7185;

[0813] (l) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7200, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7186;

[0814] (m) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7201, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7187;

[0815] (n) The polynucleotide sequence of the negative selection tag is as shown in SEQ ID NO: 7202, and the polynucleotide sequence of the negative selection probe is as shown in SEQ ID NO: 7188,

[0816] 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, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, to at least a part of any of the two side sequences outside both ends of the target sequence near the two ends. The complementarity between the first positive selection probe and any part of the uncircularized target sequence is less than 60%, preferably less than 70%, 80%, 90%, or 95%. Further preferably, the sequence of the second positive selection probe is selected from TT, GATT, TTTC.

[0817] 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 a group II intron in the unpaired region into two fragments.

[0818] 362. The method according to item 361, wherein the 5' intron fragment and the 3' intron fragment are obtained by cleaving a Group II intron at the loop region of the stem-loop structure of domain 1, domain 2, domain 3, domain 4, domain 5, or domain 6; or

[0819] the 5' intron fragment and the 3' intron fragment are obtained by cleaving a Group II intron at the linear region 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.

[0820] 363. The method according to item 361 or 362, wherein the Group II intron contains one or more nucleotide modifications relative to its wild-type form, and the modifications are selected from one or more of deletions, substitutions, and additions,

[0821] preferably the modification comprises modification of one or more EBS sequences of the Group II intron, wherein the EBS sequences are complementary paired with one or more regions of corresponding length in the target sequence at at least 60% of the nucleotide positions.

[0822] 364. The method according to item 363, wherein the modification is modification of two EBS sequences (such as EBS1 and EBS3) of the Group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably the two regions are located at both ends of the target sequence respectively.

[0823] 365. The method according to item 363, wherein the modification is modification of two EBS sequences (such as EBS1' and EBS3') of the Group II intron, wherein the EBS sequences are complementary paired with two regions of corresponding length in the target sequence at at least 60% of the nucleotide positions; preferably the two regions are located at both ends of the target sequence respectively.

[0824] 366. The method according to item 364 or 365, wherein the modification is modification of the EBS1 and / or δ sequence of the Group II intron, or modification of the EBS1' and / or δ" sequence, wherein the EBS1 and / or δ sequence is complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides, optionally the modification is modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream are complementary paired with a region of corresponding length in the target sequence at at least 60% of the nucleotides,

[0825] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to the corresponding length region in the target sequence. Optionally, the modification is 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 are complementary to the corresponding length region in the target sequence.

[0826] 367. The method according to item 366, wherein the modification includes the partial or complete deletion of domain 4, such as the deletion of the intron-encoded protein (IEP) sequence in domain 4, preferably the complete deletion of domain 4; or

[0827] The modification includes the deletion of the open reading frame (ORF).

[0828] 368. The method according to item 364, wherein the precursor RNA is capable of forming a near-scarless circRNA of the target sequence. Preferably, the near-scarless circRNA has a scar region with a 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.

[0829] 369. The method according to item 365, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0830] 370. The method according to any one of items 351 - 369, wherein the Group II intron is a Group II intron derived from a microorganism. Preferably, the Group II intron is Cte1. More preferably, the Group II intron contains a polynucleotide sequence selected from SEQ ID NO: 33 - 41.

[0831] 371. The method according to any one of items 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

[0832] 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.

[0833] 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;

[0834] 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.

[0835] 373. The method according to item 372, wherein the length of E1, E2 or both is 0 nucleotides.

[0836] 374. The method according to any one of items 352-373, wherein the target sequence is a non-coding sequence selected from the group consisting of: the spacer sequences of SEQ ID NO:4-6, polyA sequence, poly-A-C sequence, polyC sequence, polyU sequence, IRES, ribosome binding site, aptamer sequence, RNA scaffold, riboswitch, ribozymes other than self-splicing ribozymes, antisense oligonucleotides (ASO), scaffold, small RNA binding site, translation regulatory sequence and protein binding site.

[0837] 375. The method according to any one of items 352-373, 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 encodes a therapeutic product.

[0838] 376. The method according to any one of items 349-375, wherein the precursor RNA comprises modified RNA nucleotides and / or modified nucleosides.

[0839] 377. The method according to item 376, wherein at least one of the modified RNA nucleotides and / or modified nucleosides 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'-0-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-hydroxy-norvalylcarbamoyladenosine), ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine), 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 (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 (wybutosine), oayW (peroxywybutosine), OHyW (hydroxywybutosine), OHyW* (under-modified hydroxywybutosine), imG (wyosine), mimG (methylwyosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archaeosine), 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 (methyl ester of uridine 5-oxyacetic acid), chm5U (5-(carboxyhydroxymethyl)uridine), mchm5U (methyl ester of 5-(carboxyhydroxymethyl)uridine), mcm5U (5-methoxycarbonylmethyluridine), mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyluridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyluridine), ncm5Um (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 (1,2'-O-dimethylguanosine), m'Am (1,2'-O-dimethyladenosine), rm 5U (5-tauromethyluridine), τm5s2U (5-tauromethyl-2-thiouridine), imG-14 (4-demethylwyeoside), imG2 (isowyeoside), or ac6A (N6-acetyladenosine).

[0840] 378. The method according to any one of items 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.

[0841] The method according to any one of items 352 - 378 further comprises a solid surface,

[0842] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate or paper,

[0843] More preferably, the solid surface is a magnetic bead, agarose bead, streptavidin - coated bead, colloidal bead, glass bead, polymer bead, methacrylate bead, polystyrene bead, latex bead or silica bead, or

[0844] More preferably, the solid surface is a resin containing agarose, carbohydrate - based material, polymer filler, silica, glass particles, perfusion - based material or monolithic support, and the polymer filler is optionally selected from polystyrene - divinylbenzene or polymethacrylate.

[0845] 380. The method according to item 379, wherein the negative selection probe is immobilized on the solid surface by physical adsorption, covalent immobilization or affinity immobilization, and preferably the covalent immobilization is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups,

[0846] The positive selection probe is immobilized on the solid surface by physical adsorption, covalent immobilization or affinity immobilization, and preferably the covalent immobilization is achieved by covalent linkage of NH2 and NHS - modified carboxyl groups.

[0847] 381. The method according to item 379 or 380, wherein the solid surface is a magnetic bead,

[0848] Preferably, the magnetic bead is a magnetic bead modified with a functional group, more preferably an amino - modified magnetic bead, a carboxyl - modified magnetic bead, an NHS - modified magnetic bead, a maleimide - modified magnetic bead, a CNBr - modified magnetic bead, a magnetic bead modified with an affinity or specific group, or a combination of two or more of the above, and more preferably an NHS - modified magnetic bead, or

[0849] Preferably, the magnetic bead is a hydrophilic magnetic bead; or

[0850] Preferably, the magnetic bead has a diameter in the range of 0.1 μm to 10 μm, more preferably 0.5 μm to 5 μm, more preferably 0.8 μm to 1.2 μm, and more preferably 1 μm.

[0851] 382. The method according to item 379 or 380, wherein the solid surface is a chromatography packing containing agarose, and preferably the chromatography packing has a diameter of more than 5 mm and a packing height of more than 20 cm, more preferably the chromatography packing has a diameter of more than 10 mm and a packing height of more than 30 cm, and more preferably the chromatography packing has a diameter of 16 mm and a packing height of 40 cm,

[0852] Furthermore, the agarose chromatography packing material is an agarose chromatography packing material with amino modification on the surface, carboxyl modification, NHS modification, maleimide modification, CNBr modification, agarose chromatography packing material with affinity or specific group modification, or a combination of two or more of the above, and is further preferably an agarose chromatography packing material with NHS modification.

[0853] 383. The method according to any one of items 379 - 382, wherein the 3'-end of the negative selection probe is covalently fixed to the solid surface; or the 5'-end of the negative selection 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 carboxyl groups modified by NHS.

[0854] The 3'-end of the positive selection probe is covalently fixed to the solid surface; or the 5'-end of the positive selection 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 carboxyl groups modified by NHS.

[0855] The negative selection probe and the positive selection probe are fixed on different solid surfaces.

[0856] 384. The method according to any one of items 379 - 383, wherein the 3'-end and / or 5'-end of the negative selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto.

[0857] The 3'-end and / or 5'-end of the positive selection probe is further modified with a group selected from the following: amino, carboxyl, NHS-carboxyl, mercapto.

[0858] 385. The composition according to any one of items 349 - 384, wherein the minimum free energy (MFE) value of the negative selection tag ranges from -2 to 2, and the change in the MFE value (ΔMFE) of the precursor nucleic acid caused by including the tag ranges from -2 to 2.

[0859] Preferably, the positive selection probe includes a second positive selection probe, a first positive selection probe, and a second positive selection probe connected in sequence, wherein the 3'-end of one of the second positive selection probes is covalently fixed to the solid surface; or the 5'-end of the other second positive selection 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 carboxyl groups modified by NHS.

[0860] 386. The composition according to item 385, wherein the MFE value of the negative selection tag is about 0.

[0861] Preferably, the positive selection probe includes a second positive selection probe, a first positive selection probe, and a second positive selection probe connected in sequence. Among them, the 3'-end of one of the second positive selection probes and / or the 5'-end of the other second positive selection probe is further modified with a group selected from the following: amino group, carboxyl group, NHS-carboxyl group, and mercapto group.

[0862] 387. The composition according to item 385 or 386, wherein the range of the ΔMFE value of the precursor nucleic acid caused by containing the negative selection tag is 0-2, and preferably the ΔMFE value of the precursor nucleic acid caused by containing the negative selection tag is about 0.

[0863] Preferably, the positive selection probe has a polynucleotide sequence shown in any one of SEQ ID NO.7205-7206.

[0864] 388. The method according to any one of items 349-387, wherein the sample containing the circular nucleic acid to be purified is contacted with the negative selection probe or the positive selection 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

[0865] the sample is contacted with the negative selection probe or the positive selection probe at a temperature of 0°C-60°C; or

[0866] the sample is contacted with the negative selection probe or the positive selection probe in a binding solution, and the binding solution includes salts. Preferably, the binding solution further includes a buffering substance, a chelating agent, and optionally includes a stabilizer. Further preferably, the salts include cations selected from the following group: Ba 2+ 、Ca 2+ 、Mg 2+ 、Mn 2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、Cd 2+ 、Pb 2+ 、Li + 、Cs + 、Na + 、K + 、Rb + and NH 4 + ,and / or anions selected from the following group: PO 4 3- 、SO 4 3- 、CH 3 CO 3 - 、Cl- , Br - , NO 3 3- , ClO 4 3- , I - and SCN - or a combination thereof. Preferably, the concentration of the salts in the binding solution is 50 mM - 5 M.

[0867] More preferably, the salts are salts with cations being Li + or Na + .

[0868] 389. The method according to item 388, wherein the binding solution comprises 0.1 M - 0.5 M 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.

[0869] Preferably, the method further comprises a step of eluting a part of the negatively selected probe bound to the solid surface with an eluent to recover the solid surface conjugated with the negatively selected probe for reuse.

[0870] 390. The method according to any one of items 349 - 389, wherein the concentration of the 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 NH 4 + , and / or anions selected from the group consisting of: PO 4 3- , SO 4 3- , CH 3 CO 3 - , Cl - , Br - , NO 3 3- , ClO 4 3- , I - and SCN -or a combination thereof, preferably the salt has a cation of Li + or Na + and the salt is

[0871] Preferably, the eluent further comprises a buffering substance, a chelating agent, and optionally a stabilizer.

[0872] 391. The method according to item 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

[0873] the pH range of the eluent is 2.0 - 4.0, preferably the pH range is 2.5 - 3.0, and preferably the eluent is glycine - HCl with a pH range of 2.5 - 3.0.

[0874] 392. The method according to any one of items 349 - 391, wherein the method is scalable and may include a continuous flow process, preferably carried out using a liquid chromatograph (LC) or a microplate reader, and further preferably the LC is a fast protein liquid chromatograph (FPLC) or a high - performance liquid chromatograph (HPLC).

[0875] 393. A method for purifying circular nucleic acids from a sample, comprising the following steps:

[0876] Coupling magnetic beads with a negative selection probe;

[0877] Coupling magnetic beads with a positive selection probe;

[0878] Incubating an in vitro transcription (IVT) reaction solution containing the circular nucleic acid with the magnetic beads coupled with the negative selection probe;

[0879] Collecting the magnetic beads and eluting to recover the magnetic beads coupled with the negative selection probe;

[0880] Collecting the unbound fraction,

[0881] Incubating the unbound fraction with the magnetic beads coupled with the positive selection probe;

[0882] Collecting the magnetic beads and eluting to obtain the eluted circular nucleic acid, and recovering the magnetic beads coupled with the positive selection probe;

[0883] wherein the negative selection probe is at least 90% complementary to the negative selection tag, and the circular nucleic acid is generated by self - splicing of a precursor nucleic acid containing an oligonucleotide negative selection tag, and the negative selection tag is removed during the self - splicing process 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 further preferably 100% complementary;

[0884] The circular nucleic acid is generated by self-splicing of a precursor nucleic acid, wherein the positive selection probe is complementary to at least 60% of at least a part of the circular nucleic acid generated by self-splicing of the precursor nucleic acid, at least a part of the circular nucleic acid spans the splicing part of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0885] 394. The method according to item 393, wherein the IVT reaction solution is incubated with magnetic beads coupled with a negative selection probe in a binding solution, and the magnetic beads are eluted with an eluent to recover the magnetic beads coupled with the negative selection probe.

[0886] The IVT reaction solution is incubated with magnetic beads coupled with a positive selection probe in a binding solution, the magnetic beads are eluted with an eluent to obtain the eluted circular nucleic acid, and the magnetic beads coupled with the positive selection probe are recovered.

[0887] 395. The method according to item 394, wherein the eluent and the binding solution are the eluent and the binding solution involved in any one of items 388-391, and further preferably, the negative selection probe, the negative selection tag and the positive selection probe are the negative selection probe, the negative selection tag and the positive selection probe involved in any one of items 349-392.

[0888] 396. The method according to any one of items 349-395, or the composition containing oligonucleotide probes for purifying circular nucleic acids according to any one of items 301-339, or the circular nucleic acid purified by the kit according to any one of items 340-348.

[0889] 397. A composition having the circular nucleic acid according to item 396, wherein the cyclization percentage (PC) of the composition is at least 90%, at least 95%, at least 98% or at least 99%; or

[0890] The total ring purity of the composition is above 90.0%, above 91%, above 92%, above 93%, above 94%, above 95%, and preferably the multimer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%.

[0891] Further preferably, the size of the circular nucleic acid is 1000 nt - 5000 nt.

[0892] 398. A composition for generating a circular nucleic acid, comprising:

[0893] A linearized plasmid for generating a precursor nucleic acid;

[0894] The generated precursor nucleic acid;

[0895] Generated circular nucleic acid;

[0896] Uncircularized precursor nucleic acid;

[0897] Residual impurities in the in vitro transcription reaction, wherein the precursor nucleic acid and the circular nucleic acid are the precursor nucleic acid and the circular nucleic acid involved in any one of items 301-348,

[0898] Preferably, the total ring purity of the composition ranges from 20% to 50%, and the multimer content ranges from 10% to 20%.

[0899] 399. The composition according to item 398, which is used as a sample or purified as a sample via the composition comprising an oligonucleotide anion selection probe for purifying circular nucleic acid according to any one of items 301-339 or using the kit according to any one of items 340-348 in the method according to any one of items 349-396.

[0900] 400. The composition according to item 399, wherein the total ring purity of the circular nucleic acid composition after purification is above 90.0%, above 91%, above 92%, above 93%, above 94%, above 95%,

[0901] Preferably, the multimer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%. More preferably, the size of the circular nucleic acid is 1000 nt - 5000 nt. Description of the Drawings

[0902] Figure 1A-1D Schematic diagram of the self-splicing of a precursor RNA with group II intron self-splicing activity and the use of the tags disclosed herein for purifying circRNA products. Figure 1A Schematic diagram of a near scarless system designed based on the interaction between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The self-catalytic self-splicing group II intron is split into two fragments at the D4 domain, and a custom exon containing E1, E2, and the target sequence is inserted between the split introns. The arrows indicate the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3.

[0903] Figure 1B Schematic diagram of a near scarless system designed based on the interaction between δ and IBS3. The self-catalytic self-splicing group II intron is split into two fragments at the D4 domain, and a custom exon containing E1, E2, and the target sequence is inserted between the split introns. The arrows indicate the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and δ.

[0904] Figure 1C It is a schematic diagram of a scarless system designed based on the interaction between IBS1’ and EBS1. The self-catalytic and self-splicing group II intron is split into two fragments at the D4 domain, and the target sequence is inserted between the split introns. The arrows indicate the interactions between IBS1' and EBS1, and between IBS3' and EBS3.

[0905] Figure 1D It is a schematic diagram of a scarless system designed based on the interaction between δ and IBS3’. The self-catalytic and self-splicing group II intron is split into two fragments at the D4 domain, and the target sequence is inserted between the split introns. The arrows indicate the interactions between IBS1’ and EBS1, and between IBS3’ and δ.

[0906] Figure 2 The results of an exemplary purification process are shown. As shown, the IVT product is the reaction product of precursor RNA generated by in vitro transcription and subjected to cyclization conditions. FPLC purification as disclosed herein yields a P1 fraction containing highly purified circRNA (results of Example 2).

[0907] Figure 3A A schematic diagram of the negative selection system of the present application is provided. Figure 3A It 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-catalytic and self-splicing group II intron is split into two fragments at the D4 domain, and a custom exon containing E1, E2, and the target sequence is inserted between the split introns. The arrows indicate the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The negative selection tags are located at both ends of the D4 domain split into two fragments.

[0908] Figure 3B A schematic diagram of the positive selection system of the present application is provided. Figure 3B It 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-catalytic and self-splicing group II intron is split into two fragments at the D4 domain, and a custom exon containing E1, E2, and the target sequence is inserted between the split introns. The arrows indicate the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The positive selection tags are located on IBS1 and IBS3. In some embodiments, part of the positive selection tag sequence is also located on the target sequence.

[0909] Figure 3CA schematic diagram of the solid surface (such as magnetic beads or agarose chromatography packing material) conjugated with the negative selection probe of the present application is provided.

[0910] Figure 3D A schematic diagram of the solid surface (such as magnetic beads or agarose chromatography packing material) conjugated with the first negative selection probe and the second negative selection probe of the present application is provided.

[0911] Figure 3E A schematic diagram of the solid surface (such as magnetic beads or agarose chromatography packing material) conjugated with the positive selection probe of the present application is provided.

[0912] Figure 4 The results graphs before and after chromatographic purification are shown. As Figure 4 shown, after undergoing chromatographic purification, most of the impurities are removed and the purity of the circular RNA is improved.

[0913] Figure 5A and 5B respectively show the capillary electrophoresis results graphs before and after negative selection magnetic bead purification. As shown in the figure, after undergoing negative selection magnetic bead purification, the introns and precursors are removed and the purity of the circular RNA is improved.

[0914] Figure 6 The HPLC results graph for detecting the content of multimers after purification is shown. As shown in the figure, the content of multimers is extremely low after purification.

[0915] Figure 7 A and Figure 7 B respectively show the agarose electrophoresis detection results graphs after negative selection magnetic bead purification and positive selection magnetic bead purification. As shown in the figure, after negative selection magnetic bead purification and positive selection magnetic bead purification, most of the impurities are removed and the purity of the circular RNA is improved.

[0916] Figure 8 The results graph for translating Gluc and Fluc after purification is shown. As shown in the figure, G1 - G7 are Gluc and F1 - F12 are Fluc, both are samples after purification, and the main band obtained is circular RNA. It can be seen from the figure that the impurity bands above and below are basically invisible.

[0917] Figure 9 It is a schematic diagram of the type II intron structure.

[0918] Figure 10 It is a schematic diagram of the type II intron with IBS1, IBS2, IBS3, EBS1, EBS2, EBS3, δ (shown in bold).

[0919] Figure 11 The amplification of the generation of circRNA is shown.

[0920] Figure 12Shows the capillary electrophoresis analysis of four batches of CVB3-Gluc circRNA purified by HPLC using an Agilent 2100 Bioanalyzer.

[0921] Figure 13 Is a schematic diagram of the CircRNA-LNP complex and the particle size of CircRNAGluc-LNP.

[0922] Figure 14 Shows the Gaussia luciferase activity measured from mouse serum 24 hours after injection of CircRNAGluc-LNP with different formulations.

[0923] Figure 15 Shows representative IVIS images of BALB / c mice administered 20 μg of CircRNAGluc-LNP with two formulations via the intramuscular (i.m.) route. The relative luminescence map is shown, and the scale of luminescence is indicated.

[0924] Figure 16 Shows a schematic diagram of the RNA high-throughput screening workstation of the present application.

[0925] Figure 17 Is an image showing the in vivo imaging system (IVIS) spectrum of protein expression after intravenous injection of an exemplary circular RNA having a combination of a native IRES and an IRES-like sequence of the present disclosure.

[0926] Figure 18A Provides a schematic diagram of a circular RNA, which includes in the following order: a 5' tag, a 3' intron fragment, a translation initiation element, a target sequence, a 5' intron fragment, and a 3' tag.

[0927] Figure 18B Provides a schematic diagram of a circular RNA, which includes in the following order: a 3' intron fragment, a translation initiation element, a target sequence, and a 5' intron fragment. Wherein partial sequences on the translation initiation element and the target sequence are used as positive selection tags.

[0928] Figure 18C Provides a schematic diagram of a circular RNA, which includes in the following order: a 5' tag, a 3' intron fragment, a target sequence, a translation initiation element, a 5' intron fragment, and a 3' tag.

[0929] Figure 18D Provides a schematic diagram of a circular RNA, which includes in the following order: a 3' intron fragment, a target sequence, a translation initiation element, and a 5' intron fragment. Wherein partial sequences on the translation initiation element and the target sequence are used as positive selection tags.

[0930] Figure 19AA schematic diagram of circular RNA is provided, which includes, in the following order: a 5' tag, a 3' intron fragment, an adaptor sequence 2, a translation initiation element, a target sequence, an adaptor sequence 1, a 5' intron fragment, and a 3' tag.

[0931] Figure 19B A schematic diagram of circular RNA is provided, which includes, in the following order: a 3' intron fragment, an adaptor sequence 2, a translation initiation element, a target sequence, an adaptor sequence 1, and a 5' intron fragment. Partial sequences on the adaptor sequence 1 and the adaptor sequence 2 serve as positive selection tags.

[0932] Figure 19C A schematic diagram of circular RNA is provided, which includes, in the following order: a 5' tag, a 3' intron fragment, a target sequence, an adaptor sequence 1, an adaptor sequence 2, a translation initiation element, a 5' intron fragment, and a 3' tag.

[0933] Figure 19D A schematic diagram of circular RNA is provided, which includes, in the following order: a 3' intron fragment, a target sequence, an adaptor sequence 1, an adaptor sequence 2, a translation initiation element, and a 5' intron fragment. Partial sequences on the translation initiation element and the target sequence serve as positive selection tags. 7. DETAILED DESCRIPTION OF THE INVENTION

[0935] Methods and systems for purifying circular nucleic acids are provided herein. Circular nucleic acids are single-stranded nucleic acids that are joined end-to-end. As is known in the art, circular nucleic acids can be generated in vitro using chemical means or through enzymatic activity. For example, the 5' and 3' ends of a linear nucleic acid can be chemically ligated by the catalysis of bromine cyanide and morpholine derivatives, or joined in a head-to-tail manner through the activity of a nucleic acid ligase. As used herein, a "precursor nucleic acid" is a linear nucleic acid molecule that directly gives rise to a circular nucleic acid, regardless of the cyclization method.

[0936] Circular nucleic acids can include ribonucleotides, deoxyribonucleotides, and modified nucleotides and / or their analogs. In some embodiments, the circular nucleic acid is circular DNA. In some embodiments, the circular nucleic acid is circular RNA.

[0937] In some embodiments, circular RNAs (also referred to as "circRNAs" or "cRNAs") can be generated by splicing. When a linear precursor undergoes splicing, a portion of the molecule is excised, producing a circRNA with fewer total nucleotides than the linear precursor. In some embodiments, a negative selection tag can be included in the intron portion of the linear precursor, which can then be used to negatively select circRNAs 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, where the precursor containing the negative selection tag or any other impurity containing the negative selection tag 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 impurity containing the tag.

[0938] In some embodiments, a positive selection probe can be selected to be at least 60% complementary to at least a portion of the circular nucleic acid formed, but its complementarity to the precursor nucleic acid is less than 60%, that is, there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe. Thus, the circular nucleic acid product can be directly bound from the IVT reaction solution by the positive selection probe, thereby achieving the purification of the circular nucleic acid product.

[0939] As is known in the art, RNA molecules with enzymatic activity are called ribozymes. circRNAs can also be generated by ribozyme-catalyzed RNA splicing. In addition, some ribozymes can catalyze self-splicing independently of the spliceosome, and these ribozymes are called "ribozymes with self-splicing activity", "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 a circular RNA after self-splicing. Such engineered ribozymes are also referred to as "cRNAzymes" herein.

[0940] Thus, in some embodiments, the present disclosure provides a composition for purifying circular nucleic acids comprising an oligonucleotide negative selection probe, wherein the circular nucleic acid is generated by self-splicing of a precursor nucleic acid comprising an oligonucleotide negative selection tag, wherein the oligonucleotide negative selection tag is complementary to the negative selection probe and is removed during the self-splicing of the precursor nucleic acid. The present disclosure also provides a method for purifying circular nucleic acids from a sample, wherein the circular nucleic acid is generated by self-splicing of a precursor nucleic acid, and wherein the precursor nucleic acid has an oligonucleotide negative selection tag that is removed during self-splicing, the method comprising: (i) contacting the sample and an oligonucleotide negative selection probe complementary to the negative selection tag under conditions that permit binding of the negative selection tag to the probe, wherein the negative selection probe is immobilized on a solid surface; and (ii) collecting the unbound portion of the sample.

[0941] In some embodiments, provided herein are compositions comprising oligonucleotide positive selection probes for purifying circular nucleic acids, wherein the circular nucleic acids are produced by self-splicing of precursor nucleic acids, wherein the positive selection probes are at least 60% complementary to at least a portion of the circular nucleic acids produced by self-splicing of the precursor nucleic acids, at least a portion of the circular nucleic acids spans the splicing portion of the circular nucleic acids, and there is no sequence on the precursor nucleic acids that is at least 60% complementary to the positive selection probes. The methods include: (i) contacting a sample comprising the circular nucleic acids produced by self-splicing of the precursor nucleic acids with a positive selection probe that is at least 60% complementary to at least a portion of the circular nucleic acids under conditions that permit binding of the circular nucleic acids produced by self-splicing of the precursor nucleic acids to the positive selection probe, wherein the positive selection probe is immobilized on a solid surface; and (ii) collecting the portion that binds to the positive selection probe on the solid surface, at least a portion of the circular nucleic acids spans the splicing portion of the circular nucleic acids, and there is no sequence on the precursor nucleic acids that is at least 60% complementary to the positive selection probes.

[0942] Before further describing the disclosure, it is to be understood that the disclosure is not limited to the specific embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

[0943] 7.1 Definitions

[0944] 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. In addition, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art.

[0945] As used herein in the specification, "a" or "an" can mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the words "a" or "an" can mean one or more than one.

[0946] As used herein, the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports definitions that refer only to alternatives and "and / or." As used herein, "another" or "additional" can mean at least a second or more.

[0947] As used herein, the term "about" is used to indicate a value that includes the inherent error variations of the device, the method for determining the value, or the variations that exist among study subjects. The term "about" encompasses the exact numbers being referred to. In some embodiments, "about" means within plus or minus 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".

[0948] As used herein, with respect to a particular component, "substantially free of" as used herein means that no particular component is intentionally formulated into the composition, and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any unintentional contamination of the composition is far less than 0.1%, preferably less than 0.05%, more preferably less than 0.01%. The most preferred compositions are those in which the amount of the designated component cannot be detected by standard analytical methods.

[0949] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein and refer to polymeric forms of contiguous amino acids that include at least two or more chemically or biochemically modified or derivatized amino acids. The term "peptide" as used herein refers to a class of short polypeptides. The term peptide can refer to a polymer of amino acids (naturally or non-naturally occurring) that is up to about 100 amino acids in length. 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, about 75 to about 100 amino acid residues. In some embodiments, the length of a 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, about 5000 amino acid residues.

[0950] The terms "nucleic acid", "polynucleotide", and "oligonucleotide" are used interchangeably herein and refer to polymers or oligomers of nucleotides of any length. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases (such as methylated, hydroxymethylated or glycosylated), unnatural nucleotides, non-nucleotide building blocks that exhibit similar structures and / or functions 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 heterogeneous or homogeneous in composition, can be isolated from naturally occurring sources, or can be produced artificially or synthetically. Additionally, nucleic acids can be DNA or RNA, or a mixture thereof, and can exist permanently or transiently 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 (PNAs), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 4(14):4503-4510 (2002) and U.S. Patent US5,034,506), locked nucleic acids (LNAs; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, Am. Chem. Soc., 122:8595-8602 (2000)), and / or ribozymes.

[0951] When referring to polynucleotide or protein sequences, the term "identity" is used to denote 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), implementation of these algorithms on a computer (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the best-fit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), or by inspection. Another algorithm is the BLAST algorithm, described in Altschul et al., J Mol 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 available from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); blast.wustl / edu / blast / README.html. WU-BLAST-2 uses a number of search parameters, which can be set to default values if so desired. The parameters are dynamic values and are established by the program itself according to the composition of the particular sequence and the particular database against which the sequence of interest is being searched; however, these values can be adjusted to increase sensitivity. In addition, another useful algorithm is gapped BLAST, as reported in Altschul et al, (1997) Nucleic Acids Res. 25, 3389-3402. Unless otherwise stated, the percent identity herein is determined using the algorithm obtainable from the Internet address (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0952] The terms "coding sequence", "coding sequence region", "coding region", and "CDS", when referring to a polynucleotide sequence, are used interchangeably herein to refer to a portion of a DNA or RNA sequence that is, for example, or can be translated into a protein. The terms "reading frame", "open reading frame", and "ORF" are used interchangeably herein and 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, and thus, all CDSs are ORFs, but not all ORFs are CDSs.

[0953] 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 traditional Watson-Crick base pairing or other non-traditional types of pairing. Two DNA / RNA strands with complementary sequences bind to form a duplex that follows the Watson-Crick base pairing rules: 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 a second polynucleotide sequence (e.g., about 50%, about 60%, about 70%, about 80%, about 90%, and 100% complementary). Two polynucleotide sequences are "fully complementary" if all consecutive nucleotides of the polynucleotide sequence will form hydrogen bonds with the same number of consecutive nucleotides in the second polynucleotide sequence. Two polynucleotide sequences are "substantially complementary" if the degree of complementarity between the two polynucleotide sequences 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 the entire 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 high stringency conditions. Exemplary moderate stringency conditions include: incubation overnight at 37°C in a solution containing 20% formamide, 5% SSC (150 mM NaCl, 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA, followed by washing the filter in 1*SSC at about 37 - 50°C; or under substantially similar conditions, such as the moderate stringency conditions described in Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th edition (June 15, 2012).High-stringency conditions are used, for example: (1) low ionic strength and high-temperature washes, such as 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate (SDS) at 50°C, (2) use of a denaturing agent such as formamide during hybridization at 42°C, such as 50% (v / v) formamide with 0.1% bovine serum albumin (BSA) / 0.1% Ficoll / 0.1% polyvinylpyrrolidone (PVP) / 50 mM sodium phosphate buffer (pH 6.5), along with 750 mM sodium chloride and 75 mM sodium citrate, or (3) at 42°C, use of 50% formamide, 5xSSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xDenhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, (i) wash at 42°C in 0.2*SSC, (ii) wash at 55°C in 50% formamide, and (iii) wash at 55°C in 0.1*SSC (optionally in combination with EDTA). Other details and explanations of hybridization reaction stringency are provided, for example, in Sambrook, supra, and Ausubel et al., eds., Short Protocols in Molecular Biology, 5th ed., John Wiley & Sons, Inc., Hoboken, N.J. (2002).

[0954] When referring to polynucleotide sequences, the terms "hybridization" or "hybridized" refer to the formation of a binding between and / or within sequences that have complementarity.

[0955] The term "homology" refers to the percentage of identity between the nucleic acid residues of two polynucleotides or the amino acid residues of two polypeptides. The correspondence between one sequence and another can be determined by techniques known in the art. For example, homology can be determined by aligning sequence information and using readily available computer programs to directly compare the sequence information between two polypeptides. As determined using the above methods, two polynucleotides (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, respectively, match over a defined length of the molecule.

[0956] The term "scar" refers to the length of the region in the circular product that does not include the target sequence. Scarless circRNA contains a 0-nucleotide scar sequence. Near-scarless circRNA contains a scar sequence with a length equal to or less than 20 nucleotides.

[0957] The term "Tris" refers to tris(hydroxymethyl)aminomethane, which is widely used in the preparation of buffers for biochemical and molecular biology experiments. Its full name is Tris(hydroxymethyl)aminomethane.

[0958] The term "EDTA" refers to ethylenediaminetetraacetic acid. Its full name is Ethylenediaminetetraacetic acid.

[0959] The term "rpm" refers to revolutions per minute, also known as rotations per minute. Its English abbreviation is RPM, and its full name is Revolution(s) Per Minute.

[0960] The term "Tween-20" refers to polysorbate-20.

[0961] The term "PCR" refers to polymerase chain reaction, which is a molecular biology technique used to amplify specific DNA fragments. Its full name is Polymerase Chain Reaction.

[0962] The term "ATP" refers to adenosine triphosphate. Its full name is Adenosine triphosphate; the term "GTP" refers to guanosine triphosphate. Its full name is Guanosine triphosphate; the term "CTP" refers to cytidine triphosphate. Its full name is Cytidine triphosphate; the term "UTP" refers to uridine triphosphate. Its full name is Uridine triphosphate.

[0963] The term "DNase", "Deoxyribonuclease" refers to deoxyribonuclease, also known as DNA enzyme, which is an enzyme that can cut the phosphodiester bonds on the DNA backbone through hydrolysis. DNA enzyme is one of the nucleases. Currently, many different types of DNA enzymes are known, and the main differences lie in substrate specificity, chemical reaction mechanism, and biological functions.

[0964] The term "RNase" refers to ribonuclease or RNA enzyme. Its full name is ribonuclease, which is a nuclease that can hydrolyze RNA into small molecule components.

[0965] The term "CIP", "Calf Intestinal Alkaline Phosphatase" refers to calf intestinal alkaline phosphatase, which is an alkaline phosphatase commonly used to remove 5'-phosphate groups from DNA, RNA, ribose, and deoxyribonucleoside triphosphates.

[0966] The term "DEPC water" refers to ultrapure water that has been treated with DEPC (diethyl pyrocarbonate) and autoclaved.

[0967] The term "PBS" refers to phosphate-buffered saline, which is short for Phosphate buffered saline and is a commonly used buffer in biological research. It is a salt solution containing sodium phosphate with water as the solvent, and potassium chloride and potassium phosphate are added in some formulations. The osmotic concentration and ionic concentration of this solution are consistent with those in the human body.

[0968] The term "circular total purity" refers to the proportion of circular RNA content in the total RNA of the sample being tested.

[0969] The term "oligomer content" refers to the proportion of oligomer content in the total RNA of the sample being tested.

[0970] The term "Gaussia luciferase" or "Gluc" refers to the luciferase derived from the marine copepod Gaussia princeps, which has the advantages of being secretable, easy to monitor, highly sensitive, short half-life, no need for ATP participation in the reaction, strong tolerance to temperature, pH value, etc., and can be used for real-time monitoring of living cells or organisms.

[0971] The term "Firefly luciferase" or "Fluc" refers to firefly luciferase, which catalyzes the oxidation of firefly luciferin and requires oxygen and ATP.

[0972] The term "Lipid nanoparticle" or "LNP" refers to nanoparticles composed of lipids, which is a new type of drug delivery route.

[0973] The term "Fast protein liquid chromatography" or "FPLC" refers to fast protein liquid chromatography, which is a liquid chromatography method commonly used for analyzing or purifying protein mixtures.

[0974] The term "High performance liquid chromatography" or "HPLC" refers to high performance liquid chromatography, which is a chromatographic analysis technique used to separate mixtures to identify and quantify the proportions of individual components.

[0975] The terms "autocatalytic self-cleavage", "autocatalytic self-splicing", "self-splicing", or "self-cleavage" refer to self-splicing catalyzed independently of the spliceosome. Some ribozymes can catalyze self-splicing independently of the spliceosome, and these ribozymes are called "ribozymes with self-splicing activity", "self-splicing ribozymes", or "self-splicing introns".

[0976] The term "Intravenous injection(IV)" or "iv" refers to intravenous injection, a medical method of directly injecting liquid substances such as blood, medicine solution, nutrient solution, etc. into a vein.

[0977] The term "Intramuscular injection(IM)" or "im" refers to intramuscular injection, a method of injecting substances directly into muscles, which is one of the drug administration routes in medicine.

[0978] The term "Intraperitoneal injection(IP)" or "ip" refers to intraperitoneal injection, which is more used in non-human animals compared to humans.

[0979] The term "DSPC" or "Distearoylphosphatidylcholine" refers to distearoylphosphatidylcholine, a kind of phosphatidylcholine and a natural component of cell membranes.

[0980] The term "PEG" is polyethylene glycol.

[0981] The term "DMG-PEG" is a synthetic lipid formed by PEGylation of myristoyl diglycerol.

[0982] The term "nt" refers to "nucleotide", the number of nucleotides, which is used to describe the length of nucleotides.

[0983] The nomenclature of nucleotides, nucleic acids, nucleosides and amino acids used herein is consistent with the International Union of Pure and Applied Chemistry (IUPAC) standards (see, for example, bioinformatics.org / smsylupac.html). Exemplary genes and polypeptides are described herein with reference to GenBank accession numbers, GI numbers, and / or SEQ ID NOS. It should be understood that those skilled in the art can easily identify homologous sequences by referring to sequence sources, including but not limited to: Uniprot (https: / / www.uniprot.org / ), GenBank (ncbi.nlm.nih.gov / genbank / ), and EMBL (embl.org / ).

[0984] The term "domain 1" or "D1" is used herein to refer to the stem-loop structure of domain 1 of group II introns. The term "domain 2" or "D2" is used herein to refer to the stem-loop structure of domain 2 of group II introns. The term "domain 3" or "D3" is used herein to refer to the stem-loop structure of domain 3 of group II introns. The term "domain 4" or "D4" is used herein to refer to the stem-loop structure of domain 4 of group II introns. The term "domain 5" or "D5" is used herein to refer to the stem-loop structure of domain 5 of group II introns. The term "domain 6" or "D6" is used herein to refer to the stem-loop structure of domain 6 of group II introns. A stem-loop structure is a type of RNA secondary structure that can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of minimum Gibbs free energy. An example of such an algorithm is mFold, which is described by Zuker and Stiegler (Nucleic Acids Res. 9(1981), 133-148). Another exemplary folding algorithm is the online web server RNAfold developed by the Institute of Theoretical Chemistry at the University of Vienna using the 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). Additional algorithms can be found in U.S. Provisional Patent Application No. 61 / 836,080 (Attorney Docket No. 44790.11.2022; Broad reference number BI-2013 / 004A), which is incorporated herein by reference. Group II introns mainly include six stem-loop structures, called domains 1-6 (D1-D6), which are arranged in sequence and contain multiple exon-binding sequences (EBS), such as EBS1, EBS2, and EBS3. These EBS sequences interact with the intron-binding sequences (IBS) in the exon region, such as complementary pairing, and rely on the hydroxyl group within the EBS nucleic acid sequence itself to trigger splicing.

[0985] As used herein, EBS1', EBS2', and EBS3' are used to refer to regions on the target sequence that are functionally similar to EBS1, EBS2, and EBS3, respectively.

[0986] As used herein, the term "group II intron" is used herein to refer to an RNA molecule encoded by a group II intron that has a similar secondary and tertiary structure. Group II intron RNA molecules typically have six domains. See Figure 9 and Figure 10Domain 4 of group II intron RNA (also known as domain IV) contains a nucleotide sequence encoding a "group II intron-encoded protein". In some embodiments, the group II intron sequence is selected from the sequences disclosed in WO2022247943.

[0987] The term "IBS" is used herein to refer to an intron binding sequence that interacts with an exon binding sequence (EBS) to locate the splice site.

[0988] The term "IBS1" is used herein to refer to intron binding sequence 1, which interacts with exon binding sequence 1 (EBS1) to locate the splice site.

[0989] The term "IBS1'" is used herein to refer to a region on the target sequence that functions similarly to IBS1.

[0990] The term "IBS2" is used herein to refer to intron binding sequence 2, which interacts with exon binding sequence 2 (EBS2) to locate the splice site.

[0991] The term "IBS3" is used herein to refer to intron binding sequence 3, which interacts with exon binding sequence 3 (EBS3) to locate the splice site.

[0992] The term "IBS3'" is used herein to refer to a region on the target sequence that functions similarly to IBS3.

[0993] The term "δ" (delta) is used herein to refer to a region on domain 1 of a group II intron that is a single nucleotide immediately upstream of EBS1. δ pairs with IBS3 and the interaction between δ and IBS3 is called the δ-IBS3 pairing. See Figure 10 。

[0994] The term "δ"" (delta") is used herein to refer to a region on domain 1 of a group II intron that is a single nucleotide immediately upstream of EBS1'. δ" pairs with IBS3' and the interaction between δ" and IBS3' is called the δ"-IBS3' pairing. See Figure 10 。

[0995] 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 the m7G cap at the 5' end of the mRNA, initiator Met-tRNAmet, more than a dozen initiation factor proteins, directed scanning, and GTP hydrolysis to place a translationally competent ribosome at the start codon. An IRES is typically composed of a long and highly structured 5'-UTR that mediates the binding of the translation initiation complex and catalyzes the formation of a functional ribosome. In some embodiments, sequences selected from those disclosed in PCT / CN2023 / 096813, as well as sequences screened by the methods disclosed in PCT / CN2023 / 096813.

[0996] The term "IRES-like sequence" or "internal ribosome entry site-like sequence" refers to a synthetic nucleotide sequence that exhibits native IRES function. In some embodiments, the IRES-like sequence can recruit ribosomal components to mediate cap-independent translation. In some embodiments, sequences selected from those disclosed in PCT / CN2023 / 096813, as well as sequences screened by the methods disclosed in PCT / CN2023 / 096813.

[0997] Ranges: Throughout this disclosure, various aspects of the present application may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the present application. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within that range. For example, a description of a range such as 1-6 should be considered to have specifically disclosed sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of how large the range is.

[0998] 7.2 Probes and Tags

[0999] 7.2.1 Negative Selection Tags and Negative Selection Probes

[1000] The present disclosure provides compositions and methods for purifying circular nucleic acids. In some embodiments, circular nucleic acids can be generated by self-splicing of a precursor nucleic acid comprising an oligonucleotide depletion tag that is removed during self-splicing. Thus, an oligonucleotide depletion probe that is at least 60% complementary to the depletion tag can be used to capture precursors, introns, and any other impurities containing the depletion tag in the product mixture, thereby purifying circular nucleic acids lacking the depletion tag. A schematic diagram of the depletion system and method described herein is shown in Figure 3A as shown. A schematic diagram of magnetic beads conjugated with a depletion probe used in the compositions described herein is shown in Figure 3C as shown. A schematic diagram of magnetic beads conjugated with a first depletion probe and a second depletion probe used in the compositions described herein is shown in Figure 3D as shown.

[1001] In some embodiments, the depletion probe and the depletion tag can 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 depletion probe and the depletion tag can be at least 65% complementary. The depletion probe and the depletion tag can be at least 70% complementary. The depletion probe and the depletion tag can be at least 75% complementary. The depletion probe and the depletion tag can be at least 80% complementary. The depletion probe and the depletion tag can be at least 85% complementary. The depletion probe and the depletion tag can be at least 90% complementary. The depletion probe and the depletion tag can be at least 95% complementary. The depletion probe and the depletion tag can be at least 98% complementary. The depletion probe and the depletion tag can be at least 99% complementary. In some embodiments, the depletion probe and the depletion tag can 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 depletion probe and the depletion tag can be about 65% complementary. The depletion probe and the depletion tag can be about 70% complementary. The depletion probe and the depletion tag can be about 75% complementary. The depletion probe and the depletion tag can be about 80% complementary. The depletion probe and the depletion tag can be about 85% complementary. The depletion probe and the depletion tag can be about 90% complementary. The depletion probe and the depletion tag can be about 95% complementary. The depletion probe and the depletion tag can be about 98% complementary. The depletion probe and the depletion tag can be about 99% complementary. In some embodiments, the depletion probe and the depletion tag can be 100% complementary.

[1002] In some embodiments, the precursor nucleic acid can have a negative selection tag. In some embodiments, the precursor nucleic acid can have more than one negative selection tag. The negative selection tag needs to be included in the part that is removed during the self-splicing process of the precursor nucleic acid. In some embodiments, the precursor nucleic acid is a cRNA enzyme, which forms a circular nucleic acid during the self-splicing process and removes its terminal part from the circular product. Thus, in some embodiments, the precursor nucleic acid can have a negative selection tag at its 5' end or 3' end. In some embodiments, the precursor nucleic acid can have a negative selection tag at its 5' end. In some embodiments, the precursor nucleic acid can have a negative selection tag at its 3' end. In some embodiments, under the condition that both the 5' end negative selection tag and the 3' end negative selection tag are present, the 5' end negative selection tag and the 3' end negative selection tag can be the same or different.

[1003] In some embodiments, the precursor nucleic acid can have two negative selection tags. In some embodiments, the two negative selection tags on the same precursor nucleic acid are the same. In some embodiments, the two negative selection 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 can have two negative selection tags, one at its 5' end and the other at its 3' end. To purify the circular nucleic acid generated by the self-splicing of the precursor nucleic acid having two negative selection tags, one or two oligonucleotide negative selection probes can be used. In some embodiments, the two negative selection tags are the same, and the negative selection probe can be substantially complementary to the negative selection tag (i.e., at least 90% complementary). In some embodiments, the two negative selection tags are different, and one negative selection probe that is substantially complementary to at least one negative selection tag (i.e., at least 90% complementary) can be used. In some embodiments, the two negative selection tags are different, and two different negative selection probes that are each substantially complementary to one of the negative selection tags (i.e., at least 90% complementary) can be used.

[1004] In some embodiments, provided herein is a composition for purifying a circular nucleic acid, comprising a first oligonucleotide negative selection probe and a second oligonucleotide negative selection probe, wherein the circular nucleic acid is generated by the self-splicing of a precursor nucleic acid comprising a first and a second oligonucleotide negative selection tag, wherein the first negative selection tag and the second negative selection tag are at least 60% complementary to the first negative selection probe and the second negative selection probe, respectively, and are removed during the self-splicing of the precursor nucleic acid.

[1005] In some embodiments, the first negative selection tag and the second negative selection tag can 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 to the first and second negative selection probes, respectively. In some embodiments, the first negative selection tag and the second negative selection tag can be at least 65% complementary to the first negative selection probe and the second negative selection probe, respectively. The first and second negative selection tags can be at least 70% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be at least 75% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be at least 80% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be at least 85% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be at least 90% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be at least 95% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be at least 98% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be at least 99% complementary to the first and second negative selection probes, respectively. In some embodiments, the negative selection probe and the negative selection tag can 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 negative selection tags can be about 65% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be about 70% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be about 75% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be about 80% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be about 85% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags can be about 90% comp...

Claims

1. A composition comprising an oligonucleotide negative selection probe for purifying circular nucleic acids, wherein the circular nucleic acid is produced by self-splicing of a precursor nucleic acid comprising an oligonucleotide negative selection tag, wherein the negative selection tag is at least 90% complementary to the negative selection probe, wherein the negative selection tag is removed during the self-splicing process 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 further preferably 100% complementary.

2. The composition according to claim 1, wherein the precursor nucleic acid comprises the negative selection tag at its 5' end; and / or the precursor nucleic acid comprises the negative selection tag at its 3' end, Under the condition that the negative selection tag at the 5' end and the negative selection tag at the 3' end are present at the same time, the negative selection tag at the 5' end and the negative selection tag at the 3' end may be the same or different.

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

4. The composition of claim 3, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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 the group II intron, wherein the 5' intron fragment is located at the 5' side of the 3' intron fragment in the group II intron; (2) the E1 is a 5' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; (3) the E2 is a 3' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

5. The composition of claim 3, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) 3' intron fragment; (b) exon fragment 2 (E2); (c) adapter sequence; (d) target sequence; (e) adapter sequence; (f) exon fragment 1 (E1); and (g) 5' intron fragment; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of the group II intron, wherein the 5' intron fragment is located at the 5' side of the 3' intron fragment in the group II intron; (2) the E1 is a 5' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; (3) the E2 is a 3' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

6. The composition of claim 3, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 5' homology 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' homology arm; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of the group II intron, wherein the 5' intron fragment is located at the 5' side of the 3' intron fragment in the group II intron; (2) the E1 is a 5' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; (3) the E2 is a 3' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

7. The composition of claim 3, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 5' homology 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' homology arm; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of the group II intron, wherein the 5' intron fragment is located at the 5' side of the 3' intron fragment in the group II intron; (2) the E1 is a 5' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; (3) the E2 is a 3' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

8. 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, and wherein the precursor nucleic acid has an oligonucleotide negative selection tag, and the oligonucleotide negative selection tag is removed during the self-splicing process, the method comprising: (i) contacting the sample with an oligonucleotide negative selection probe that is at least 90% complementary to the negative selection tag under conditions that allow the negative selection tag to bind to the negative selection probe, wherein the negative selection probe is immobilized on a solid surface; and (ii) collecting the unbound portion of the sample, 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.

9. A method for purifying circular nucleic acids from a sample, comprising the steps of: coupling the magnetic beads to the negative selection probe; Incubate the IVT reaction solution containing the circular nucleic acid obtained by in vitro transcription with magnetic beads coupled with negative selection probes; Collecting the magnetic beads and eluting to recover the magnetic beads coupled with the negative selection probe; Collect the unbound fraction, The negative selection probe is at least 90% complementary to the negative selection tag, and the circular nucleic acid is produced by self-splicing of a precursor nucleic acid containing an oligonucleotide negative selection tag, wherein the negative selection tag is removed during the self-splicing process of the precursor nucleic acid, and 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 further preferably 100% complementary.

10. A composition for producing circular nucleic acid, comprising: Linearized plasmids for producing precursor nucleic acids; The resulting precursor nucleic acid; The generated circular nucleic acid; Uncircularized precursor nucleic acid; Residual impurities from an 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 one of claims 1 to 48, Preferably the composition has a total ring purity in the range of 20-50% and a polymer content in the range of 10-20%.

11. A composition for purifying circular nucleic acids comprising a first oligonucleotide negative selection probe and a second oligonucleotide negative selection probe, wherein the circular nucleic acid is produced by self-splicing of a precursor nucleic acid comprising a first oligonucleotide negative selection tag and a second oligonucleotide negative selection tag, wherein the first and second negative selection tags are at least 90% complementary to the first and second negative selection probes, respectively, and are removed during self-splicing of the precursor nucleic acid, preferably the first and second negative selection 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 selection probes, respectively, and further preferably 100% complementary. 12 . The composition according to claim 11 , wherein the precursor nucleic acid comprises the first negative selection tag at its 5′ end and the second negative selection tag at its 3′ end, and the first negative selection tag and the second negative selection tag may be the same or different.

13. The composition according to claim 11 or 12, wherein the circular nucleic acid is a circular RNA (circRNA), and preferably the precursor nucleic acid is an RNA having group II intron self-splicing activity.

14. The composition of claim 13, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (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 the group II intron, wherein the 5' intron fragment is located at the 5' side of the 3' intron fragment in the group II intron; (2) the E1 is a 5' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; (3) the E2 is a 3' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and (4) The target sequence does not exist, or is a protein coding sequence, a non-coding sequence, or a combination of the two. It is further preferred that the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection 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 selection tag.

15. The composition of claim 13, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) 3' intron fragment; (b) exon fragment 2 (E2); (c) adapter sequence; (d) target sequence; (e) adapter sequence; (f) exon fragment 1 (E1); and (g) 5' intron fragment; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of the group II intron, wherein the 5' intron fragment is located at the 5' side of the 3' intron fragment in the group II intron; (2) the E1 is a 5' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; (3) the E2 is a 3' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and (4) The target sequence does not exist, or is a protein coding sequence, a non-coding sequence, or a combination of the two. It is further preferred that the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection tag; (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); (g) a 5' intron fragment; and a second negative selection tag.

16. The composition of claim 13, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 5' homology 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' homology arm; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of the group II intron, wherein the 5' intron fragment is located at the 5' side of the 3' intron fragment in the group II intron; (2) the E1 is a 5' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; (3) the E2 is a 3' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and (4) The target sequence does not exist, or is a protein coding sequence, a non-coding sequence, or a combination of the two. It is further preferred that the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection tag; (a) a 5' homology 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' homology arm; and a second negative selection tag.

17. The composition of claim 13, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) a 5' homology 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' homology arm; wherein: (1) The 5' intron fragment and the 3' intron fragment are both fragments of the group II intron, wherein the 5' intron fragment is located at the 5' side of the 3' intron fragment in the group II intron; (2) the E1 is a 5' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; (3) the E2 is a 3' adjacent exon fragment of the group II intron, and its length is ≥ 0 nucleotides; and (4) The target sequence does not exist, or is a protein coding sequence, a non-coding sequence, or a combination of the two. It is further preferred that the precursor RNA comprises the following operably linked elements from 5' to 3': a first negative selection tag; (a) a 5' homology 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; (i) a 3' homology arm; and a second negative selection tag.

18. A method for purifying a circular nucleic acid from a sample, wherein the circular nucleic acid is produced by self-splicing of a precursor nucleic acid, and wherein the precursor nucleic acid has an oligonucleotide first negative selection tag and an oligonucleotide second negative selection tag, and the first negative selection tag and the second negative selection tag are removed during the self-splicing process, the method comprising: (i) contacting the sample with the first negative selection probe and the second negative selection probe, which are at least 90% complementary to the first negative selection tag and the second negative selection probe, respectively, under conditions that allow the first negative selection tag to bind to the first negative selection probe, and the second negative selection tag to bind to the second negative selection probe, wherein the first negative selection probe and the second negative selection probe are immobilized on a solid surface; and (ii) collecting the unbound portion of the sample, Preferably, the first and second negative selection 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 selection probes, respectively, and more preferably 100% complementary.

19. A method for purifying circular nucleic acids from a sample, comprising the steps of: coupling the magnetic beads to the first negative selection probe and the second negative selection probe; Incubate the IVT reaction solution containing the circular nucleic acid obtained by in vitro transcription with magnetic beads coupled with the first negative selection probe and the second negative selection probe; Collecting the magnetic beads and eluting to recover the magnetic beads coupled with the first negative selection probe and the second negative selection probe; Collect the unbound fraction, Wherein the first and second negative selection tags are at least 90% complementary to the first and second negative selection probes, respectively, and the circular nucleic acid is produced by self-splicing of a precursor nucleic acid comprising a first oligonucleotide negative selection tag and a second oligonucleotide negative selection tag, wherein the negative selection tag is removed during the self-splicing process of the precursor nucleic acid, preferably, the first and second negative selection 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 selection probes, respectively, and further preferably 100% complementary.

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