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 case is a divisional application of a case with an application date of January 10, 2024, an invention name of "Negative selection method and system 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 by Reference into the Sequence Listing

[0005] The contents of the electronic sequence listing TPG03609-sequencing list.xml (file size: 6.17 MB; created on January 10, 2024) are incorporated herein by reference in their entirety. 3. Technical field

[0006] The present application relates to the field of molecular biology, and in particular to a method and system for purifying circular nucleic acids. The circular nucleic acids to be purified by the methods provided herein can be produced by self-splicing of precursor nucleic acids. 4. Background Technology

[0007] Circular RNA (circRNA) is a type of RNA molecule formed by head-to-tail connection, which has 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 RNA, circRNA has better stability, thus providing a promising new platform for RNA drugs.

[0008] Purity is a key factor in RNA therapy, as contaminants often lead to immunogenicity. Although there are available methods to purify RNA, there is still a lack of effective methods and systems for purifying RNA, especially circRNA. The compositions, methods and systems provided herein meet this need and provide related advantages. 5. Summary of the Invention

[0010] 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.

[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 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.

[0013] 3. A composition according to item 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.

[0014] 4. A composition according to item 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) target sequence; (d) exon fragment 1 (E1); and (e) 5' intron fragment; wherein:

[0015] (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;

[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 does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0019] 5. A composition according to item 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:

[0020] (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;

[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 does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0024] 6. A composition according to item 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:

[0025] (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;

[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 does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0029] 7. A composition according to item 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:

[0030] (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;

[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 noncoding sequence, or a combination of the two.

[0034] 8. A composition according to any one of items 1-7, 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.

[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. A 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, wherein 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, wherein m is an integer from 2335 to 3544 or from 4755 to 5964.

[0037] 11. The composition according to any one of items 1 to 9, 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0053] 13. The composition according to any one of items 4 to 7, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron of the unpaired region into two fragments.

[0054] 14. A composition according to item 13, 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

[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. A composition according to item 13 or 14, wherein the group II intron comprises a modification of one or more nucleotides relative to its wild-type form, and the modification is selected from one or more of a deletion, a substitution and an addition,

[0057] Preferably, the modification comprises modification of 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, respectively.

[0058] 16. A composition according to item 15, wherein the modification is the modification of two EBS sequences (such as EBS1 and EBS3) of the group II intron, wherein the EBS sequence is 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.

[0059] 17. A composition according to item 15, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of the group II intron, wherein the EBS sequence is 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.

[0060] 18. A composition according to item 15 or 16, 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 to a region of corresponding length in the target sequence 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 sequence are complementary to a region of corresponding length in the target sequence at least 60% of the nucleotides,

[0061] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to a region of corresponding length in the target sequence, and 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 100% of the nucleotides upstream thereof are complementary to a region of corresponding length in the target sequence.

[0062] 19. A composition according to item 14, wherein the modification comprises a partial or complete deletion of domain 4, such as a deletion of an intron-encoded protein (IEP) sequence in domain 4, preferably a complete deletion of domain 4; or

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

[0064] 20. A composition according to item 16, wherein the precursor RNA is capable of forming a nearly scarless circRNA of the target sequence, preferably the nearly scarless circRNA has a scar region having 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. A composition according to item 17, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0066] 22. A composition according to any one of items 3 to 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 comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0067] 23. A 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 is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 75-88.

[0069] 24. A composition according to any one of items 4 to 23, wherein E1 and / or E2 has a length of 0-20 nucleotides, preferably a length of 0-10 nucleotides, such as a length of 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. A composition according to item 24, wherein E1, E2 or both are 0 nucleotides in length.

[0072] 26. A composition according to any one of items 4-25, wherein the target sequence is a non-coding sequence selected from the following group: a spacer sequence of SEQ ID NO:4-6, a polyA sequence, a poly-AC sequence, a polyC sequence, a polyU sequence, an IRES, a ribosome binding site, an adapter sequence, an RNA scaffold, a riboswitch, a ribozyme other than a self-splicing ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translation regulatory sequence and a protein binding site.

[0073] 27. A 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 natural IRES sequence, and a protein coding sequence, preferably the protein coding sequence encodes a therapeutic product.

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

[0075] 29. A composition according to claim 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'-0-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio-N6-isopentenyl adenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine), g6A (N6-glycylaminoformyl adenosine), t6A (N6-threonylaminoformyl adenosine), ms2t6A (2-methylthio-N6-threonylaminoformyl adenosine), m6t6A (N6-methyl-N6-threonylaminoformyl adenosine), hn6A (N6-hydroxynorvaline acylaminoformyladenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine), Ar(p) (2'-0-ribosyladenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-0-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'-0-methylguanosine), m22G (N2, N2-dimethylguanosine), m2Gm (N2, 2'-O-dimethylguanosine), m2aGm (N2, N2, 2'-O-trimethylguanosine), Gr (p) (2'-0-ribosylguanosine (phosphate)), yW (whitinosine), oayW (peroxywhitinosine), OH yW (hydroxy wyoside), OHyW* (undermodified hydroxy wyoside), imG (wyoside), mimG (methyl wyoside), Q (braid), oQ (epoxybraid), galQ (galactosyl-braid), manQ (mannosyl-braid), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archauridine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-0-methyluridine), acp3U (3-(3-amino-3-carboxypropyl) uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl) uridine), mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester), mcm5U (5-methoxycarbonylmethyl uridine), mcm5Um (5-methoxycarbonylmethyl-2'-0-methyl uridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyl uridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine), cmnm5U (5-carboxymethylaminomethyl uridine), cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyl uridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6, N6-dimethyladenosine), Im (2'-0-methylinosine), m4C (N4-methylcytidine), m4Cm (N4, 2'-0-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6, 2'-O-dimethyladenosine), m6 2Am (N6,N6,0-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-0-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-0-methylcytidine), m'Gm (l,2'-0-dimethylguanosine), m'Am (l,2'-0-dimethyladenosine), rm 5U (5-taurine methyluridine), τm5s2U (5-taurine methyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine). ,

[0076] 30. A 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. A composition according to any one of items 1 to 30, further comprising a solid surface,

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

[0079] It is further preferred that the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads or silica beads, or

[0080] It is further preferred that the solid surface is a resin containing agarose, a carbohydrate-based material, a polymer filler, silica, glass particles, a cast-based material or a monolithic support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

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

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

[0083] The magnetic beads are preferably functional group-modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleimide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specificity groups, or a combination of two or more thereof, more preferably NHS-modified magnetic beads, or

[0084] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0085] Preferably, the magnetic beads have a diameter 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. A composition according to item 31 or 32, wherein the solid surface is an agarose chromatography medium,

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

[0088] Further, the agarose chromatography filler is an agarose chromatography filler with amino modification on the surface, a carboxyl modified agarose chromatography filler, an NHS modified agarose chromatography filler, a maleamide modified agarose chromatography filler, a CNBr modified agarose chromatography filler, an agarose chromatography filler modified with affinity or specificity groups, or a combination of two or more of the above, and further preferably an NHS modified agarose chromatography filler.

[0089] 35. A 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 by covalent connection of NH2 and NHS-modified carboxyl groups.

[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 by a group selected from the following: amino group, carboxyl group, NHS-carboxyl group, thiol group.

[0091] 37. A 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 resulting from the inclusion of the tag ranges from -2 to 2.

[0092] 38. A composition according to claim 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 ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative selection tag ranges from 0 to 2, preferably the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative selection tag is about 0.

[0094] 40. A negative selection kit for purifying circular nucleic acids, comprising: a composition comprising oligonucleotide negative selection probes for purifying circular nucleic acids as described in any one of items 1 to 39, a binding solution, and an elution solution.

[0095] 41. A kit according to item 40, wherein the binding solution comprises a salt comprising a cation selected from the group consisting of: Ba 2+ , Ca 2+ Mg 2+ , Mn 2+ , Fe 2+ , Cu 2+ 、Zn 2+ 、Cd 2+ , Pb 2+ , Li + , Cs + 、Na+ , K + , Rb + and 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,

[0096] It is further preferred that the salt is a cation of Li + Or Na + of salts.

[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 buffer substance, a chelating agent, and optionally a stabilizer.

[0099] 43. A kit according to item 41 or 42, 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.

[0100] 44. A kit according to any one of items 40-43, 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 CO 3 - , Cl - Br - 、NO 3 3- , ClO 4 3- ,I - and SCN - or a combination thereof, preferably the salt is a cation of Li + Or Na + of salts,

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

[0102] 45. A 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 negative selection probes, an elution fluid and a binding fluid, wherein the negative selection probe is at least 90% complementary to a 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.

[0105] 47. A 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 binding solution are the eluent and binding solution involved in any one of items 40-45.

[0107] 48. A 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 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 negative selection tag, and the oligonucleotide negative selection tag is removed during the self-splicing process, the method comprising:

[0109] (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

[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 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.

[0114] 51. A method according to item 49 or 50, 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.

[0115] 52. A 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) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0116] (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;

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

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

[0119] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0120] 53. A method according to item 51, 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:

[0121] (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;

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

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

[0124] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0125] 54. A method according to item 51, 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:

[0126] (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;

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

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

[0129] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0130] 55. A method according to claim 51, 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:

[0131] (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;

[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 does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0135] 56. A 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. A 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. A 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, wherein 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, wherein m is an integer from 2335 to 3544 or from 4755 to 5964.

[0138] 59. A method according to any one of items 49-58, 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0154] 61. A method according to any one of items 49-60, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron of the unpaired region into two fragments.

[0155] 62. A method according to item 61, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting 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

[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. A method according to item 61 or 62, wherein the group II intron comprises a modification of one or more nucleotides relative to its wild-type form, and the modification is selected from one or more of a deletion, a substitution and an addition,

[0158] Preferably, the modification comprises modification of 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, respectively.

[0159] 64. A method according to item 63, wherein the modification is the modification of two EBS sequences (such as EBSl and EBS3) of the group II intron, wherein the EBS sequence is complementarily 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.

[0160] 65. A method according to item 63, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of the group II intron, wherein the EBS sequence is complementarily 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.

[0161] 66. A method according to item 64 or 65, 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 to a region of corresponding length in the target sequence 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 sequence are complementary to a region of corresponding length in the target sequence at least 60% of the nucleotides,

[0162] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to a region of corresponding length in the target sequence, and 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 100% of the nucleotides upstream thereof are complementary to a region of corresponding length in the target sequence.

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

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

[0165] 68. A method according to item 64, wherein the precursor RNA is capable of forming a nearly scarless circRNA of the target sequence, and preferably the nearly scarless circRNA has a scar region having 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. A method according to item 65, wherein the precursor RNA is capable of forming 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 further preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0168] 71. A 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 is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 75-88.

[0170] 72. The method according to any one of items 52 to 71, wherein E1 and / or E2 has a length of 0-20 nucleotides, preferably a length of 0-10 nucleotides, such as a length of 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. A method according to item 72, wherein E1, E2 or both are 0 nucleotides in length.

[0173] 74. A method according to any one of items 52-73, wherein the target sequence is a non-coding sequence selected from the following group: an intervening sequence of SEQ ID NO:4-6, a polyA sequence, a poly-AC sequence, a polyC sequence, a polyU sequence, an IRES, a ribosome binding site, an adapter sequence, an RNA scaffold, a riboswitch, a ribozyme other than a self-splicing ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translation regulatory sequence and a protein binding site.

[0174] 75. A 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 natural IRES sequence, and a protein coding sequence, preferably the protein coding sequence encodes a therapeutic product.

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

[0176] 77. A method according to claim 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'-0-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio-N6-isopentenyl adenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine), g6A (N6-glycylaminoformyl adenosine), t6A (N6-threonylaminoformyl adenosine), ms2t6A (2-methylthio-N6-threonylaminoformyl adenosine), m6t6A (N6-methyl-N6-threonylaminoformyl adenosine), hn6A (N6-hydroxynorvaline acylaminoformyl adenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyl adenosine), Ar(p) (2'-0-ribosyl adenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-0-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'-0-methylguanosine), m2 2G (N2, N2-dimethylguanosine), m2Gm (N2, 2'-O-dimethylguanosine), m2aGm (N2, N2, 2'-O-trimethylguanosine), Gr(p) (2'-0-ribosylguanosine (phosphate)), yW (wybutin), oayW (peroxywybutin), OHyW (hydroxywybutin), OHyW* (undermodified hydroxywybutin), imG (wyother), mimG (methylwyother), Q (braid), oQ (epoxybraid), galQ (galactosyl-braid), manQ (mannosyl-braid), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archauridine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-0-methyluridine), acp3U (3-(3-amino-3-carboxypropyl) uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl) uridine), mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester), mcm5U (5-methoxycarbonylmethyl uridine), mcm5Um (5-methoxycarbonylmethyl-2'-0-methyl uridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyl uridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine), cmnm5U (5-carboxymethylaminomethyl uridine), cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyl uridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6, N6-dimethyladenosine), Im (2'-0-methylinosine), m4C (N4-methylcytidine), m4Cm (N4, 2'-0-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6, 2'-O-dimethyladenosine), m6 2Am (N6,N6,0-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-0-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-0-methylcytidine), m'Gm (l,2'-0-dimethylguanosine), m'Am (l,2'-0-dimethyladenosine), rm 5U (5-taurine methyluridine), τm5s2U (5-taurine methyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine). ,

[0177] 78. A 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 comprising a solid surface,

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

[0180] It is further preferred that the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads or silica beads, or

[0181] It is further preferred that the solid surface is a resin containing agarose, a carbohydrate-based material, a polymer filler, silica, glass particles, a cast-based material or a monolithic support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

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

[0183] 81. The method of claim 79 or 80, wherein the solid surface is a magnetic bead,

[0184] The magnetic beads are preferably functional group-modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleimide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specificity groups, or a combination of two or more thereof, more preferably NHS-modified magnetic beads, or

[0185] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0186] Preferably, the magnetic beads have a diameter 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.

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

[0188] Further, the agarose chromatography filler is an agarose chromatography filler with amino modification on the surface, a carboxyl modified agarose chromatography filler, an NHS modified agarose chromatography filler, a maleamide modified agarose chromatography filler, a CNBr modified agarose chromatography filler, an agarose chromatography filler modified with affinity or specificity groups, or a combination of two or more of the above, and further preferably an NHS modified agarose chromatography filler.

[0189] 83. A 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, and preferably the covalent fixation is achieved by covalent connection of NH2 and NHS-modified carboxyl groups.

[0190] 84. A 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 by a group selected from the following: amino group, carboxyl group, NHS-carboxyl group, thiol group.

[0191] 85. A 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 resulting from the inclusion of the tag ranges from -2 to 2.

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

[0193] 87. A composition according to item 85 or 86, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative selection tag ranges from 0 to 2, preferably the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative selection tag is approximately 0.

[0194] 88. A 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, 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+ Mg2+ , 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,

[0197] It is further preferred that the salt is a cation of Li + Or Na + of salts.

[0198] 89. The method according to item 88, 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,

[0199] Preferably, the method further comprises the step of eluting the negative selection probe bound to the solid surface with an elution solution to recover the solid surface coupled with the negative selection probe for reuse.

[0200] 90. A method according to any one of items 49-89, wherein the concentration of the salt in the eluent is 0M, and the salt comprises 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 CO 3 - , Cl - Br - 、NO 3 3- , ClO 4 3- ,I - and SCN - or a combination thereof, preferably the salt is a cation of Li + Or Na + of salts,

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

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

[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. A method according to any one of items 49-91, wherein the method is scalable and may include a continuous flow process, preferably performed using a liquid chromatograph (LC) or an ELISA reader, and further preferably the LC is a fast liquid chromatograph (FPLC) or a high performance liquid chromatograph (HPLC).

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

[0206] coupling the magnetic beads to the negative selection probe;

[0207] Incubate the IVT reaction solution containing the circular nucleic acid obtained by in vitro transcription with magnetic beads coupled with negative selection probes;

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

[0209] Collect the unbound fraction,

[0210] 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.

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

[0212] 95. A method according to item 94, wherein the elution liquid and the binding liquid are the elution liquid and the binding liquid involved in any one of items 88-91, and further preferably, the negative selection probe and the negative selection label are the negative selection probe and the negative selection label involved in any one of items 49-92.

[0213] 96. Circular nucleic acids purified according to the method described in any one of items 49-95, or using the composition comprising oligonucleotide negative selection probes for purifying circular nucleic acids described in any one of items 1-39, or using the kit described in any one of items 40-48.

[0214] 97. A composition having a circular nucleic acid as described in item 96, wherein the circularization 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%, and the polymer content is preferably 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 producing circular nucleic acids, comprising:

[0218] Linearized plasmids for producing precursor nucleic acids;

[0219] The resulting precursor nucleic acid;

[0220] The generated circular nucleic acid;

[0221] Uncircularized precursor nucleic acid;

[0222] 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 items 1 to 48,

[0223] Preferably the composition has a total ring purity in the range of 20-50% and a polymer content in the range of 10-20%.

[0224] 99. A composition according to item 98, which is used as a sample for the method described in any one of items 49-96 or as a sample purified via a composition comprising an oligonucleotide negative selection probe for purifying circular nucleic acids described in any one of items 1-39 or using a kit described in any one of items 40-48.

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

[0226] The preferred polymer content 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 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.

[0229] 102. A composition according to item 101, 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.

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

[0231] 104. A composition according to item 103, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) 3' intron fragment; (b) exon fragment 2 (E2); (c) target sequence; (d) exon fragment 1 (E1); and (e) 5' intron fragment; wherein:

[0232] (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;

[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 does not exist, or is a protein coding sequence, a non-coding sequence, or a combination of the two.

[0236] 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.

[0237] 105. A composition according to item 103, 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:

[0238] (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;

[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 does not exist, or is a protein coding sequence, a non-coding sequence, or a combination of the two.

[0242] 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.

[0243] 106. A composition according to item 103, 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:

[0244] (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;

[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 does not exist, or is a protein coding sequence, a non-coding sequence, or a combination of the two.

[0248] 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.

[0249] 107. A composition according to item 103, 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:

[0250] (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;

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

[0252] (3) the 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] 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.

[0255] 108. A 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. A 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. A composition according to any one of items 101-109, wherein

[0258] When the first negative selection tag and negative selection probe pair are the same as the second negative selection tag and 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, wherein 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; wherein n is an integer from 2335 to 3544.

[0260] 111. A composition according to any one of items 101-109, 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 selected from any one of SEQ ID NOs: 7189-7202; or the first negative selection probe and the second negative selection probe have a polynucleotide sequence selected from any one of 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 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0276] 113. A composition according to any one of items 104-107, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron of the unpaired region into two fragments.

[0277] 114. A composition according to item 113, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting 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

[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. A composition according to item 113 or 114, wherein the group II intron comprises a modification of one or more nucleotides relative to its wild-type form, and the modification is selected from one or more of a deletion, a substitution and an addition,

[0280] Preferably, the modification comprises modification of 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, respectively.

[0281] 116. A 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 sequence is complementarily 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.

[0282] 117. A 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 sequence is complementarily 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.

[0283] 118. A composition according to item 115 or 116, 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 to a region of corresponding length in the target sequence at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence at least 60% of the nucleotides,

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

[0285] 119. A composition according to item 114, wherein the modification comprises a partial or complete deletion of domain 4, such as a deletion of an intron-encoded protein (IEP) sequence in domain 4, preferably a complete deletion of domain 4; or

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

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

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

[0289] 122. A 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 comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0290] 123. A composition according to any one of items 104-122, wherein the 3' intron fragment is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 42-52; and / or

[0291] The 5' intron fragment is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 75-88.

[0292] 124. A composition according to any one of items 104 to 123, wherein E1 and / or E2 has a length of 0-20 nucleotides, preferably a length of 0-10 nucleotides, such as a length of 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. A composition according to item 124, wherein E1, E2 or both are 0 nucleotides in length.

[0295] 126. A composition according to any one of items 104-125, wherein the target sequence is a non-coding sequence selected from the following group: an intervening sequence of SEQ ID NO:4-6, a polyA sequence, a poly-AC sequence, a polyC sequence, a polyU sequence, an IRES, a ribosome binding site, an adapter sequence, an RNA scaffold, a riboswitch, a ribozyme other than a self-splicing ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translation regulatory sequence and a protein binding site.

[0296] 127. A 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 natural IRES sequence, and a protein coding sequence, preferably the protein coding sequence encodes a therapeutic product.

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

[0298] 129. A composition according to claim 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'-0-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio-N6-isopentenyl adenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine), g6A (N6-glycylaminoformyl adenosine), t6A (N6-threonylaminoformyl adenosine), ms2t6A (2-methylthio-N6-threonylaminoformyl adenosine), m6t6A (N6-methyl-N6-threonylaminoformyl adenosine), hn6A (N6-hydroxynorvaline acylaminoformyladenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine), Ar(p) (2'-0-ribosyladenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-0-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'-0-methylguanosine), m22G (N2, N2-dimethylguanosine), m2Gm (N2, 2'-O-dimethylguanosine), m2aGm (N2, N2, 2'-O-trimethylguanosine), Gr (p) (2'-0-ribosylguanosine (phosphate)), yW (whitinosine), oayW (peroxywhitinosine), OH yW (hydroxy wyoside), OHyW* (undermodified hydroxy wyoside), imG (wyoside), mimG (methyl wyoside), Q (braid), oQ (epoxybraid), galQ (galactosyl-braid), manQ (mannosyl-braid), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archauridine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-0-methyluridine), acp3U (3-(3-amino-3-carboxypropyl) uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl) uridine), mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester), mcm5U (5-methoxycarbonylmethyl uridine), mcm5Um (5-methoxycarbonylmethyl-2'-0-methyl uridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyl uridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine), cmnm5U (5-carboxymethylaminomethyl uridine), cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyl uridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6, N6-dimethyladenosine), Im (2'-0-methylinosine), m4C (N4-methylcytidine), m4Cm (N4, 2'-0-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6, 2'-O-dimethyladenosine), m6 2Am (N6,N6,0-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-0-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-0-methylcytidine), m'Gm (l,2'-0-dimethylguanosine), m'Am (l,2'-0-dimethyladenosine), rm 5U (5-taurine methyluridine), τm5s2U (5-taurine methyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine). ,

[0299] 130. A 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. A composition according to any one of items 101-130, further comprising a solid surface,

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

[0302] It is further preferred that the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads or silica beads, or

[0303] It is further preferred that the solid surface is a resin containing agarose, a carbohydrate-based material, a polymer filler, silica, glass particles, a cast-based material or a monolithic support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

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

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

[0306] The magnetic beads are preferably functional group-modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleimide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specificity groups, or a combination of two or more thereof, more preferably NHS-modified magnetic beads, or

[0307] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0308] Preferably, the magnetic beads have a diameter 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.

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

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

[0311] Further, the agarose chromatography filler is an agarose chromatography filler with amino modification on the surface, a carboxyl modified agarose chromatography filler, an NHS modified agarose chromatography filler, a maleamide modified agarose chromatography filler, a CNBr modified agarose chromatography filler, an agarose chromatography filler modified with affinity or specificity groups, or a combination of two or more of the above, and further preferably an NHS modified agarose chromatography filler.

[0312] 135. A 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 the covalent fixation is preferably achieved by covalent linkage of NH2 and NHS-modified carboxyl groups.

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

[0314] 137. A composition according to any one of items 101-136, wherein the minimum free energy (MFE) values ​​of the first negative selection tag and the second negative selection tag range from -2 to 2, and the change in MFE value (ΔMFE) of the precursor nucleic acid resulting from the inclusion of the tags ranges from -2 to 2.

[0315] 138. A composition according to item 137, wherein the MFE value of the first negative selection tag and the second negative selection tag is about 0.

[0316] 139. A composition according to item 137 or 138, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the first negative selection tag and the second negative selection tag is in the range of 0-2, preferably the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the first negative selection tag and the second negative selection tag is about 0.

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

[0318] 141. A kit according to item 140, wherein the binding solution comprises a salt comprising a cation selected from the group consisting of: Ba 2+ , Ca 2+ Mg 2+ , Mn 2+ , Fe 2+ , Cu 2+、Zn 2+ 、Cd 2+ , Pb 2+ , Li + , Cs + 、Na + , K + , Rb + and 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,

[0319] It is further preferred that the salt is a cation of Li + Or Na + of salts.

[0320] 142. A 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 buffer substance, a chelating agent, and optionally a stabilizer.

[0322] 143. A kit according to item 141 or 142, 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.

[0323] 144. A kit according to any one of items 140-143, 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 CO 3 - , Cl - Br - 、NO 3 3- , ClO 4 3- ,I - and SCN - or a combination thereof, preferably the salt is a cation of Li + Or Na + of salts,

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

[0325] 145. A 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 coupled with negative selection probes, an elution fluid and a binding fluid, wherein the negative selection probe is at least 90% complementary to a 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.

[0328] 147. A kit according to item 146, 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 101-139, or

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

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

[0331] 149. 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, the first negative selection tag and the second negative selection tag being removed during the self-splicing process, the method comprising:

[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 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

[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. A method according to item 149, 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.

[0336] 151. A method according to item 149 or 150, 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.

[0337] 152. A 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) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0338] (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;

[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 the two.

[0342] 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.

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

[0344] (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;

[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 the two.

[0348] 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.

[0349] 154. A method according to item 151, 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:

[0350] (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;

[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 does not exist, or is a protein coding sequence, a non-coding sequence, or a combination of the two.

[0354] 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.

[0355] 155. A method according to item 151, 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:

[0356] (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;

[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 does not exist, or is a protein coding sequence, a non-coding sequence, or a combination of the two.

[0360] 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.

[0361] 156. A 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. A 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. A method according to any one of items 149-157, wherein

[0364] When the first negative selection tag and negative selection probe pair are the same as the second negative selection tag and 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, wherein n is an integer from 135 to 1234; or

[0365] 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; wherein n is an integer from 2335 to 3544.

[0366] 159. A method according to any one of items 49-58, 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 selected from any one of SEQ ID NOs: 7189-7202; or the first negative selection probe and the second negative selection probe have a polynucleotide sequence selected from any one of SEQ ID NOs: 7175-7188.

[0367] 160. A 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 shown in SEQ ID NO: 7189, and the polynucleotide sequence of the first or second negative selection probe is shown in SEQ ID NO: 7175;

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

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

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

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

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

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

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

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

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

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

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

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

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

[0382] 161. A method according to any one of items 149-160, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron of the unpaired region into two fragments.

[0383] 162. A method according to item 161, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting 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

[0384] 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.

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

[0386] Preferably, the modification comprises modification of 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, respectively.

[0387] 164. A method according to item 163, wherein the modification is the modification of two EBS sequences (such as EBSl and EBS3) of the group II intron, wherein the EBS sequence is complementarily 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.

[0388] 165. A method according to item 163, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of the group II intron, wherein the EBS sequence is complementarily 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.

[0389] 166. A method according to item 164 or 165, 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 to a region of corresponding length in the target sequence 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 sequence are complementary to a region of corresponding length in the target sequence at least 60% of the nucleotides,

[0390] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to a region of corresponding length in the target sequence, and 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 100% of the nucleotides upstream thereof are complementary to a region of corresponding length in the target sequence.

[0391] 167. A method according to item 166, wherein the modification comprises a partial or complete deletion of domain 4, such as a deletion of an intron-encoded protein (IEP) sequence in domain 4, preferably a complete deletion of domain 4; or

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

[0393] 168. A method according to item 164, wherein the precursor RNA is capable of forming a nearly scarless circRNA of the target sequence, and preferably the nearly scarless circRNA has a scar region having 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. A method according to item 165, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0395] 170. A 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. A 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 is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 75-88.

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

[0399] 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.

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

[0401] 174. A method according to any one of items 152-173, wherein the target sequence is a non-coding sequence selected from the following group: an intervening sequence of SEQ ID NO:4-6, a polyA sequence, a poly-AC sequence, a polyC sequence, a polyU sequence, an IRES, a ribosome binding site, an adapter sequence, an RNA scaffold, a riboswitch, a ribozyme other than a self-splicing ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translation regulatory sequence and a protein binding site.

[0402] 175. A 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 natural IRES sequence, and a protein coding sequence, preferably the protein coding sequence encodes a therapeutic product.

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

[0404] 177. A method according to claim 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-isopentenyl adenosine), ms2i6A (2-methylthio-N6-isopentenyl adenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine), g6A (N6-glycylaminoformyl adenosine), t6A (N6-threonylaminoformyl adenosine), ms2t6A (2-methylthio-N6-threonylaminoformyl adenosine), m6t6A (N6-methyl-N6-threonylaminoformyl adenosine), hn6A (N6-hydroxynorvaline acylaminoformyladenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine), Ar(p) (2'-0-ribosyladenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-0-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'-0-methylguanosine), m22G (N2, N2-dimethylguanosine), m2Gm (N2, 2'-O-dimethylguanosine), m2aGm (N2, N2, 2'-O-trimethylguanosine), Gr (p) (2'-0-ribosylguanosine (phosphate)), yW (whitinosine), oayW (peroxywhitinosine), OH yW (hydroxy wyoside), OHyW* (undermodified hydroxy wyoside), imG (wyoside), mimG (methyl wyoside), Q (braid), oQ (epoxybraid), galQ (galactosyl-braid), manQ (mannosyl-braid), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archauridine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-0-methyluridine), acp3U (3-(3-amino-3-carboxypropyl) uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl) uridine), mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester), mcm5U (5-methoxycarbonylmethyl uridine), mcm5Um (5-methoxycarbonylmethyl-2'-0-methyl uridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyl uridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine), cmnm5U (5-carboxymethylaminomethyl uridine), cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyl uridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6, N6-dimethyladenosine), Im (2'-0-methylinosine), m4C (N4-methylcytidine), m4Cm (N4, 2'-0-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6, 2'-O-dimethyladenosine), m6 2Am (N6,N6,0-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-0-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-0-methylcytidine), m'Gm (l,2'-0-dimethylguanosine), m'Am (l,2'-0-dimethyladenosine), rm 5U (5-taurine methyluridine), τm5s2U (5-taurine methyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine). ,

[0405] 178. A 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] 179. The method according to any one of items 152-178, further comprising a solid surface,

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

[0408] It is further preferred that the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads or silica beads, or

[0409] It is further preferred that the solid surface is a resin containing agarose, a carbohydrate-based material, a polymer filler, silica, glass particles, a cast-based material or a monolithic support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

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

[0411] 181. The method of claim 179 or 180, wherein the solid surface is a magnetic bead,

[0412] The magnetic beads are preferably functional group-modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleimide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specificity groups, or a combination of two or more thereof, more preferably NHS-modified magnetic beads, or

[0413] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0414] Preferably, the magnetic beads have a diameter 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.

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

[0416] Further, the agarose chromatography filler is an agarose chromatography filler with amino modification on the surface, a carboxyl modified agarose chromatography filler, an NHS modified agarose chromatography filler, a maleamide modified agarose chromatography filler, a CNBr modified agarose chromatography filler, an agarose chromatography filler modified with affinity or specificity groups, or a combination of two or more of the above, and further preferably an NHS modified agarose chromatography filler.

[0417] 183. A 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 the covalent fixation is preferably achieved by covalent connection of NH2 and NHS-modified carboxyl groups.

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

[0419] 185. A method according to any one of items 149-184, wherein the minimum free energy (MFE) values ​​of the first negative selection tag and the second negative selection tag range from -2 to 2, and the change in MFE value (ΔMFE) of the precursor nucleic acid resulting from the inclusion of the tags ranges from -2 to 2.

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

[0421] 187. A method according to item 185 or 186, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the first negative selection tag and the second negative selection tag is in the range of 0-2, and preferably the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the first negative selection tag and the second negative selection tag is approximately 0.

[0422] 188. A 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 and second negative selection probes at a pH of 4.0-9.0, preferably 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, or 8.5-9.0; or

[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 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 is a cation of Li + Or Na + of salts,

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

[0430] 191. A 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. A method according to any one of items 149-191, wherein the method is scalable and may include a continuous flow process, preferably performed using a liquid chromatograph (LC) or an ELISA reader, and further preferably the LC is a fast liquid chromatograph (FPLC) or a high performance liquid chromatograph (HPLC).

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

[0434] coupling the magnetic beads to the first negative selection probe and the second negative selection probe;

[0435] 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;

[0436] Collecting the magnetic beads and eluting to recover the magnetic beads coupled with the first negative selection probe and the second negative selection probe;

[0437] Collect the unbound fraction,

[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 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.

[0439] 194. A method according to item 193, wherein the IVT reaction solution is incubated with magnetic beads coupled to a first negative selection probe and a second negative selection probe in a binding solution, and the magnetic beads are eluted with an elution solution to recover the magnetic beads coupled to the first negative selection probe and the second negative selection probe.

[0440] 195. A method according to item 194, wherein the elution fluid and the binding fluid are the elution fluid and the binding fluid involved in any one of items 188-191, and further preferably, the negative selection probe and the negative selection label are the first negative selection probe and the second negative selection probe, and the first negative selection label and the second negative selection label involved in any one of items 149-195.

[0441] 196. Circular nucleic acid purified according to the method described in any one of items 149-195, or using 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.

[0442] 197. A composition having a circular nucleic acid as described in item 196, wherein the circularization 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%, and the polymer content is preferably 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 producing a circular nucleic acid, comprising:

[0446] Linearized plasmids for producing precursor nucleic acids;

[0447] The resulting precursor nucleic acid;

[0448] The generated circular nucleic acid;

[0449] Uncircularized precursor nucleic acid;

[0450] 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 items 101-148,

[0451] Preferably the composition has a total ring purity in the range of 20-50% and a polymer content in the range of 10-20%.

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

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

[0454] The preferred polymer 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 acids, wherein the circular nucleic acid is produced by self-splicing of a precursor nucleic acid, wherein the positive selection probe is at least 60% complementary to at least a portion of the circular nucleic acid produced by self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the spliced ​​portion of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0457] 202. A 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 portion of the circular nucleic acid produced 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.

[0458] 203. A composition according to item 201 or 202, 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.

[0459] 204. A composition according to item 203, wherein the precursor RNA comprises the following operably linked elements from 5' to 3': (a) 3' intron fragment; (b) exon fragment 2 (E2); (c) target sequence; (d) exon fragment 1 (E1); and (e) 5' intron fragment; wherein:

[0460] (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;

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

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

[0463] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0464] 205. A composition according to item 203, 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:

[0465] (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;

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

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

[0468] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0469] 206. A composition according to item 203, 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:

[0470] (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;

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

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

[0473] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0474] 207. A composition according to item 203, 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:

[0475] (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;

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

[0477] (3) the 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 noncoding sequence, or a combination of the two.

[0479] 208. A composition according to any one of items 201-207, wherein the positive selection probe is 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides in length, preferably 20-30 nucleotides.

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

[0481] 210. A 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 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 target sequence that has not been circularized 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 portion of the linked portion formed by circularization of the 3' and 5' ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any portion of the target sequence that has not been circularized is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0484] 211. A 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 NOs. 7203-7206 and SEQ ID NOs. 7222-7225.

[0485] 212. A composition according to item 210, wherein the positive selection probe comprises a first positive selection probe and a second positive selection probe, the second positive selection probe is at least 60% complementary to any at least a part of the two side sequences near the outer sides of 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 target sequence that has not been cyclized is less than 60%, preferably less than 70%, 80%, 90%, or 95%, and it is further preferred that the sequence of the second positive selection probe is selected from TT, GATT, and TTTC.

[0486] 213. A composition according to any one of items 204-207, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron of the unpaired region into two fragments.

[0487] 214. A composition according to item 213, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting 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

[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. A composition according to item 213 or 214, wherein the group II intron comprises a modification of one or more nucleotides relative to its wild-type form, and the modification is selected from one or more of a deletion, a substitution and an addition,

[0490] Preferably, the modification comprises modification of 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, respectively.

[0491] 216. A composition according to item 215, wherein the modification is the modification of two EBS sequences (such as EBSl and EBS3) of the group II intron, wherein the EBS sequence is complementarily 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.

[0492] 217. A composition according to item 215, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of the group II intron, wherein the EBS sequence is complementarily 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.

[0493] 218. A composition according to item 215 or 216, 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 to a region of corresponding length in the target sequence at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence at least 60% of the nucleotides,

[0494] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to a region of corresponding length in the target sequence, and 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 100% of the nucleotides upstream thereof are complementary to a region of corresponding length in the target sequence.

[0495] 219. A composition according to item 214, wherein the modification comprises a partial or complete deletion of domain 4, such as a deletion of an intron-encoded protein (IEP) sequence in domain 4, preferably a complete deletion of domain 4; or

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

[0497] 220. A composition according to item 216, wherein the precursor RNA is capable of forming a nearly scarless circRNA of the target sequence, preferably the nearly scarless circRNA has a scar region 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 in length.

[0498] 221. A composition according to item 217, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0499] 222. A 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, and further preferably the group II intron comprises a polynucleotide sequence selected from SEQ IDNO:33-41.

[0500] 223. A composition according to any one of items 204-222, wherein the 3' intron fragment is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 42-52; and / or

[0501] The 5' intron fragment is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 75-88.

[0502] 224. A composition according to any one of items 204 to 223, wherein E1 and / or E2 has a length of 0-20 nucleotides, preferably a length of 0-10 nucleotides, such as a length of 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. A composition according to item 224, wherein E1, E2 or both are 0 nucleotides in length.

[0505] 226. A composition according to any one of items 204-225, wherein the target sequence is a non-coding sequence selected from the following group: an intervening sequence of SEQ ID NO:4-6, a polyA sequence, a poly-AC sequence, a polyC sequence, a polyU sequence, an IRES, a ribosome binding site, an adapter sequence, an RNA scaffold, a riboswitch, a ribozyme other than a self-splicing ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translation regulatory sequence and a protein binding site.

[0506] 227. A 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 natural IRES sequence, and a protein coding sequence, preferably the protein coding sequence encodes a therapeutic product.

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

[0508] 229. A 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'-0-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio-N6-isopentenyl adenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine), g6A (N6-glycylaminoformyl adenosine), t6A (N6-threonylaminoformyl adenosine), ms2t6A (2-methylthio-N6-threonylaminoformyl adenosine), m6t6A (N6-methyl-N6-threonylaminoformyl adenosine), hn6A (N6-hydroxynorvaline acylaminoformyladenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine), Ar(p) (2'-0-ribosyladenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-0-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'-0-methylguanosine), m22G (N2, N2-dimethylguanosine), m2Gm (N2, 2'-O-dimethylguanosine), m2aGm (N2, N2, 2'-O-trimethylguanosine), Gr (p) (2'-0-ribosylguanosine (phosphate)), yW (whitinosine), oayW (peroxywhitinosine), OH yW (hydroxy wyoside), OHyW* (undermodified hydroxy wyoside), imG (wyoside), mimG (methyl wyoside), Q (braid), oQ (epoxybraid), galQ (galactosyl-braid), manQ (mannosyl-braid), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archauridine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-0-methyluridine), acp3U (3-(3-amino-3-carboxypropyl) uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl) uridine), mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester), mcm5U (5-methoxycarbonylmethyl uridine), mcm5Um (5-methoxycarbonylmethyl-2'-0-methyl uridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyl uridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine), cmnm5U (5-carboxymethylaminomethyl uridine), cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyl uridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6, N6-dimethyladenosine), Im (2'-0-methylinosine), m4C (N4-methylcytidine), m4Cm (N4, 2'-0-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6, 2'-O-dimethyladenosine), m6 2Am (N6,N6,0-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-0-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-0-methylcytidine), m'Gm (l,2'-0-dimethylguanosine), m'Am (l,2'-0-dimethyladenosine), rm 5U (5-taurine methyluridine), τm5s2U (5-taurine methyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine). ,

[0509] 230. A 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] 231. The composition of 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] It is further preferred that the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads or silica beads, or

[0513] It is further preferred that the solid surface is a resin containing agarose, a carbohydrate-based material, a polymer filler, silica, glass particles, a cast-based material or a monolithic support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

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

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

[0516] The magnetic beads are preferably functional group-modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleimide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specificity groups, or a combination of two or more thereof, more preferably NHS-modified magnetic beads, or

[0517] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0518] Preferably, the magnetic beads have a diameter 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.

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

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

[0521] Further, the agarose chromatography filler is an agarose chromatography filler with amino modification on the surface, a carboxyl modified agarose chromatography filler, an NHS modified agarose chromatography filler, a maleamide modified agarose chromatography filler, a CNBr modified agarose chromatography filler, an agarose chromatography filler modified with affinity or specificity groups, or a combination of two or more of the above, and further preferably an NHS modified agarose chromatography filler.

[0522] 235. A 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 linkage of NH2 and NHS-modified carboxyl groups.

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

[0524] 237. A composition according to item 235, 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 another of the second positive selection probes is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and the covalent fixation is preferably achieved by covalent connection of NH2 and NHS-modified carboxyl groups.

[0525] 238. A composition according to item 236, 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 another of the second positive selection probes are further modified by a group selected from the following: amino, carboxyl, NHS-carboxyl, thiol.

[0526] 239. A composition according to item 237 or 238, wherein the positive selection probe has a polynucleotide sequence shown in any one of SEQ ID NOs. 7205-7206.

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

[0528] 241. A kit according to item 240, wherein the binding solution comprises a salt comprising a cation selected from the group consisting of: Ba 2+ , Ca 2+ Mg2+ , Mn 2+ , Fe 2+ , Cu 2+ 、Zn 2+ 、Cd 2+ , Pb 2+ , Li + , Cs + 、Na + , K + , Rb + and 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,

[0529] It is further preferred that the salt is a cation of Li + Or Na + of salts.

[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 buffer substance, a chelating agent, and optionally a stabilizer.

[0532] 243. A kit according to item 241 or 242, 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.

[0533] 244. A kit according to any one of items 240-243, 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+ , 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 is a cation of Li + Or Na + of salts,

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

[0535] 245. A 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 coupled with positive selection probes, an elution fluid and a binding fluid, wherein the circular nucleic acid is produced by self-splicing of a precursor nucleic acid, the positive selection probe is at least 60% complementary to at least a portion of the circular nucleic acid produced by self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the spliced ​​portion of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0538] 247. A 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 elution liquid and the binding liquid are the elution liquid and the binding liquid involved in any one of items 240-245.

[0539] 248. A kit according to item 246 or 247, wherein the magnetic beads having the positive selection probes fixed thereto can be reused.

[0540] 249. 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, the method comprising:

[0541] (i) contacting a sample comprising the circular nucleic acid produced by self-splicing of the precursor nucleic acid with a positive selection probe that is at least 60% complementary to at least a portion of the circular nucleic acid under conditions that allow the circular nucleic acid produced 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

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

[0543] At least a portion of the circular nucleic acid spans a spliced ​​portion of the circular nucleic acid, and a sequence that is at least 60% complementary to the positive selection probe is not present on the precursor nucleic acid.

[0544] 250. A method according to claim 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 portion of the circular nucleic acid produced 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. A method according to item 249 or 250, 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.

[0546] 252. A 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 target sequence; (d) 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 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;

[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 noncoding sequence, or a combination of the two.

[0551] 253. A method according to item 251, 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:

[0552] (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;

[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 noncoding sequence, or a combination of the two.

[0556] 254. A method according to item 251, 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:

[0557] (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;

[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 noncoding sequence, or a combination of the two.

[0561] 255. A method according to item 251, 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:

[0562] (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;

[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 noncoding sequence, or a combination of the two.

[0566] 256. A method according to any one of items 249-255, wherein the positive selection probe has a length of 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides, preferably 20-30 nucleotides.

[0567] 257. According to the method described in any one of items 249-256, 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 to the linker fragment formed by E1 and E2 of the circular nucleic acid, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the target sequence that has not been circularized 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 portion of the linked portion formed by circularization of the 3' and 5' ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any portion of the target sequence that has not been circularized is less than 60%, preferably less than 70%, 80%, 90%, or 95%.

[0571] 259. A 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 NOs. 7203-7206 and SEQ ID NOs. 7222-7225.

[0572] 260. A method according to item 259, wherein the positive selection probe comprises a first positive selection probe and a second positive selection probe, the second positive selection probe is at least 60% complementary to any at least a part of the two side sequences near the outer sides of 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 target sequence that has not been cyclized is less than 60%, preferably less than 70%, 80%, 90%, or 95%, and it is further preferred that the sequence of the second positive selection probe is selected from TT, GATT, and TTTC.

[0573] 261. A method according to any one of items 249-260, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron of the unpaired region into two fragments.

[0574] 262. A method according to item 261, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting 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

[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. A method according to item 261 or 262, wherein the group II intron comprises a modification of one or more nucleotides relative to its wild-type form, and the modification is selected from one or more of a deletion, a substitution and an addition,

[0577] Preferably, the modification comprises modification of 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, respectively.

[0578] 264. A method according to item 263, wherein the modification is the modification of two EBS sequences (such as EBSl and EBS3) of the group II intron, wherein the EBS sequence is complementarily 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.

[0579] 265. A method according to item 263, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of the group II intron, wherein the EBS sequence is complementarily 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.

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

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

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

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

[0584] 268. A method according to item 264, wherein the precursor RNA is capable of forming a nearly scarless circRNA of the target sequence, and preferably the nearly scarless circRNA has a scar region having 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. A method according to item 265, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0586] 270. A 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 further preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0587] 271. A 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 is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 75-88.

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

[0590] 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.

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

[0592] 274. A method according to any one of items 252-273, wherein the target sequence is a non-coding sequence selected from the following group: an intervening sequence of SEQ ID NO:4-6, a polyA sequence, a poly-AC sequence, a polyC sequence, a polyU sequence, an IRES, a ribosome binding site, an adapter sequence, an RNA scaffold, a riboswitch, a ribozyme other than a self-splicing ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translation regulatory sequence and a protein binding site.

[0593] 275. A 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 natural IRES sequence, and a protein coding sequence, preferably the protein coding sequence encodes a therapeutic product.

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

[0595] 277. A method according to claim 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'-0-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio-N6-isopentenyl adenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine), g6A (N6-glycylaminoformyl adenosine), t6A (N6-threonylaminoformyl adenosine), ms2t6A (2-methylthio-N6-threonylaminoformyl adenosine), m6t6A (N6-methyl-N6-threonylaminoformyl adenosine), hn6A (N6-hydroxynorvaline acylaminoformyladenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine), Ar(p) (2'-0-ribosyladenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-0-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'-0-methylguanosine), m22G (N2, N2-dimethylguanosine), m2Gm (N2, 2'-O-dimethylguanosine), m2aGm (N2, N2, 2'-O-trimethylguanosine), Gr (p) (2'-0-ribosylguanosine (phosphate)), yW (whitinosine), oayW (peroxywhitinosine), OH yW (hydroxy wyoside), OHyW* (undermodified hydroxy wyoside), imG (wyoside), mimG (methyl wyoside), Q (braid), oQ (epoxybraid), galQ (galactosyl-braid), manQ (mannosyl-braid), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archauridine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-0-methyluridine), acp3U (3-(3-amino-3-carboxypropyl) uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl) uridine), mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester), mcm5U (5-methoxycarbonylmethyl uridine), mcm5Um (5-methoxycarbonylmethyl-2'-0-methyl uridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyl uridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine), cmnm5U (5-carboxymethylaminomethyl uridine), cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyl uridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6, N6-dimethyladenosine), Im (2'-0-methylinosine), m4C (N4-methylcytidine), m4Cm (N4, 2'-0-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6, 2'-O-dimethyladenosine), m6 2Am (N6,N6,0-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-0-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-0-methylcytidine), m'Gm (l,2'-0-dimethylguanosine), m'Am (l,2'-0-dimethyladenosine), rm 5U (5-taurine methyluridine), τm5s2U (5-taurine methyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine). ,

[0596] 278. A 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] 279. The method according to any one of items 252-278, further comprising a solid surface,

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

[0599] It is further preferred that the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads or silica beads, or

[0600] It is further preferred that the solid surface is a resin containing agarose, a carbohydrate-based material, a polymer filler, silica, glass particles, a cast-based material or a monolithic support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

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

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

[0603] The magnetic beads are preferably functional group-modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleimide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specificity groups, or a combination of two or more thereof, more preferably NHS-modified magnetic beads, or

[0604] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0605] Preferably, the magnetic beads have a diameter 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.

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

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

[0608] 283. A 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 linkage of NH2 and NHS-modified carboxyl groups.

[0609] 284. A 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 by a group selected from the following: amino group, carboxyl group, NHS-carboxyl group, thiol group.

[0610] 285. A 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 another of the second positive selection probes is covalently fixed to the solid surface, preferably the solid surface is a magnetic bead, and the covalent fixation is preferably achieved by covalent connection of NH2 and NHS-modified carboxyl groups.

[0611] 286. A 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 another of the second positive selection probes are further modified by a group selected from the following: amino, carboxyl, NHS-carboxyl, thiol.

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

[0613] 288. A 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-60°C; or

[0615] The sample is contacted with the positive 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,

[0616] It is further preferred that the salt is a cation of Li + Or Na + of salts.

[0617] 289. The method according to item 288, 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,

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

[0619] 290. A method according to any one of items 249-289, 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 CO 3 - , Cl - Br - 、NO 3 3- , ClO 4 3- ,I - and SCN - or a combination thereof, preferably the salt is a cation of Li + Or Na + of salts,

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

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

[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. A method according to any one of items 249-291, wherein the method is scalable and may include a continuous flow process, preferably performed using a liquid chromatograph (LC) or an ELISA reader, and further preferably the LC is a fast liquid chromatograph (FPLC) or a high performance liquid chromatograph (HPLC).

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

[0625] coupling the magnetic beads to the positive selection probe;

[0626] Incubate the IVT reaction solution containing the circular nucleic acid obtained by in vitro transcription with magnetic beads coupled with positive selection probes;

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

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

[0630] 295. A method according to item 294, wherein the elution liquid and the binding liquid are the elution liquid and the binding liquid 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. Circular nucleic acids purified according to the method described in any one of items 249-295, or using the composition comprising oligonucleotide positive selection probes for purifying circular nucleic acids described in any one of items 201-239, or using the kit described in any one of items 240-248.

[0632] 297. A composition having a circular nucleic acid as described in item 296, wherein the circularization 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 the polymer content is preferably below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%,

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

[0635] 298. A composition for producing a circular nucleic acid, comprising:

[0636] Linearized plasmids for producing precursor nucleic acids;

[0637] The resulting precursor nucleic acid;

[0638] The generated circular nucleic acid;

[0639] Uncircularized precursor nucleic acid;

[0640] 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 items 201-248,

[0641] Preferably the composition has a total ring purity in the range of 20-50% and a polymer content in the range of 10-20%.

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

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

[0644] The preferred polymer 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 comprising oligonucleotide probes for purifying circular nucleic acids, wherein the probes comprise negative selection probes and positive selection probes,

[0647] 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, further preferably 100% complementary, and

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

[0649] 302. A composition according to item 301, 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,

[0650] 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 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 portion of the circular nucleic acid produced 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.

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

[0653] 304. A 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 target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0654] (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;

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

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

[0657] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0658] 305. A composition according to item 303, 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:

[0659] (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;

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

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

[0662] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0663] 306. A composition according to item 303, 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:

[0664] (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;

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

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

[0667] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0668] 307. A composition according to item 303, 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:

[0669] (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;

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

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

[0672] (4) The target sequence does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[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 positive selection probe has a length of 10-50, 10-40, 10-30, 20-50, 20-40, or 20-30 nucleotides, preferably 20-30 nucleotides.

[0675] 309. A 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; and 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, wherein 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, wherein m is an integer from 2335 to 3544 or from 4755 to 5964,

[0677] Preferably, the positive selection probe comprises 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 linker fragment formed by E1 and E2 of the circular nucleic acid, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the target sequence that has not been circularized 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 portion of the linked portion formed by circularization of the 3' and 5' ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any portion of the target sequence that has not been circularized 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 shown in 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 possessed by the composition are selected from any one of the following combinations:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0697] Preferably, the positive selection probe includes a first positive selection probe and a second positive selection probe, and the second positive selection probe is at least 60% complementary to any at least a part of the two side sequences close to the outside of 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 target sequence that has not been cyclized is less than 60%, preferably less than 70%, 80%, 90%, or 95%. It is further preferred that the sequence of the second positive selection probe is selected from TT, GATT, and TTTC.

[0698] 313. A composition according to any one of items 304-307, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron of the unpaired region into two fragments.

[0699] 314. A composition according to item 313, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting 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

[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. A composition according to item 313 or 314, wherein the group II intron comprises a modification of one or more nucleotides relative to its wild-type form, and the modification is selected from one or more of a deletion, a substitution and an addition,

[0702] Preferably, the modification comprises modification of 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, respectively.

[0703] 316. A composition according to item 315, wherein the modification is the modification of two EBS sequences (such as EBSl and EBS3) of the group II intron, wherein the EBS sequence is complementarily 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.

[0704] 317. A 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 sequence is complementarily 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.

[0705] 318. A composition according to item 315 or 316, 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 to a region of corresponding length in the target sequence at least 60% of the nucleotides, optionally the modification is a modification of the EBS1 and / or δ sequence and its upstream sequence, wherein the EBS1 and / or δ sequence and its upstream sequence are complementary to a region of corresponding length in the target sequence at least 60% of the nucleotides,

[0706] Preferably, 100% of the nucleotides of the EBS1 and / or δ sequence are complementary to a region of corresponding length in the target sequence, and 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 100% of the nucleotides upstream thereof are complementary to a region of corresponding length in the target sequence.

[0707] 319. A composition according to item 314, wherein the modification comprises a partial or complete deletion of domain 4, such as a deletion of an intron-encoded protein (IEP) sequence in domain 4, preferably a complete deletion of domain 4; or

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

[0709] 320. A composition according to item 316, wherein the precursor RNA is capable of forming a nearly scarless circRNA of the target sequence, preferably the nearly scarless circRNA has a scar region 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 in length.

[0710] 321. A composition according to item 317, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0711] 322. A 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, and further preferably the group II intron comprises a polynucleotide sequence selected from SEQ IDNO:33-41.

[0712] 323. A composition according to any one of items 304-322, wherein the 3' intron fragment is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 42-52; and / or

[0713] The 5' intron fragment is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 75-88.

[0714] 324. A composition according to any one of items 304 to 323, wherein E1 and / or E2 has a length of 0-20 nucleotides, preferably a length of 0-10 nucleotides, such as a length of 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. A composition according to item 324, wherein E1, E2 or both are 0 nucleotides in length.

[0717] 326. A composition according to any one of items 304-325, wherein the target sequence is a non-coding sequence selected from the following group: an intervening sequence of SEQ ID NO:4-6, a polyA sequence, a poly-AC sequence, a polyC sequence, a polyU sequence, an IRES, a ribosome binding site, an adapter sequence, an RNA scaffold, a riboswitch, a ribozyme other than a self-splicing ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translation regulatory sequence and a protein binding site.

[0718] 327. A 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 natural IRES sequence, and a protein coding sequence, preferably the protein coding sequence encodes a therapeutic product.

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

[0720] 329. A composition according to claim 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'-0-methyladenosine), ms2 m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio-N6-isopentenyl adenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine), g6A (N6-glycylaminoformyl adenosine), t6A (N6-threonylaminoformyl adenosine), ms2t6A (2-methylthio-N6-threonylaminoformyl adenosine), m6t6A (N6-methyl-N6-threonylaminoformyl adenosine), hn6A (N6-hydroxynorvaline acylaminoformyladenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine), Ar(p) (2'-0-ribosyladenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-0-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'-0-methylguanosine), m22G (N2, N2-dimethylguanosine), m2Gm (N2, 2'-O-dimethylguanosine), m2aGm (N2, N2, 2'-O-trimethylguanosine), Gr (p) (2'-0-ribosylguanosine (phosphate)), yW (whitinosine), oayW (peroxywhitinosine), OH yW (hydroxy wyoside), OHyW* (undermodified hydroxy wyoside), imG (wyoside), mimG (methyl wyoside), Q (braid), oQ (epoxybraid), galQ (galactosyl-braid), manQ (mannosyl-braid), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archauridine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-0-methyluridine), acp3U (3-(3-amino-3-carboxypropyl) uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl) uridine), mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester), mcm5U (5-methoxycarbonylmethyl uridine), mcm5Um (5-methoxycarbonylmethyl-2'-0-methyl uridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyl uridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine), cmnm5U (5-carboxymethylaminomethyl uridine), cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyl uridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6, N6-dimethyladenosine), Im (2'-0-methylinosine), m4C (N4-methylcytidine), m4Cm (N4, 2'-0-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6, 2'-O-dimethyladenosine), m6 2Am (N6,N6,0-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-0-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-0-methylcytidine), m'Gm (l,2'-0-dimethylguanosine), m'Am (l,2'-0-dimethyladenosine), rm 5U (5-taurine methyluridine), τm5s2U (5-taurine methyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine). ,

[0721] 330. A 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] 331. A composition according to any one of items 301-330, further comprising a solid surface,

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

[0724] It is further preferred that the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads or silica beads, or

[0725] It is further preferred that the solid surface is a resin containing agarose, a carbohydrate-based material, a polymer filler, silica, glass particles, a cast-based material or a monolithic support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

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

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

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

[0729] The magnetic beads are preferably functional group-modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleimide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specificity groups, or a combination of two or more thereof, more preferably NHS-modified magnetic beads, or

[0730] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0731] Preferably, the magnetic beads have a diameter 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.

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

[0733] Preferably, the diameter of the chromatography packing is more than 5 mm, and the packing height is more than 20 cm. More preferably, the diameter of the chromatography packing is more than 10 mm, and the packing height is more than 30 cm. More preferably, the diameter of the chromatography packing is more than 16 mm, and the packing height is more than 40 cm.

[0734] Further, the agarose chromatography filler is an agarose chromatography filler with amino modification on the surface, a carboxyl modified agarose chromatography filler, an NHS modified agarose chromatography filler, a maleamide modified agarose chromatography filler, a CNBr modified agarose chromatography filler, an agarose chromatography filler modified with affinity or specificity groups, or a combination of two or more of the above, and further preferably an NHS modified agarose chromatography filler.

[0735] 335. A 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, and preferably the covalent fixation is achieved by covalent bonding of NH2 and NHS-modified carboxyl groups,

[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, and the covalent fixation is preferably achieved by covalent bonding of NH2 and NHS-modified carboxyl groups.

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

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

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

[0740] 337. A 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 the inclusion of the tag ranges from -2 to 2,

[0741] 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 another 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.

[0742] 338. A 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 another of the second positive selection probes are further modified by a group selected from the following: amino, carboxyl, NHS-carboxyl, thiol.

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

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

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

[0747] 341. A kit according to item 340, wherein the binding solution comprises a salt comprising a cation selected from the group consisting of: Ba 2+ , Ca 2+ Mg 2+ , Mn 2+ , Fe 2+ , Cu 2+ 、Zn 2+ 、Cd 2+ , Pb 2+ , Li + , Cs + 、Na + , K + , Rb + and NH 4 + , and / or an anion selected from the group consisting of: 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 salt is a cation of Li + Or Na + of salts,

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

[0754] 345. A 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 negative selection probes, magnetic beads coupled with positive selection probes, 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 produced 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 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.

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

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

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

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

[0762] 349. 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, 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:

[0763] (i) contacting the sample with 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 a sample comprising the circular nucleic acid produced by self-splicing of the precursor nucleic acid with a positive selection probe that is at least 60% complementary to at least a portion of the circular nucleic acid under conditions that allow the circular nucleic acid produced 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 moieties bound to the positive selection probe on the solid surface,

[0767] At least a portion of the circular nucleic acid spans a spliced ​​portion of the circular nucleic acid, and a sequence that is at least 60% complementary to the positive selection probe is absent from the precursor nucleic acid,

[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. A 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 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 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 portion of the circular nucleic acid produced 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.

[0772] 351. A method according to item 349 or 350, 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.

[0773] 352. A 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) exon fragment 2 (E2); (c) a target sequence; (d) exon fragment 1 (E1); and (e) a 5' intron fragment; wherein:

[0774] (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;

[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 does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0778] 353. A method according to item 351, 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:

[0779] (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;

[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 does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0783] 354. A method according to item 351, 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:

[0784] (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;

[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 does not exist or is a protein coding sequence, a noncoding sequence, or a combination of the two.

[0788] 355. A method according to item 351, 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:

[0789] (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;

[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 noncoding sequence, or a combination of the two.

[0793] 356. A method according to any one of items 349-355, 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.

[0794] 357. A 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, wherein 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, wherein m is an integer from 2335 to 3544 or from 4755 to 5964,

[0796] Preferably, the positive selection probe comprises a first positive selection probe and an optional second positive selection probe,

[0797] The first positive selection probe is at least 60% complementary to the linker fragment formed by E1 and E2 of the circular nucleic acid, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any part of the target sequence that has not been circularized 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 portion of the linked portion formed by circularization of the 3' and 5' ends of the target sequence, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementary, or 100% complementary, and the complementarity between the first positive selection probe and any portion of the target sequence that has not been circularized 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 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,

[0800] Preferably, the positive selection probe has a polynucleotide sequence shown in any one of SEQ ID NOs. 7203-7206 and SEQ ID NOs. 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 shown in SEQ ID NO: 7189, and the polynucleotide sequence of the negative selection probe is shown in SEQ ID NO: 7175;

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

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

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

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

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

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

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

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

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

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

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

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

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

[0816] Preferably, the positive selection probe includes a first positive selection probe and a second positive selection probe, and the second positive selection probe is at least 60% complementary to any at least a part of the two side sequences close to the outside of 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 target sequence that has not been cyclized is less than 60%, preferably less than 70%, 80%, 90%, or 95%. It is further preferred that the sequence of the second positive selection probe is selected from TT, GATT, and TTTC.

[0817] 361. A method according to any one of items 349-360, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting the group II intron of the unpaired region into two fragments.

[0818] 362. A method according to item 361, wherein the 5' intron fragment and the 3' intron fragment are obtained by cutting 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 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.

[0820] 363. A method according to item 361 or 362, wherein the group II intron comprises a modification of one or more nucleotides relative to its wild-type form, and the modification is selected from one or more of a deletion, a substitution and an addition,

[0821] Preferably, the modification comprises modification of 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, respectively.

[0822] 364. A method according to item 363, wherein the modification is the modification of two EBS sequences (such as EBSl and EBS3) of the group II intron, wherein the EBS sequence is complementarily 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.

[0823] 365. A method according to item 363, wherein the modification is the modification of two EBS sequences (such as EBS1' and EBS3') of the group II intron, wherein the EBS sequence is complementarily 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.

[0824] 366. A 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 to a region of corresponding length in the target sequence 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 sequence are complementary to a region of corresponding length in the target sequence at least 60% of the nucleotides,

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

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

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

[0828] 368. A method according to item 364, wherein the precursor RNA is capable of forming a nearly scarless circRNA of the target sequence, and preferably the nearly scarless circRNA has a scar region having 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. A method according to item 365, wherein the precursor RNA is capable of forming a scarless circRNA of the target sequence.

[0830] 370. A 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, and further preferably the group II intron comprises a polynucleotide sequence selected from SEQ ID NO: 33-41.

[0831] 371. A method according to any one of items 352-370, wherein the 3' intron fragment is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NO:42-52; and / or

[0832] The 5' intron fragment is at least 95%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from SEQ ID NOs: 75-88.

[0833] 372. The method according to any one of items 352 to 371, wherein E1 and / or E2 is 0-20 nucleotides in length, preferably 0-10 nucleotides in length, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length;

[0834] 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.

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

[0836] 374. A method according to any one of items 352-373, wherein the target sequence is a non-coding sequence selected from the following group: an intervening sequence of SEQ ID NO:4-6, a polyA sequence, a poly-AC sequence, a polyC sequence, a polyU sequence, an IRES, a ribosome binding site, an adapter sequence, an RNA scaffold, a riboswitch, a ribozyme other than a self-splicing ribozyme, an antisense oligonucleotide (ASO), a scaffold, a small RNA binding site, a translation regulatory sequence and a protein binding site.

[0837] 375. A 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 natural IRES sequence, and a protein coding sequence, preferably the protein coding sequence encodes a therapeutic product.

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

[0839] 377. A method according to claim 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-isopentenyl adenosine), ms2i6A (2-methylthio-N6-isopentenyl adenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine), g6A (N6-glycylaminoformyl adenosine), t6A (N6-threonylaminoformyl adenosine), ms2t6A (2-methylthio-N6-threonylaminoformyl adenosine), m6t6A (N6-methyl-N6-threonylaminoformyl adenosine), hn6A (N6-hydroxynorvaline acylaminoformyladenosine), ms2hn6A (2-methylthio-N6-hydroxynorvalylaminoformyladenosine), Ar(p) (2'-0-ribosyladenosine (phosphate)), I (inosine), m1I (1-methylinosine), m1hn (1,2'-O-dimethylinosine), m3C (3-methylcytidine), Cm (2'-0-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'-0-methylguanosine), m22G (N2, N2-dimethylguanosine), m2Gm (N2, 2'-O-dimethylguanosine), m2aGm (N2, N2, 2'-O-trimethylguanosine), Gr (p) (2'-0-ribosylguanosine (phosphate)), yW (whitinosine), oayW (peroxywhitinosine), OH yW (hydroxy wyoside), OHyW* (undermodified hydroxy wyoside), imG (wyoside), mimG (methyl wyoside), Q (braid), oQ (epoxybraid), galQ (galactosyl-braid), manQ (mannosyl-braid), preQo (7-cyano-7-deazaguanosine), preQi (7-aminomethyl-7-deazaguanosine), G+ (archauridine), D (dihydrouridine), m5Um (5,2'-0-dimethyluridine), s4U (4-thiouridine), m5s2U (5-methyl-2-thiouridine), s2Um (2-thio-2'-0-methyluridine), acp3U (3-(3-amino-3-carboxypropyl) uridine), ho5U (5-hydroxyuridine), mo5U (5-methoxyuridine), cmo5U (uridine 5-oxyacetic acid), mcmo5U (uridine 5-oxyacetic acid methyl ester), chm5U (5-(carboxyhydroxymethyl) uridine), mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester), mcm5U (5-methoxycarbonylmethyl uridine), mcm5Um (5-methoxycarbonylmethyl-2'-0-methyl uridine), m cm5s2U (5-methoxycarbonylmethyl-2-thiouridine), nm5S2U (5-aminomethyl-2-thiouridine), mnm5U (5-methylaminomethyl uridine), mnm5s2U (5-methylaminomethyl-2-thiouridine), mnm5se2U (5-methylaminomethyl-2-selenouridine), ncm5U (5-carbamoylmethyl uridine), ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine), cmnm5U (5-carboxymethylaminomethyl uridine), cmnm5Um (5-carboxymethylaminomethyl-2'-0-methyl uridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), m6 2A (N6, N6-dimethyladenosine), Im (2'-0-methylinosine), m4C (N4-methylcytidine), m4Cm (N4, 2'-0-dimethylcytidine), hm5C (5-hydroxymethylcytidine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), m6Am (N6, 2'-O-dimethyladenosine), m6 2Am (N6,N6,0-2'-trimethyladenosine), m2,7G (N2,7-dimethylguanosine), m2,2,7G (N2,N2,7-trimethylguanosine), m3Um (3,2'-0-dimethyluridine), m5D (5-methyldihydrouridine), f5Cm (5-formyl-2'-0-methylcytidine), m'Gm (l,2'-0-dimethylguanosine), m'Am (l,2'-0-dimethyladenosine), rm 5U (5-taurine methyluridine), τm5s2U (5-taurine methyl-2-thiouridine), imG-14 (4-demethylwyosine), imG2 (isowyosine), or ac6A (N6-acetyladenosine). ,

[0840] 378. A 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] 379. The method according to any one of items 352-378, further comprising a solid surface,

[0842] Preferably, the solid surface is a bead, resin, tube, nanoparticle, chip, plate or paper,

[0843] It is further preferred that the solid surface is magnetic beads, agarose beads, streptavidin-coated beads, colloidal beads, glass beads, polymer beads, methacrylate beads, polystyrene beads, latex beads or silica beads, or

[0844] It is further preferred that the solid surface is a resin containing agarose, a carbohydrate-based material, a polymer filler, silica, glass particles, a cast-based material or a monolithic support, wherein the polymer filler is optionally selected from polystyrene-divinylbenzene or polymethacrylate.

[0845] 380. A method according to item 379, wherein the negative selection probe is fixed on the solid surface by physical adsorption, covalent fixation or affinity fixation, preferably covalent fixation is achieved by covalent linkage of NH2 and NHS-modified carboxyl groups,

[0846] The positive selection probe is immobilized on the solid surface by physical adsorption, covalent immobilization or affinity immobilization, preferably covalent immobilization is achieved by covalent linkage of NH2 and NHS-modified carboxyl groups.

[0847] 381. The method of item 379 or 380, wherein the solid surface is a magnetic bead,

[0848] The magnetic beads are preferably functional group-modified magnetic beads, more preferably amino-modified magnetic beads, carboxyl-modified magnetic beads, NHS-modified magnetic beads, maleimide-modified magnetic beads, CNBr-modified magnetic beads, magnetic beads modified with affinity or specificity groups, or a combination of two or more thereof, more preferably NHS-modified magnetic beads, or

[0849] Preferably, the magnetic beads are hydrophilic magnetic beads; or

[0850] Preferably, the magnetic beads have a diameter 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 filler containing agarose, preferably the diameter of the chromatography filler is more than 5 mm, and the filler height is more than 20 cm, more preferably the diameter of the chromatography filler is more than 10 mm, and the filler height is more than 30 cm, more preferably the diameter of the chromatography filler is 16 mm, and the filler height is 40 cm,

[0852] Further, the agarose chromatography filler is an agarose chromatography filler with amino modification on the surface, a carboxyl modified agarose chromatography filler, an NHS modified agarose chromatography filler, a maleamide modified agarose chromatography filler, a CNBr modified agarose chromatography filler, an agarose chromatography filler modified with affinity or specificity groups, or a combination of two or more of the above, and further preferably an NHS modified agarose chromatography filler.

[0853] 383. A 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 the covalent fixation is preferably achieved by covalent bonding of NH2 and NHS-modified carboxyl groups,

[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 the covalent fixation is preferably achieved by covalent bonding of NH2 and NHS-modified carboxyl groups.

[0855] The negative selection probe and the positive selection probe are immobilized on different solid surfaces.

[0856] 384. A method according to any one of items 379-383, wherein the 3' end and / or the 5' end of the negative selection probe is further modified by a group selected from the following: amino, carboxyl, NHS-carboxyl, thiol,

[0857] The 3' end and / or 5' end of the positive selection probe is further modified by a group selected from the following: amino, carboxyl, NHS-carboxyl, thiol.

[0858] 385. A 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 resulting from the inclusion of 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 another 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.

[0860] 386. A composition according to item 385, wherein the MFE value of the negative selection tag is about 0,

[0861] 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 another of the second positive selection probes are further modified by a group selected from the following: amino, carboxyl, NHS-carboxyl, thiol.

[0862] 387. The composition according to item 385 or 386, wherein the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative selection tag is in the range of 0-2, preferably the ΔMFE value of the precursor nucleic acid resulting from the inclusion of the negative selection tag is about 0,

[0863] Preferably, the positive selection probe has a polynucleotide sequence shown in any one of SEQ ID NOs. 7205-7206.

[0864] 388. A 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, 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,

[0867] It is further preferred that the salt is a cation of Li + Or Na + of salts.

[0868] 389. The method according to item 388, 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,

[0869] Preferably, the method further comprises the step of eluting the negative selection probe bound to the solid surface with an elution solution to recover the solid surface coupled with the negative selection probe for reuse.

[0870] 390. A method according to any one of items 349-389, 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 CO 3 - , Cl - Br - 、NO 3 3- , ClO 4 3- ,I - and SCN -or a combination thereof, preferably the salt is a cation of Li + Or Na + of salts,

[0871] Preferably, the eluent further comprises a buffer substance, a chelating agent, and optionally a stabilizer.

[0872] 391. A 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. A method according to any one of items 349-391, wherein the method is scalable and may include a continuous flow process, preferably performed using a liquid chromatograph (LC) or an ELISA reader, and further preferably the LC is a fast liquid chromatograph (FPLC) or a high performance liquid chromatograph (HPLC).

[0875] 393. A method for purifying circular nucleic acids from a sample, comprising the steps of:

[0876] coupling the magnetic beads to the negative selection probe;

[0877] coupling the magnetic beads to the positive selection probe;

[0878] Incubate the IVT reaction solution containing the circular nucleic acid obtained by in vitro transcription with magnetic beads coupled with negative selection probes;

[0879] Collecting the magnetic beads and eluting to recover the magnetic beads coupled with the negative selection probe;

[0880] Collect the unbound fraction,

[0881] Incubating the unbound fraction with magnetic beads coupled with positive selection probes;

[0882] collecting the magnetic beads for elution 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, 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 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 further preferably 100% complementary;

[0884] The circular nucleic acid is produced by self-splicing of a precursor nucleic acid, wherein the positive selection probe is at least 60% complementary to at least a portion of the circular nucleic acid produced by self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the spliced ​​portion of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0885] 394. The method according to item 393, wherein the IVT reaction solution is incubated with magnetic beads coupled to a negative selection probe in a binding solution, and the magnetic beads coupled to the negative selection probe are eluted with an elution solution to recover the magnetic beads,

[0886] The IVT reaction solution is incubated with the magnetic beads coupled with the positive selection probe in the binding solution, the magnetic beads are eluted with the elution solution to obtain the eluted circular nucleic acid, and the magnetic beads coupled with the positive selection probe are recovered.

[0887] 395. A method according to item 394, wherein the elution fluid and the binding fluid are the elution fluid and the binding fluid involved in any one of items 388-391, and further preferably, the negative selection probe, the negative selection label and the positive selection probe are the negative selection probe, the negative selection label and the positive selection probe involved in any one of items 349-392.

[0888] 396. Circular nucleic acids purified according to the method described in any one of items 349-395, or using the composition comprising oligonucleotide probes for purifying circular nucleic acids described in any one of items 301-339, or using the kit described in any one of items 340-348.

[0889] 397. A composition having a circular nucleic acid as described in item 396, wherein the circularization 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 the polymer content is preferably below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, below 1.5%,

[0891] More preferably, the size of the circular nucleic acid is 1000 nt-5000 nt.

[0892] 398. A composition for producing a circular nucleic acid, comprising:

[0893] Linearized plasmids for producing precursor nucleic acids;

[0894] The resulting precursor nucleic acid;

[0895] The generated circular nucleic acid;

[0896] Uncircularized precursor nucleic acid;

[0897] 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 items 301-348,

[0898] Preferably the composition has a total ring purity in the range of 20-50% and a polymer content in the range of 10-20%.

[0899] 399. A composition according to item 398, which is used as a sample for the method described in any of items 349-396 or as a sample purified via a composition comprising an oligonucleotide negative selection probe for purifying circular nucleic acids described in any of items 301-339 or using a kit described in any of items 340-348.

[0900] 400. The composition according to item 399, wherein the total 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 polymer content is below 2.0%, below 1.9%, below 1.8%, below 1.7%, below 1.6%, and below 1.5%. More preferably, the size of the circular nucleic acid is 1000nt-5000nt. 6. Description of the drawings

[0902] Figures 1A-1D Provided is a schematic diagram of the self-splicing of a precursor RNA having group II intron self-splicing activity and the use of the tags disclosed herein for purifying circRNA products. Figure 1A Schematic diagram of a nearly scarless system designed based on the interaction between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The autocatalytic self-splicing group II intron is split into two fragments at the D4 domain, and a customized exon containing E1, E2 and target sequences is inserted between the split introns. Arrows indicate the interaction between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3.

[0903] Figure 1B Schematic diagram of a nearly scarless system designed based on the interaction between δ and IBS3. The autocatalytic self-splicing group II intron is split into two fragments at the D4 domain, and a customized exon containing E1, E2 and target sequence is inserted between the split introns. Arrows indicate the interactions between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and δ.

[0904] Figure 1C Schematic diagram of the scarless system designed based on the interaction between IBS1' and EBS1. The autocatalytic self-splicing group II intron is split into two fragments at the D4 domain, and the target sequence is inserted between the split introns. Arrows indicate the interactions between IBS1' and EBS1, and IBS3' and EBS3.

[0905] Figure 1D Schematic diagram of the scarless system designed based on the interaction between δ and IBS3'. The autocatalytic self-splicing group II intron is split into two fragments at the D4 domain, and the target sequence is inserted between the split introns. Arrows indicate the interactions between IBS1' and EBS1, and IBS3' and δ.

[0906] Figure 2 Results of an exemplary purification process are shown. As shown, the IVT product is the reaction product of a precursor RNA produced by in vitro transcription and subjected to circularization conditions. FPLC purification as disclosed herein produces 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 Schematic diagram of a nearly scarless system designed based on the interaction between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The autocatalytic self-splicing group II intron is split into two fragments at the D4 domain, and a custom exon containing E1, E2 and a target sequence is inserted between the split introns. Arrows indicate the interaction between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The negative selection tags are located at both ends of the D4 domain that is 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 nearly scarless system designed based on the interaction between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. The self-catalytic self-splicing II group intron is split into two fragments at the D4 domain, and a custom exon containing E1, E2 and a target sequence is inserted between the split introns. Arrows represent the interaction between IBS1 and EBS1, IBS2 and EBS2, and IBS3 and EBS3. Wherein the positive selection tag is located on IBS1 and IBS3. In some embodiments, the positive selection tag also has a partial sequence located on the target sequence.

[0909] Figure 3CA schematic diagram of a solid surface (such as magnetic beads or agarose chromatography filler) coupled with a negative selection probe in the present application is provided.

[0910] Figure 3D A schematic diagram of a solid surface (such as magnetic beads or agarose chromatography filler) coupled with a first negative selection probe and a second negative selection probe is provided in the present application.

[0911] Figure 3E A schematic diagram of a solid surface (such as a magnetic bead or agarose chromatography filler) coupled with a cationic selection probe in the present application is provided.

[0912] Figure 4 The results before and after chromatography purification are shown. Figure 4 As shown, after chromatography purification, most of the impurities were removed and the purity of circular RNA was improved.

[0913] Figure 5A and 5B The capillary electrophoresis results before and after negative selection magnetic bead purification are shown. As shown in the figure, after negative selection magnetic bead purification, introns and precursors are removed, and the purity of circular RNA is improved.

[0914] Figure 6 The HPLC result of detecting the aggregate content after purification is shown. As shown in the figure, the aggregate content is extremely low after purification.

[0915] Figure 7 A and Figure 7 B shows the agarose electrophoresis detection results 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 circular RNA is improved.

[0916] Figure 8 The results of translating Gluc and Fluc after purification are shown. As shown in the figure, G1-G7 is Gluc, and F1-F12 is Fluc. They are all purified samples. 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] Fig. 9 This is a schematic diagram of the structure of a group II intron.

[0918] Fig.10 Schematic diagram of the structure of the group II intron with IBS1, IBS2, IBS3, EBS1, EBS2, EBS3, and δ (shown in bold).

[0919] Fig.11 Amplification of the generation of circRNA is shown.

[0920] Fig.12Shown is the analysis of four batches of CVB3-Gluc circRNA purified from HPLC using capillary electrophoresis using an Agilent 2100 Bioanalyzer.

[0921] Fig.13 Schematic diagram of the CircRNA-LNP complex and the particle size of CircRNAGluc-LNP.

[0922] Fig.14 Shown are Gaussia luciferase activities measured from mouse serum 24 hours after injection of CircRNAGluc-LNPs in different formulations.

[0923] Fig.15 Representative IVIS images of BALB / c mice administered 20 ug CircRNAGluc-LNPs with two formulations by intramuscular (im) route are shown. Relative luminescence graphs are shown, and the scale of luminescence is indicated.

[0924] Fig.16 A schematic diagram of the RNA high-throughput screening workstation of the present application is shown.

[0925] Fig.17 is an image showing an in vivo imaging system (IVIS) profile of protein expression following IV injection of an exemplary circular RNA of the present disclosure having a combination of native IRES and IRES-like sequences.

[0926] Fig.18A A schematic diagram of a circular RNA is provided, 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] Fig.18B A schematic diagram of a circular RNA is provided, which comprises, in the following order: a 3' intron fragment, a translation initiation element, a target sequence, and a 5' intron fragment, wherein the translation initiation element and a partial sequence on the target sequence serve as positive selection tags.

[0928] Fig.18C A schematic diagram of a circular RNA is provided, 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] Fig.18D A schematic diagram of a circular RNA is provided, which comprises, in the following order: a 3' intron fragment, a target sequence, a translation initiation element, and a 5' intron fragment, wherein the translation initiation element and a partial sequence on the target sequence serve as positive selection tags.

[0930] Fig.19AA schematic diagram of a circular RNA is provided, which includes, in the following order: a 5' tag, a 3' intron fragment, a linker sequence 2, a translation initiation element, a target sequence, a linker sequence 1, a 5' intron fragment, and a 3' tag.

[0931] Fig.19B A schematic diagram of a circular RNA is provided, which includes, in the following order: a 3' intron fragment, a linker sequence 2, a translation initiation element, a target sequence, a linker sequence 1, and a 5' intron fragment, wherein the linker sequence 1 and a partial sequence on the linker sequence 2 are used as positive selection tags.

[0932] Fig.19C A schematic diagram of a circular RNA is provided, which includes, in the following order: a 5' tag, a 3' intron fragment, a target sequence, a linker sequence 1, a linker sequence 2, a translation initiation element, a 5' intron fragment, and a 3' tag.

[0933] Fig.19D A schematic diagram of a circular RNA is provided, which includes, in the following order: a 3' intron fragment, a target sequence, a linker sequence 1, a linker sequence 2, a translation initiation element, and a 5' intron fragment, wherein the translation initiation element and a partial sequence on the target sequence serve as positive selection tags. 7. Detailed description of the invention

[0935] Provided herein are methods and systems for purifying circular nucleic acids. Circular nucleic acids are single-stranded nucleic acids that are linked end to end. As known in the art, circular nucleic acids can be produced in vitro using chemical means or by enzymatic activity. For example, the 5' end and 3' end of a linear nucleic acid can be chemically linked by catalysis of bromine cyanide and morpholinyl derivatives, or linked in an end-to-end manner by the activity of a nucleic acid ligase. As used herein, a "precursor nucleic acid" is a linear nucleic acid molecule that directly produces a circular nucleic acid, regardless of the cyclization method.

[0936] Circular nucleic acids can include ribonucleotides, deoxyribonucleotides, and modified nucleotides and / or their analogs. In some embodiments, the circular nucleic acid is a circular DNA. In some embodiments, the circular nucleic acid is a circular RNA.

[0937] In some embodiments, circular RNA (also referred to as "circRNA" or "cRNA") can be produced 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 contained in an intron portion of a linear precursor, which can then be used to negatively select circRNA from precursors and introns. Specifically, a sample containing circular RNA, such as a product of a splicing reaction starting from a labeled linear precursor, can be mixed with a negative selection probe immobilized on a solid surface, wherein a precursor containing a negative selection tag or any other impurity containing a negative selection tag can bind to the negative selection probe and be removed from the solution, producing a circRNA solution substantially free of precursors, introns, and any other impurities containing tags.

[0938] In some embodiments, a positive selection probe can be selected to be at least 60% complementary to at least a portion of the formed circular nucleic acid, but its complementarity with 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, so that the positive selection probe can directly bind to the circular nucleic acid product from the IVT reaction solution, thereby achieving purification of the circular nucleic acid product.

[0939] As known in the art, RNA molecules with enzymatic activity are referred to as ribozymes. CircRNA can also be produced by RNA splicing catalyzed by ribozymes. In addition, some ribozymes can catalyze self-splicing independently of the spliceosome, and these ribozymes are referred to as "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 "cRNA enzymes" herein.

[0940] Therefore, in some embodiments, the present invention provides 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 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 invention also provides a method for purifying circular nucleic acids 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 negative selection tag that is removed during self-splicing, the method comprising: (i) contacting the sample with an oligonucleotide negative selection probe complementary to the negative selection tag under conditions that allow the negative selection tag to bind to the probe, wherein the negative selection probe is fixed to a solid surface; and (ii) collecting the unbound portion of the sample.

[0941] In some embodiments, the present invention provides a composition comprising an oligonucleotide positive selection probe for purifying a circular nucleic acid, wherein the circular nucleic acid is produced by self-splicing of a precursor nucleic acid, wherein the positive selection probe is at least 60% complementary to at least a portion of the circular nucleic acid produced by self-splicing of the precursor nucleic acid, at least a portion of the circular nucleic acid spans the spliced ​​portion of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe. The method comprises: (i) contacting a sample comprising the circular nucleic acid produced by self-splicing of the precursor nucleic acid with a positive selection probe that is at least 60% complementary to at least a portion of the circular nucleic acid under conditions that allow the circular nucleic acid produced 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 (ii) collecting a portion bound to the positive selection probe on the solid surface, wherein at least a portion of the circular nucleic acid spans the spliced ​​portion of the circular nucleic acid, and there is no sequence on the precursor nucleic acid that is at least 60% complementary to the positive selection probe.

[0942] Before the present disclosure is further described, it is to be understood that the present disclosure is not limited to the particular embodiments set forth herein and it is to be further 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 those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. 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" may refer to one or more. As used herein in the clauses, when used in conjunction with the word "comprising", the word "a" or "an" may refer to one or more than one.

[0946] As used herein, the term "or" in an item is used to mean "and / or" unless explicitly indicated to refer to only alternatives, or the alternatives are mutually exclusive, although the disclosure supports definitions and "and / or" referring only to alternatives. As used herein, "another" or "additional" may mean at least a second or more.

[0947] As used herein, the term "about" is used to indicate a certain value, which includes the inherent error variation of the device, the method for determining the value, or the variation between the research subjects. The term "about" covers the exact number quoted. 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" 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 much less than 0.1%, preferably less than 0.05%, and more preferably less than 0.01%. The most preferred composition is one in which the amount of the specified component cannot be detected by standard analytical methods.

[0949] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to a polymeric form of consecutive amino acids comprising at least two or more chemically or biochemically modified or derivatized amino acids. The term "peptide" as used herein refers to a class of short polypeptides. The term peptide can refer to an amino acid (natural or non-naturally occurring) polymer of 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 the peptide can be: about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1250, about 1500, about 1750, about 2000, about 2250, about 2500, about 2750, about 3000, about 3250, about 3500, about 3750, about 4000, about 4250, about 4500, about 4750, 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. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases (such as methylated, hydroxymethylated or glycosylated), non-natural nucleotides, non-nucleotide components that exhibit structures and / or functions similar to natural nucleotides (i.e., "nucleotide analogs"), and / or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Nucleic acids or polynucleotides can be heterologous or homologous in composition, can be isolated from naturally occurring sources, or can be artificially or synthetically produced. In addition, nucleic acids can be DNA or RNA, or mixtures thereof, and can exist permanently or temporarily in single-stranded or double-stranded forms (including homoduplexes, heteroduplexes and hybrid states). Nucleic acid structures also include, for example, DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, for example, Braasch and Corey, Biochemistry, 4(14):4503-4510 (2002) and U.S. Pat. No. 5,034,506), locked nucleic acids (LNAs; see Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, Am. Chem. Soc., 122:8595-8602 (2000)), and / or ribozymes.

[0951] When referring to a polynucleotide sequence or a protein sequence, the term "identity" is used to represent the similarity between two sequences. Sequence similarity or identity can be determined using standard techniques known in the art, including but not limited to: Smith & Waterman, Adv. Appl. Math. 2, 482 (1981) local sequence identity algorithm, Needleman & Wunsch, J Mol. Biol. 48, 443 (1970) sequence identity comparison algorithm, Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988) similarity search method, by computer implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), Devereux et al., Nucl. Acid Res. 12, 387-395 (1984) described best fit sequence program 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 that can be optionally set to default values. The parameters are dynamic values ​​and are established by the program itself based on the composition of the particular sequence and the composition of the particular database being searched for the sequence of interest; however, these values ​​can be adjusted to increase sensitivity. In addition, another useful algorithm is Gapped BLAST as reported by Altschul et al, (1997) Nucleic Acids Res. 25, 3389-3402. Unless otherwise stated, percent identity herein is determined using an algorithm available 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, e.g., that is or can be translated into a protein. The terms "reading frame", "open reading frame" and "ORF" are used interchangeably herein to refer to a nucleotide sequence that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG or TGA) in some embodiments. An open reading frame may contain introns and exons, and therefore, 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, either 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 a 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 a polynucleotide sequence will form hydrogen bonds with the same number of consecutive nucleotides in a second polynucleotide sequence. Two polynucleotide sequences are "substantially complementary" if the degree of complementarity between the 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 a region of at least 8 nucleotides (e.g., at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or more nucleotides) or if the two polynucleotide sequences hybridize under at least moderate conditions, or in some embodiments, under high stringency conditions. Exemplary moderate stringency conditions include: incubation overnight at 37°C in a solution comprising 20% ​​formamide, 5% SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured 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 use, for example: (1) low ionic strength and high temperature washing, 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) with 750 mM sodium chloride and 75 mM sodium citrate. , or (3) at 42° C., using 50% formamide, 5×SSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 pg / ml), 0.1% SDS, and 10% dextran sulfate, (i) 42° C. washes in 0.2*SSC, (ii) 55° C. washes in 50% formamide, and (iii) 55° C. washes in 0.1*SSC (optionally in combination with EDTA). Additional details and explanations of hybridization 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, NJ (2002).

[0954] The terms "hybridization" or "hybridized" when referring to polynucleotide sequences refer to the binding formed between and / or between sequences that have complementarity.

[0955] The term "homology" refers to the percent identity between the nucleic acid residues of two polynucleotides or the amino acid residues of two polypeptides. The correspondence between one sequence and another sequence can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information between two polypeptides by aligning the sequence information and using readily available computer programs. 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 circRNAs contain a scar sequence of 0 nucleotides. Nearly scarless circRNAs contain a scar sequence that is equal to or less than 20 nucleotides in length.

[0957] The term "Tris" refers to tris(hydroxymethyl)aminomethane, which is widely used in the preparation of buffers for biochemistry and molecular biology experiments.

[0958] The term "EDTA" refers to ethylenediaminetetraacetic acid.

[0959] The term "rpm" refers to the rotation speed per minute, or the number of revolutions per minute, RPM, the 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.

[0962] The term "ATP" refers to adenosine triphosphate; the term "GTP" refers to guanosine triphosphate; the term "CTP" refers to cytidine triphosphate; the term "UTP" refers to uridine triphosphate.

[0963] The term "DNase" 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 a type of nuclease. There are many different types of DNA enzymes known so far, and the main differences are substrate specificity, chemical reaction mechanism, and biological function.

[0964] The term "RNase" refers to ribonuclease or RNA enzyme, which is a nuclease that can hydrolyze RNA into small molecules.

[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 and ribose and deoxyribonucleoside triphosphates.

[0966] The term "DEPC water" refers to ultrapure water that has been treated with DEPC (diethyl pyrocarbonate) and sterilized by high temperature and high pressure.

[0967] The term "PBS" refers to phosphate buffered saline, which is a commonly used buffer in biological research. It is a salt solution containing sodium phosphate in water, with potassium chloride and potassium phosphate added in some formulations. The osmotic pressure and ion concentration of this solution are consistent with those in the human body.

[0968] The term "total circular purity" refers to the ratio of the content of circular RNA to the total RNA of the sample tested.

[0969] The term "polymer content" refers to the ratio of the polymer content to the total amount of RNA in the sample tested.

[0970] The term "Gaussia luciferase" or "Gluc" refers to the luciferase derived from the marine copepod Gaussiaprinceps, which has the advantages of being secretable, easy to monitor, extremely sensitive, short half-life, reaction without ATP participation, and strong tolerance to temperature, pH, 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, an enzyme that catalyzes the oxidation of firefly luciferin and requires oxygen and ATP.

[0972] The term “Lipid nanoparticle” or “LNP” refers to nanoparticles composed of lipids and is a new type of drug delivery route.

[0973] The term "Fast protein liquid chromatography" or "FPLC" refers to fast protein liquid chromatography, a type of liquid chromatography that is often used to analyze or purify protein mixtures.

[0974] The term "High performance liquid chromatography" or "HPLC" refers to high performance liquid chromatography, a chromatographic technique used to separate mixtures in order to identify and quantify the proportions of the individual components.

[0975] The terms "autocatalytic autocleavage", "autocatalytic self-splicing", "self-splicing" or "self-cleavage" refer to self-splicing independent of spliceosomal catalysis. Some ribozymes can catalyze self-splicing independent of the spliceosome, and these ribozymes are referred to as "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 procedure in which liquid substances such as blood, medicine, and nutrients are injected directly into a vein.

[0977] The term "Intramuscular injection (IM)" or "im" refers to a muscle injection, a method of injecting a substance directly into the muscle, which is one of the routes of drug administration in medicine.

[0978] The term "Intraperitoneal injection (IP)" or "ip" refers to an injection into the peritoneum, which is more often used in non-human animals than in humans.

[0979] The term "DSPC" or "Distearoylphosphatidylcholine" refers to distearoylphosphatidylcholine, a type of phosphatidylcholine that is 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", nucleotide number, and is used to describe nucleotide length.

[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, e.g., bioinformatics.org / smsylupac.html). Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and / or SEQ ID NOS. It should be understood that one skilled in the art can readily identify homologous sequences by reference 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 a type II intron. The term "domain 2" or "D2" is used herein to refer to the stem-loop structure of domain 2 of a type II intron. The term "domain 3" or "D3" is used herein to refer to the stem-loop structure of domain 3 of a type II intron. The term "domain 4" or "D4" is used herein to refer to the stem-loop structure of domain 4 of a type II intron. The term "domain 5" or "D5" is used herein to refer to the stem-loop structure of domain 5 of a type II intron. The term "domain 6" or "D6" is used herein to refer to the stem-loop structure of domain 6 of a type II intron. The 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 and 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 of the University of Vienna using a centroid structure prediction algorithm (e.g., AR Gruber et al., 2008, Cell 106). (1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62). Additional algorithms can be found in U.S. Provisional Patent Application No. 61 / 836,080, which is incorporated herein by reference. Type II introns mainly include 6 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 intron binding sequences (IBS) in the exon region, such as complementary pairing, and trigger splicing by the hydroxyl group within the EBS nucleic acid sequence itself.

[0985] As used herein, EBS1', EBS2', and EBS3' are used to refer to regions on a target sequence that have functions similar to EBS1, EBS2, and EBS3, respectively.

[0986] As used herein, the term "group II intron" is used herein to refer to RNA molecules encoded by group II introns that have similar secondary and tertiary structures. Group II intron RNA molecules typically have six domains. See Fig. 9 and Fig.10 . Domain 4 (also referred to as domain IV) of the type II intron RNA comprises a nucleotide sequence encoding a "type II intron encoded protein". In some embodiments, the type 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, which interacts with an exon binding sequence (EBS) to position splice sites.

[0988] The term "IBS1" is used herein to refer to intron binding sequence 1, which interacts with exon binding sequence 1 (EBS1) to locate splice sites.

[0989] The term "IBS1'" is used herein to refer to a region of a 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 splice sites.

[0991] The term "IBS3" is used herein to refer to intron binding sequence 3, which interacts with exon binding sequence 3 (EBS3) to locate splice sites.

[0992] The term "IBS3'" is used herein to refer to a region of a target sequence that functions similarly to IBS3.

[0993] The term "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. Delta pairs with IBS3 and the interaction between delta and IBS3 is referred to as delta-IBS3 pairing. See Fig.10 .

[0994] The term "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 referred to as δ"-IBS3' pairing. See Fig.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 an m7G cap at the 5' end of the mRNA, an initiator Met-tRNAmet, more than a dozen initiation factor proteins, directional scanning and GTP hydrolysis to place translation-competent ribosomes at the start codon. 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 functional ribosomes. In some embodiments, the sequences are selected from the sequences disclosed in PCT / CN2023 / 096813, and the 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 the function of a natural IRES. In some embodiments, an IRES-like sequence can recruit ribosomal components to mediate cap-independent translation. In some embodiments, the sequence is selected from the sequence disclosed in PCT / CN2023 / 096813, and the sequence screened by the method disclosed in PCT / CN2023 / 096813.

[0997] Range: 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 hard limit to the scope of the present application. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual values ​​within the range. For example, a description of a range such as 1-6 should be considered to have specifically disclosed subranges, such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies no matter how large the range is.

[0998] 7.2 Probes and Labels

[0999] 7.2.1 Negative Selection Labels and Negative Selection Probes

[1000] Provided herein are compositions and methods for purifying circular nucleic acids. In some embodiments, circular nucleic acids can be produced by self-splicing of a precursor nucleic acid containing an oligonucleotide negative selection tag, which is removed during self-splicing. Therefore, an oligonucleotide negative selection probe that is at least 60% complementary to the negative selection tag can be used to capture precursors, introns, and any other impurities containing the negative selection tag in the product mixture, thereby purifying circular nucleic acids lacking the negative selection tag. A schematic diagram of the negative selection system and method involved in this article is shown in FIG. Figure 3A The schematic diagram of the magnetic beads coupled with the negative selection probe used in the composition involved in this article is as shown in Figure 3C As shown, the schematic diagram of the magnetic beads coupled with the first negative selection probe and the second negative selection probe used in the composition involved in this article is as shown Figure 3D shown.

[1001] In some embodiments, the negative selection probe and the 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. In some embodiments, the negative selection probe and the negative selection tag can be at least 65% complementary. The negative selection probe and the negative selection tag can be at least 70% complementary. The negative selection probe and the negative selection tag can be at least 75% complementary. The negative selection probe and the negative selection tag can be at least 80% complementary. The negative selection probe and the negative selection tag can be at least 85% complementary. The negative selection probe and the negative selection tag can be at least 90% complementary. The negative selection probe and the negative selection tag can be at least 95% complementary. The negative selection probe and the negative selection tag can be at least 98% complementary. The negative selection probe and the negative selection tag can be at least 99% complementary. In some embodiments, the negative selection probe and the negative selection tag may be about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% complementary. In some embodiments, the negative selection probe and the negative selection tag may be about 65% complementary. The negative selection probe and the negative selection tag may be about 70% complementary. The negative selection probe and the negative selection tag may be about 75% complementary. The negative selection probe and the negative selection tag may be about 80% complementary. The negative selection probe and the negative selection tag may be about 85% complementary. The negative selection probe and the negative selection tag may be about 90% complementary. The negative selection probe and the negative selection tag may be about 95% complementary. The negative selection probe and the negative selection tag may be about 98% complementary. The negative selection probe and the negative selection tag may be about 99% complementary. In some embodiments, the negative selection probe and the negative selection tag may be 100% complementary.

[1002] In some embodiments, the precursor nucleic acid may have a negative selection tag. In some embodiments, the precursor nucleic acid may have more than one negative selection tag. The negative selection tag needs to be included in the portion removed during the self-splicing process of the precursor nucleic acid. In some embodiments, the precursor nucleic acid is a cRNA enzyme, which forms a circular nucleic acid during the self-splicing process and removes its terminal portion from the circular product. Therefore, in some embodiments, the precursor nucleic acid may have a negative selection tag at its 5' end or 3' end. In some embodiments, the precursor nucleic acid may have a negative selection tag at its 5' end. In some embodiments, the precursor nucleic acid may have a negative selection tag at its 3' end. In some embodiments, under the condition that the negative selection tag at the 5' end and the negative selection tag at the 3' end are simultaneously present, the negative selection tag at the 5' end and the negative selection tag at the 3' end may be the same or different.

[1003] In some embodiments, the precursor nucleic acid may 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 may have two negative selection tags, one at its 5' end and the other at the 3' end. In order to purify the circular nucleic acid produced by the self-splicing of the precursor nucleic acid with 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 a 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, the present invention provides 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 produced by 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 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 and second negative selection probes, 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 may be at least 98% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags may be at least 99% complementary to the first and second negative selection probes, respectively. In some embodiments, the negative selection probes and the negative selection tags may be about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% complementary. In some embodiments, the first and second negative selection tags may be about 65% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags may be about 70% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags may be about 75% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags may be about 80% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags may be about 85% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags may be about 90% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags may be about 95% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags may be about 98% complementary to the first and second negative selection probes, respectively. The first and second negative selection tags may be about 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 may be 100% complementary to the first and second negative selection probes, respectively.

[1006] Although all substantially complementary negative selection probes and negative selection tag pairs can be used to remove precursors, intron fragments and / or other impurities containing negative selection tags from circular nucleic acids without negative selection tags, it is important that the inclusion of the negative selection tag in the precursor nucleic acid has minimal effect on the secondary structure of the precursor nucleic acid so that it does not negatively affect the self-splicing activity of the precursor nucleic acid. It was found that the oligonucleotide negative selection tags that can be used in the compositions and methods disclosed herein should meet the...

Claims

1. 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 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 100% complementary to the first and second negative selection probes, respectively, and are removed during self-splicing of the precursor nucleic acid; The polynucleotide sequence of the first or second negative selection tag of the composition is shown in SEQ ID NO: 7201, and the polynucleotide sequence of the first or second negative selection probe is shown in SEQ ID NO: 7187. wherein the circular nucleic acid is a circular RNA (circRNA), The precursor nucleic acid is an RNA having group II intron self-splicing activity. 2 . The composition according to claim 1 , 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.

3. The composition of claim 1, 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.

4. The composition of claim 3, wherein 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.

5. The composition of claim 1, 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 according to claim 5, wherein 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.

7. The composition of claim 1, 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.

8. A composition according to claim 7, wherein 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.

9. The composition of claim 1, 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.

10. A composition according to claim 9, wherein 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.

11. 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 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.

12. 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.

13. A composition for producing circular nucleic acids, 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%.

14. 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 first negative selection tag and an oligonucleotide second negative selection tag, the first negative selection tag and the second negative selection tag being 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.

15. 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.

16. 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.

17. The composition according to claim 16, 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.

18. The composition according to claim 16 or 17, 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.

19. The composition of claim 18, 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.

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