Construction body for preparing circular RNA, kit, preparation method, circular RNA and application

Abstract

The invention belongs to the technical field of biology, and discloses a construct for preparing circular RNA, a kit, a preparation method, circular RNA and application. The construction body provided by the invention comprises an adenine-rich marker sequence, the length of the marker sequence is 20-120nt, and the content of adenine or complementary basic groups of the adenine is 75-100%. Due to the introduction of the marker sequence, the space structure of the construct can be optimized, the self-shearing catalytic activity of the construct is improved, and the cyclization efficiency is effectively improved; meanwhile, due to the arrangement of the marker sequence, the circular RNA and other non-cyclized RNA can be distinguished through ligo (dT) or ligo (dA), so that positive screening or negative screening of the circular RNA can be carried out by only utilizing a fixing agent comprising ligo (dT) or ligo (dA) in the preparation of the circular RNA, rapid and efficient enrichment of the circular RNA is realized, and the marker sequence has the potential of being applied to large-scale industrial production of the circular RNA and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a construct, a kit, a preparation method, and circular RNA for preparing circular RNA and applications. Background Art

[0002] Circular RNA (circRNA) is a type of single-stranded closed circular RNA molecule that can translate and express any target protein with the assistance of an internal ribosome entry site (IRES) and can be developed as a candidate vaccine or drug.

[0003] The enrichment degree of circRNA is an important factor affecting the preparation of high-purity circRNA by in vitro synthesis. Currently, RNase R is mainly used to digest uncircularized linear RNA to achieve the enrichment of circRNA. However, since the complementary base pairing inside linear RNA will form a complex secondary structure, the RNase R digestion efficiency is low, and a high concentration of RNase R is required. However, RNase R is expensive, and circRNA is not completely resistant to RNase R. High concentrations of RNase R will also lead to the degradation of circRNA, which cannot achieve low-cost and large-scale enrichment of circRNA, and has great limitations.

[0004] Therefore, it is of great significance to obtain an efficient and economical circRNA enrichment method and apply it to the industrial-scale enrichment of circRNA to promote the research and application of mRNA drugs and vaccines. Summary of the invention

[0005] The first object of the present invention is to provide a construct for preparing circular RNA, wherein the construct includes an adenine-rich marker sequence, and the length of the marker sequence is 20 to 120 nt. The introduction of the marker sequence can optimize the spatial structure of the construct and improve its self-cleavage catalytic activity, thereby promoting the circularization process of the construct in in vitro transcription, and effectively improving the circularization efficiency; at the same time, the position of the marker sequence on the construct can be selected so that the marker sequence is retained or not retained in the final circular RNA. At this time, a fixative including oligo (dT) can be used to achieve positive screening or negative screening of circular RNA, thereby achieving rapid and efficient enrichment of circular RNA, which has broad application prospects.

[0006] The second object of the present invention is to provide a method for preparing circular RNA.

[0007] The third object of the present invention is to provide a kit for preparing circular RNA.

[0008] The fourth object of the present invention is to provide a circular RNA.

[0009] The fifth object of the present invention is to provide the use of the above-mentioned construct, kit and / or circular RNA in the preparation of mRNA products.

[0010] Specifically, the construct for preparing circular RNA provided by the present invention includes, in sequence from 5' to 3', 3'-intron, with or without exon 2, IRES sequence, target gene, with or without exon 1 and 5'-intron, and the construct includes a marker sequence, the length of the marker sequence is 20 to 120 nt, and the content of adenine or its complementary base in the marker sequence is 50 to 100%.

[0011] Furthermore, the marker sequence is located at the 5' end of the IRES sequence and / or the 3' end of the target gene, and the marker sequence is retained in the circular RNA prepared by the construct.

[0012] Furthermore, the construct includes, in the 5' to 3' direction, a 3'-intron, with or without exon 2, a marker sequence, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence.

[0013] Furthermore, the construct includes, in the 5' to 3' direction, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, a marker sequence, with or without exon 1 and a 5'-intron connected in sequence.

[0014] Furthermore, the construct includes, in the 5' to 3' direction, a 3'-intron, with or without exon 2, a marker sequence, an IRES sequence, a target gene, a marker sequence, with or without exon 1 and a 5'-intron connected in sequence.

[0015] Furthermore, the marker sequence is located at the 5' end of the 3'-intron, the 3' end of the 5'-intron, inside the 3'-intron and / or inside the 5'-intron, and the circular RNA prepared by the construct does not retain the marker sequence.

[0016] Furthermore, the construct includes a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence along the 5' to 3' direction.

[0017] Furthermore, the construct includes a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence along the 5' to 3' direction, and a marker sequence is also inserted into the 3'-intron and / or the 5'-intron.

[0018] Furthermore, the construct includes, in the 5' to 3' direction, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron and a marker sequence connected in sequence.

[0019] Furthermore, the construct includes a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron and a marker sequence connected in sequence along the 5' to 3' direction, and a marker sequence is also inserted into the 3'-intron and / or the 5'-intron.

[0020] Furthermore, the construct includes a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron and a marker sequence connected in sequence along the 5' to 3' direction.

[0021] Furthermore, the construct includes a 3'-intron, a marker sequence, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron and a marker sequence connected in sequence along the 5' to 3' direction, and a marker sequence is also inserted into the 3'-intron and / or the 5'-intron.

[0022] Furthermore, the construct includes a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence along the 5' to 3' direction, and a marker sequence is inserted into the 3'-intron and / or the 5'-intron.

[0023] Further, the construct is derived from one or more of the type I intron of Microcystis aeruginosa, the type I intron of the cob gene of Fusarium oxysporum, and the type I intron of Anabaena tRNA.

[0024] Furthermore, the nucleotide sequence of the 3'-intron is shown in SEQ ID NO:2, the nucleotide sequence of exon 2 is shown in SEQ ID NO:3, the nucleotide sequence of exon 1 is shown in SEQ ID NO:7, and the nucleotide sequence of the 5'-intron is shown in SEQ ID NO:8.

[0025] Furthermore, the nucleotide sequence of the 3'-intron is shown in SEQ ID NO:12, and the nucleotide sequence of the 5'-intron is shown in SEQ ID NO:13.

[0026] Furthermore, the nucleotide sequence of the 3'-intron is shown in SEQ ID NO:14, the nucleotide sequence of exon 2 is shown in SEQ ID NO:15, the nucleotide sequence of exon 1 is shown in SEQ ID NO:16, and the nucleotide sequence of the 5'-intron is shown in SEQ ID NO:17.

[0027] Furthermore, the IRES sequence is selected from one or more of a Coxsackie virus IRES sequence, an encephalomyocarditis virus IRES sequence, a polio virus IRES sequence, a Kaposi's sarcoma virus IRES sequence and a herpes virus IRES sequence.

[0028] Furthermore, the nucleotide sequence of the IRES sequence is shown in SEQ ID NO:5.

[0029] The preparation method of the circular RNA provided by the present invention comprises: taking the above-mentioned construct for in vitro transcription to obtain an in vitro transcription product; taking the in vitro transcription product for oligo (dW) enrichment treatment to obtain the circular RNA.

[0030] Furthermore, the oligo(dW) enrichment treatment method is selected from oligo(dT) enrichment treatment and / or oligo(dA) enrichment treatment.

[0031] The kit for preparing circular RNA provided by the present invention comprises the above construct and an oligo (dW) fixative.

[0032] The circular RNA provided by the present invention is prepared by the above circular RNA preparation method.

[0033] The present invention also provides the use of the above construct, kit and / or circular RNA in preparing RNA products. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A schematic diagram of the structure of the construct provided in Example 1 of the present invention and its cyclization process;

[0035] Figure 2 A schematic diagram of the structure of the construct provided in Example 4 of the present invention and its cyclization process;

[0036] Figure 3 A schematic diagram of the structure of the construct provided in Example 5 of the present invention and its cyclization process;

[0037] Figure 4 A schematic diagram of the structure of the construct provided for Comparative Example 1 and its cyclization process;

[0038] Figure 5A schematic diagram of the structure of the construct provided in Example 13 of the present invention and its cyclization process;

[0039] Figure 6 A schematic diagram of the structure of the construct provided in Example 14 of the present invention and its cyclization process;

[0040] Figure 7 This is a schematic diagram of the structure of the construct provided in Example 15 of the present invention and its cyclization process. DETAILED DESCRIPTION

[0041] Based on the existing known construct structure with self-cleavage catalytic activity for preparing circular RNA, the inventors of the present invention have found through in-depth and extensive research that, for the purpose of improving the cyclization efficiency of the construct and realizing large-scale enrichment of circular RNA, introducing a sequence with a length of 20 to 120 nt and rich in adenine or its complementary base into the construct can optimize the spatial structure of the construct, thereby improving the self-cleavage catalytic activity of the construct, and the introduced sequence can be used as a target sequence for separating circular RNA from other non-circularized RNAs. Circular RNA and other non-circularized RNAs can be separated by only using an oligo (dW) fixative, thereby realizing enrichment of circular RNA, thereby obtaining the technical solution of the present invention.

[0042] In the present invention, the construct specifically includes 3'-intron, presence or absence of exon 2, IRES sequence, target gene, presence or absence of exon 1 and 5'-intron in sequence along the 3' to 5' direction.

[0043] In the present invention, the "exon 2 or not" and "exon 1 or not" specifically include the following situations: the construct may have both exon 2 and exon 1; or, the construct only includes exon 1; or, the construct only includes exon 2; or, the construct does not include exon 2 and exon 1. That is, exon 2 and exon 1 in the construct are not necessarily present fragments.

[0044] In the present invention, the construct includes a marker sequence, and the length of the marker sequence is 20 to 120 nt, such as 20 nt, 23 nt, 28 nt, 30 nt, 35 nt, 40 nt, 50 nt, 80 nt, 100 nt, 120 nt or any value therebetween; the content of adenine or its complementary base in the marker sequence is 50 to 100%, such as 50%, 60%, 70%, 75%, 78%, 80%, 87.3%, 90%, 100% or any value therebetween. Wherein, the complementary base of adenine refers to thymine and / or uracil.

[0045] In the present invention, the position of the marker sequence on the construct is not particularly limited, and can be, but is not limited to, one or more positions selected from the group consisting of: inside the 3'-intron, at the 5' end of the 3'-intron, between exon 2 and the IRES sequence, between the target gene and exon 1, inside the 5'-intron, and at the 3' end of the 5'-intron.

[0046] In the present invention, the marker sequence is preferably located at the 5' end of the IRES sequence and / or the 5' end of the target gene. In this case, the circular RNA prepared by the construct retains the marker sequence, and positive screening of the marker sequence can be performed to achieve enrichment of the circular RNA.

[0047] In some specific embodiments, the construct includes, in sequence from 5' to 3', a 3'-intron, with or without exon 2, a marker sequence, an IRES sequence, a target gene, with or without exon 1, and a 5'-intron.

[0048] In some specific embodiments, the construct includes, in sequence from 5' to 3', a 3'-intron, with or without exon 2, an IRES sequence, a target gene, a marker sequence, with or without exon 1, and a 5'-intron.

[0049] In some specific embodiments, the construct includes, in sequence from 5' to 3', a 3'-intron, with or without exon 2, a marker sequence, an IRES sequence, a target gene, a marker sequence, with or without exon 1 and a 5'-intron.

[0050] In the present invention, the marker sequence is preferably located at the 5' end of the 3'-intron, the 3' end of the 5'-intron, the interior of the 3'-intron and / or the interior of the 5'-intron, that is, the marker sequence is located in the cut-off region (or non-circularized region) in the construct. At this time, the circular RNA prepared by the construct does not retain the marker sequence, and the enrichment of the circular RNA can be achieved by negative screening of the marker sequence.

[0051] In some specific embodiments, the construct includes a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence along the 5' to 3' direction, and a marker sequence is inserted in the 3'-intron.

[0052] In some specific embodiments, the construct includes a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence along the 5' to 3' direction, and a marker sequence is inserted into the 5'-intron. In some specific embodiments, the construct includes a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence along the 5' to 3' direction, and a marker sequence is inserted into the 3'-intron and the 5'-intron. Wherein, the marker sequences inserted in the 3'-intron and the 5'-intron can be completely identical, partially identical or completely different.

[0053] In some specific embodiments, the construct includes a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron linked sequentially along the 5' to 3' direction.

[0054] In some specific embodiments, the construct includes a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence along the 5' to 3' direction, and a marker sequence is also inserted into the 3'-intron and / or the 5'-intron.

[0055] In some specific embodiments, the construct includes, in the 5' to 3' direction, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron, and a marker sequence connected sequentially.

[0056] In some specific embodiments, the construct includes a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron and a marker sequence connected in sequence along the 5' to 3' direction, and a marker sequence is also inserted into the 3'-intron and / or the 5'-intron.

[0057] In some specific embodiments, the construct includes a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron and a marker sequence connected sequentially along the 5' to 3' direction.

[0058] In some specific embodiments, the construct includes a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron and a marker sequence connected in sequence along the 5' to 3' direction, and a marker sequence is also inserted into the 3'-intron and / or the 5'-intron.

[0059] It should be noted that in the above-mentioned specific embodiments in which a marker sequence is further inserted into the 3'-intron and / or the 5'-intron, the sequence of the marker sequence and the insertion position may be completely identical, partially identical or completely different.

[0060] In the present invention, the construct has type I intron self-splicing catalytic activity, and can be specifically derived from but not limited to: one or more of the type I intron of Microcystis aeruginosa, the type I intron of the cob gene of Fusarium oxysporum, and the type I intron of Anabaena tRNA.

[0061] In some specific embodiments, the nucleotide sequence of the 3'-intron is preferably as shown in SEQ ID NO:2, the nucleotide sequence of exon 2 is preferably as shown in SEQ ID NO:3, the nucleotide sequence of exon 1 is preferably as shown in SEQ ID NO:7, and the nucleotide sequence of the 5'-intron is as shown in SEQ ID NO:8. At this time, the construct has excellent circularization efficiency, and only a small amount of exogenous sequence is retained in the resulting circular RNA. In some more preferred embodiments, the 3'-intron is as shown in SEQ ID NO:12, and the 5'-intron is as shown in SEQ ID NO:13. At this time, while the construct has good circularization efficiency, only a very small amount of exogenous nucleotides remain in the resulting circRNA. In some specific embodiments, the nucleotide sequence of the 3'-intron is as shown in SEQ ID NO:14, the nucleotide sequence of exon 2 is as shown in SEQ ID NO:15, the nucleotide sequence of exon 1 is as shown in SEQ ID NO:16, and the nucleotide sequence of the 5'-intron is as shown in SEQ ID NO:17. At this time, the construct has excellent circularization efficiency, and only a small amount of exogenous sequences are retained in the resulting circular RNA.

[0062] In the present invention, the specific type of the IRES is not particularly limited. The IRES sequence refers to an endogenous ribosome entry site sequence, which allows ribosomes to directly bind to the internal position of mRNA and initiate translation without relying on traditional promoters or regulatory sequences. Specific examples include but are not limited to: one or more of a Coxsackievirus IRES sequence, an encephalomyocarditis virus IRES sequence, a poliovirus IRES sequence, a Kaposi's sarcoma virus IRES sequence, and a herpes virus IRES sequence.

[0063] In some specific embodiments, the nucleotide sequence of the IRES sequence is preferably as shown in SEQ ID NO:5.

[0064] Based on the above construct, the present invention also provides a method for preparing circular RNA, which specifically comprises: taking the construct for in vitro transcription to obtain an in vitro transcription product; taking the in vitro transcription product for oligo (dT) enrichment treatment to obtain the circular RNA.

[0065] In the present invention, the construct has excellent self-cleavage catalytic activity, so it can achieve circularization during the in vitro transcription process to obtain circular RNA.

[0066] In the present invention, the in vitro transcription is not particularly limited. The in vitro transcription refers to the process of converting a DNA template into RNA in vitro (i.e., in an environment outside the organism). Those skilled in the art can make adaptive choices from the reagents and conditions disclosed in the prior art as needed, or directly use a commercial in vitro transcription kit to conduct experiments.

[0067] In the present invention, the oligo(dW) enrichment treatment refers to utilizing the specific recognition effect of oligo(dW) and the marker sequence to separate circular RNA from other RNA molecules to achieve the enrichment of circular RNA in in vitro transcription products, specifically including oligo(dT) enrichment treatment and / or oligo(dA) enrichment treatment.

[0068] In some specific embodiments, specific examples of the oligo(dW) enrichment treatment include but are not limited to: one or more of oligo(dW) chromatography enrichment treatment, oligo(dW) magnetic bead enrichment treatment and oligo(dW) spin column enrichment treatment.

[0069] Based on the above-mentioned method for preparing circular RNA, the present invention also provides a kit for preparing circular RNA, which includes the raw materials for preparing circular RNA, namely the above-mentioned construct and an oligo(dW) fixative.

[0070] In the present invention, the oligo(dW) fixative is a substance including oligo(dW) to achieve specific recognition of the labeled sequence, and specific examples include but are not limited to: one or more of oligo(dA) fibers, oligo(dT) fibers, oligo(dA) magnetic beads and oligo(dT) magnetic beads.

[0071] The present invention also provides a circular RNA, which is prepared by the circular RNA preparation method.

[0072] The present invention also provides the use of the above construct, kit and / or circular RNA in the preparation of RNA products. Specific examples of the RNA products include but are not limited to: RNA drugs and / or RNA vaccines.

[0073] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0074] The constructs involved in the following examples and comparative examples were synthesized by a chemical synthesis company.

[0075] The reagents and sources involved in the following examples and comparative examples are:

[0076] T7 High-Efficiency RNA Synthesis Kit (NEB, catalog number E2040S);

[0077] oligo dT chromatography column (BIA / Sartorius, catalog number 311.1219-2);

[0078] oligo dA chromatography column (Changzhou Tiandirenhe Biotechnology, custom column);

[0079] Agilent DNF-471 RNA Kit (15 nt) (Agilent, catalog number DNF-471-0500);

[0080] The chromatographic column is PLRP-S (Agilent, catalog number PL1912-3803).

[0081] Example 1

[0082] This embodiment provides a construct, referring to Figure 1 The construct includes a T7 promoter, a 3'-intron, an exon 2, a marker sequence, an IRES sequence, a GFP encoding gene, an exon 1 and a 5'-intron connected in sequence along the 5'-3' direction. The nucleotide sequences of each fragment are shown in Table 1.

[0083] Table 1.

[0084]

[0085]

[0086] In the construct provided in this example, the length of the marker sequence is 70 nt, and the adenine content is 85.71%.

[0087] Example 2

[0088] The construct provided in this example is basically the same as that in Example 1, except that the length of the marker sequence is 30 nt and the adenine content is 100%. The specific nucleotide sequence is shown in Table 2. The other conditions are the same to obtain the construct.

[0089] Table 2.

[0090] Snippet SEQ ID NO: Nucleotide sequence Marker sequence II 9 AAAAAAAAAAAAAAAAAAAAAAAAAAA

[0091] Example 3

[0092] The construct provided in this example is basically the same as that in Example 1, except that the length of the marker sequence is 100 nt and the adenine content is 90.00%. The specific nucleotide sequence is shown in Table 3. The other conditions are the same to obtain the construct.

[0093] Table 3.

[0094]

[0095] Example 4

[0096] This embodiment provides a construct, referring to Figure 2 The construct includes a T7 promoter, a 3'-intron, an exon 2, an IRES sequence, a GFP encoding gene, a marker sequence, an exon 1 and a 5'-intron connected in sequence along the 5'-3' direction, and the nucleotide sequence of each fragment is specifically shown in Table 1.

[0097] Example 5

[0098] This embodiment provides a construct, referring to Figure 3 The construct includes a T7 promoter, a 3'-intron, an exon 2, a marker sequence, an IRES sequence, a GFP encoding gene, a marker sequence, an exon 1 and a 5'-intron connected in sequence along the 5'-3' direction. The nucleotide sequences of each fragment are specifically shown in Table 1.

[0099] Example 6

[0100] The construct provided in this example is basically the same as that in Example 1, except that the length of the marker sequence is 30 nt and the content of thymine is 100%. The specific nucleotide sequence is shown in Table 4. The other conditions are the same to obtain the construct.

[0101] Table 4.

[0102] Snippet SEQ ID NO: Nucleotide sequence Marker sequence IV 11 TTTTTTTTTTTTTTTTTTTTTTTTTTTT

[0103] Example 7

[0104] The construct provided in this example is substantially the same as that in Example 1, except that the construct does not include exon 2 and exon 1. The other conditions are the same to obtain the construct.

[0105] Example 8

[0106] The construct provided in this example is basically the same as that in Example 7, except that the nucleotide sequences of the 3'-intron and the 5'-intron are specifically shown in Table 5, and the other conditions are the same to obtain the construct.

[0107] Table 5.

[0108]

[0109] Example 9

[0110] The construct provided in this example is basically the same as that in Example 1, except that the nucleotide sequences of the 3'-intron, exon 2, exon 1 and 5'-intron in the construct are different, as shown in Table 6. The other conditions are the same to obtain the construct.

[0111] Table 6.

[0112]

[0113]

[0114] Example 10

[0115] The construct provided in this example is basically the same as that in Example 4, except that the nucleotide sequences of the 3'-intron, exon 2, exon 1 and 5'-intron in the construct are different, as shown in Table 6. The other conditions are the same to obtain the construct.

[0116] Embodiment 11

[0117] The construct provided in this example is basically the same as that in Example 5, except that the nucleotide sequences of the 3'-intron, exon 2, exon 1 and 5'-intron in the construct are different, as shown in Table 6. The other conditions are the same to obtain the construct.

[0118] Comparative Example 1

[0119] The construct provided in this comparative example is substantially the same as that in Example 1, except that Figure 4 , the construct does not include a marker sequence, and other conditions are the same to obtain a construct.

[0120] Comparative Example 2

[0121] The construct provided by this comparative example is basically the same as that of Example 7, except that the marker sequence is not included in the construct. The construct is obtained under the same other conditions.

[0122] Comparative Example 3

[0123] The construct provided in this example is basically the same as that in Example 8, except that the marker sequence is not included in the construct. The other conditions are the same to obtain the construct.

[0124] Example 12

[0125] This example is used to illustrate a method for preparing circular RNA. In the preparation method, the constructs provided in Examples 1 to 11 and Comparative Examples 1 to 3 are used for in vitro transcription and oligo(dW) enrichment treatment, specifically including:

[0126] (1) Use T7 high-efficiency RNA synthesis kit was used to perform in vitro transcription on the construct according to the instructions to obtain in vitro transcription products.

[0127] (2) Taking a portion of the in vitro transcription product, precipitating the in vitro transcription product using a 3 M LiCl solution to obtain a precipitated product, detecting the precipitated product using capillary electrophoresis (CE), and calculating the cyclization efficiency (CE, %) according to the following formula.

[0128] Circularization efficiency (CE, %) = (circular RNA + open-circular RNA) peak area / total area of ​​all peaks

[0129] The CE conditions include: using an Agilent 5200 fragment analyzer system with an Agilent DNF-471 RNA Kit (15 nt), and using the DNF-471 analysis program to detect the precipitated product.

[0130] (3) Using an oligo dW chromatography column (oligo dT chromatography column or oligo dA chromatography column) to enrich the in vitro transcription products, and eluting the oligo dW chromatography column with DEPC water, collecting the eluate to obtain the enriched product, and detecting the enriched product by reverse phase high performance liquid chromatography (RP-HPLC), the circular RNA ratio (RP-HPLC, %) is calculated according to the following formula.

[0131] Circular RNA ratio (RP-HPLC, %) = Circular RNA peak area / Total peak area Wherein, the conditions of RP-HPLC include: chromatographic column is PLRP-S (8μm, 150x2.1mm); mobile phase A is: 0.1M n-hexylamine acetate, mobile phase B is: 0.1M n-hexylamine acetate / 50% acetonitrile; flow rate is 0.5mL / min, injection volume is 12μL, column temperature is 60℃, sample plate temperature is 25℃, and detection wavelength of detector is 260nm; separation gradient program is as follows: start: 87% (v / v) mobile phase A + 13% (v / v) mobile phase B; from 21min: 20% (v / v) mobile phase A + 80% (v / v) mobile phase B; from 44.8min: mobile phase B. The results are shown in Table 7.

[0132] Table 7.

[0133] Group Ring formation efficiency (CE,%) Circular RNA percentage (RP-HPLC,%) Example 1 79.1 90.2 Example 2 78.0 89.7 Example 3 77.8 90.1 Example 4 84.3 93.2 Example 5 83.5 91.1 Example 6 75.2 86.3 Example 7 21.3 60.1 Example 8 22.1 59.9 Example 9 78.5 88.6 Example 10 79.2 88.9 Embodiment 11 79.6 88.7 Comparative Example 1 28.7 27.7** Comparative Example 2 11.7 10.8** Comparative Example 3 20.4 19.3**

[0134] ** The constructs provided in Comparative Examples 1 to 3 are unlabeled and cannot be subjected to oligo dT chromatography to enrich circular RNA. Therefore, the measured circular RNA ratio is the ratio of circular RNA in the LiCl precipitation product.

[0135] From the test results shown in Table 7, it can be seen that compared with Comparative Examples 1 to 3, the marker sequence with high adenine content or high thymine content is added to the construct provided in Examples 1 to 11 of the present invention, and the circularization efficiency is significantly improved, and the introduction of the marker sequence can not only effectively improve the circularization series of the construct, but also can be used as a positive screening marker for oligo dW chromatography column treatment, thereby obtaining a circular RNA with high purity. That is, the introduction of the marker sequence can not only promote the circularization of the construct during in vitro transcription and improve the circularization efficiency; and the marker sequence is retained in the circular RNA, so that large-scale enrichment thereof can be achieved by methods such as oligo dT chromatography column.

[0136] Embodiment 13

[0137] This embodiment provides a construct, referring to Figure 5 The construct includes a T7 promoter, a marker sequence, a 3'-intron, exon 2, an IRES sequence, a GFP encoding gene, exon 1 and a 5'-intron connected in sequence along the 5'-3' direction, and the nucleotide sequence of each fragment is specifically shown in Table 1.

[0138] Embodiment 14

[0139] This embodiment provides a construct, referring to Figure 6 The construct includes a T7 promoter, a 3'-intron, an exon 2, an IRES sequence, a GFP encoding gene, an exon 1, a 5'-intron and a marker sequence connected in sequence along the 5'-3' direction, and the nucleotide sequence of each fragment is specifically shown in Table 1.

[0140] Embodiment 15

[0141] This embodiment provides a construct, referring to Figure 7 The construct includes a T7 promoter, a marker sequence, a 3'-intron, exon 2, an IRES sequence, a GFP encoding gene, exon 1, a 5'-intron and a marker sequence connected in sequence along the 5'-3' direction, and the nucleotide sequence of each fragment is specifically shown in Table 1.

[0142] Example 16

[0143] This example is used to illustrate a method for preparing circular RNA. In this preparation method, the constructs provided in Examples 13 to 15 are used for in vitro transcription and oligo(dT) enrichment treatment, which specifically includes:

[0144] (1) Use The construct was transcribed in vitro using the T7 High-Efficiency RNA Synthesis Kit (NEB, catalog number E2040S) according to the instructions to obtain an in vitro transcription product.

[0145] (2) Taking a portion of the in vitro transcription product, precipitating the in vitro transcription product using a 3 M LiCl solution to obtain a precipitated product, detecting the precipitated product using capillary electrophoresis (CE), and calculating the cyclization efficiency (CE, %) according to the following formula.

[0146] Circularization efficiency (CE, %) = (circular RNA + open-circular RNA) peak area / total area of ​​all peaks

[0147] (3) Using an oligo dW chromatography column (oligo dT chromatography column, oligo dA chromatography column or oligo dC chromatography column) to enrich the in vitro transcription product, and eluting the oligo dW chromatography column with DEPC water, collecting the eluate to obtain the enriched product, and detecting the enriched product by reverse phase high performance liquid chromatography (RP-HPLC), the circular RNA ratio (RP-HPLC, %) is calculated according to the following formula.

[0148] Circular RNA ratio (RP-HPLC, %) = Circular RNA peak area / Total peak area

[0149] The detection methods and conditions used in CE and RP-HPLC were consistent with those in Example 10. The results are shown in Table 8.

[0150] Table 8.

[0151] Group Ring formation efficiency (CE,%) Circular RNA percentage (RP-HPLC,%) Embodiment 15 30.6 82.2 Example 16 32.5 81.6 Embodiment 17 37.1 83.4 Comparative Example 1 28.7 27.7**

[0152] **Comparative Example 1 provides a construct without a label, and oligo dT chromatography cannot be performed to enrich circular RNA. Therefore, the measured circular RNA ratio is the ratio of circular RNA in the LiCl precipitation product.

[0153] From the test results shown in Table 8, it can be seen that compared with Comparative Example 1, the marker sequence with a high adenine content added to the construct provided in Examples 13 to 15 of the present invention can be used as a negative screening marker for oligo dT chromatography enrichment, and the proportion of circular RNA in the obtained enriched product is more than 80%. That is, the introduction of the marker sequence in the cut region of the construct can make the resulting circular RNA free of marker sequence residues, and the uncircularized linear RNA or the cut sequence contains the marker sequence, and large-scale enrichment of circular RNA can be achieved by methods such as oligo dT chromatography columns.

[0154] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A construct for preparing circular RNA, the construct comprising, in sequence from 5' to 3' direction: 3'-intron, with or without exon 2, IRES sequence, target gene, with or without exon 1 and 5'-intron, characterized in that the construct includes a marker sequence, the length of the marker sequence is 20 to 120 nt, and the content of adenine or its complementary base in the marker sequence is 50 to 100%.

2. The construct for preparing circular RNA according to claim 1, characterized in that The marker sequence is located at the 5' end of the IRES sequence and / or the 3' end of the target gene, and the marker sequence is retained in the circular RNA prepared by the construct.

3. The construct for preparing circular RNA according to claim 2, characterized in that The construct includes, in the direction from 5' to 3', a 3'-intron, with or without exon 2, a marker sequence, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence; Optionally, the construct comprises, in sequence along the 5' to 3' direction, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, a marker sequence, with or without exon 1, and a 5'-intron; Optionally, the construct comprises, in order from 5' to 3' direction, a 3'-intron, with or without exon 2, a marker sequence, an IRES sequence, a gene of interest, a marker sequence, with or without exon 1 and a 5'-intron.

4. The construct for preparing circular RNA according to claim 1, characterized in that The marker sequence is located at the 5' end of the 3'-intron, the 3' end of the 5'-intron, inside the 3'-intron and / or inside the 5'-intron, and the circular RNA prepared by the construct does not retain the marker sequence.

5. The construct for preparing circular RNA according to claim 4, characterized in that The construct includes a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence along the 5' to 3' direction; Optionally, the construct comprises a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron connected in sequence along the 5' to 3' direction, and a marker sequence is further inserted into the 3'-intron and / or the 5'-intron; Optionally, the construct comprises, in the 5' to 3' direction, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron, and a marker sequence connected in sequence; Optionally, the construct comprises a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron and a marker sequence connected in sequence along the 5' to 3' direction, and a marker sequence is further inserted into the 3'-intron and / or the 5'-intron; Optionally, the construct comprises, in the 5' to 3' direction, a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron, and a marker sequence connected in sequence; Optionally, the construct comprises a marker sequence, a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1, a 5'-intron and a marker sequence connected in sequence along the 5' to 3' direction, and a marker sequence is further inserted into the 3'-intron and / or the 5'-intron; Optionally, the construct comprises a 3'-intron, with or without exon 2, an IRES sequence, a target gene, with or without exon 1 and a 5'-intron sequentially linked along the 5' to 3' direction, and a marker sequence is inserted into the 3'-intron and / or the 5'-intron.

6. The construct for preparing circular RNA according to claim 1, characterized in that The construct is derived from one or more of a Microcystis aeruginosa type I intron, a Fusarium oxysporum cob gene type I intron, and an Anabaena tRNA type I intron; Optionally, the nucleotide sequence of the 3'-intron is shown in SEQ ID NO:2, the nucleotide sequence of exon 2 is shown in SEQ ID NO:3, the nucleotide sequence of exon 1 is shown in SEQ ID NO:7, and the nucleotide sequence of the 5'-intron is shown in SEQ ID NO:8; Optionally, the nucleotide sequence of the 3'-intron is as shown in SEQ ID NO: 12, and the nucleotide sequence of the 5'-intron is as shown in SEQ ID NO: 13; Optionally, the nucleotide sequence of the 3'-intron is shown as SEQ ID NO:14, the nucleotide sequence of exon 2 is shown as SEQ ID NO:15, the nucleotide sequence of exon 1 is shown as SEQ ID NO:16, and the nucleotide sequence of the 5'-intron is shown as SEQ ID NO:

17.

7. The construct for preparing circular RNA according to claim 1, characterized in that The IRES sequence is selected from one or more of a Coxsackievirus IRES sequence, an encephalomyocarditis virus IRES sequence, a poliovirus IRES sequence, a Kaposi's sarcoma virus IRES sequence, and a herpes virus IRES sequence; Optionally, the nucleotide sequence of the IRES sequence is as shown in SEQ ID NO:

5.

8. A method for preparing circular RNA, characterized in that: The preparation method comprises: taking the construct described in any one of claims 1 to 7 and performing in vitro transcription to obtain an in vitro transcription product; The in vitro transcription product is subjected to oligo(dW) enrichment treatment to obtain the circular RNA.

9. The method for preparing circular RNA according to claim 8, characterized in that: The oligo(dW) enrichment treatment method is selected from oligo(dT) enrichment treatment and / or oligo(dA) enrichment treatment.

10. A kit for preparing circular RNA, characterized in that: The kit comprises the construct according to any one of claims 1 to 7 and an oligo(dW) fixing agent.

11. A circular RNA, characterized in that The circular RNA is prepared by the circular RNA preparation method according to claim 8 or 9.

12. Use of the construct according to any one of claims 1 to 7, the kit according to claim 10 and / or the circular RNA according to claim 11 in the preparation of RNA products.

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