A sequence, sequence combination for improving the expression efficiency of circular RNA and their uses

Abstract

The present invention discloses a sequence, sequence combination, and use thereof for improving the expression efficiency of circular RNA. By constructing a circular RNA expression plasmid using the sequence or sequence combination, the efficiency of expressing circular RNA using the plasmid can be significantly improved. The sequence, sequence combination, and circular RNA expression plasmid disclosed in the present invention can be used to prepare circular RNA drugs. By delivering the drugs into cells, the expression efficiency of the target circular RNA is improved, which is of great significance for the treatment of diseases caused by low circular RNA expression and the research and development of corresponding drugs.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biomedicine technology, and specifically relates to a sequence, a sequence combination and a use thereof for improving the expression efficiency of circular RNA. Background Art

[0002] Circular RNAs (circRNAs) are a large class of single-stranded circular RNA molecules that form loops through covalent bonds. They are common products of eukaryotic transcription and post-transcriptional RNA processing. By interacting with microRNAs (miRNAs) or proteins, circRNAs play important roles in many physiological functions and human diseases, particularly cancer, cardiovascular disease, and neurodegenerative diseases. CircRNAs lack either a 5' cap or a 3' polyadenylated tail, making them more resistant to exonuclease degradation and exhibiting greater stability and a longer half-life than mRNA. Due to the important physiological functions and greater stability of circRNAs, disease treatment strategies targeting circRNAs are increasingly being explored.

[0003] Currently, there are two main methods for manipulating circular RNA expression: one that reduces circular RNA expression through RNA interference (RNA interference), and the other that increases circular RNA expression using circular RNA overexpression plasmids. The specific method chosen is determined by the role of circular RNA in cellular life processes and the development of diseases. Studies have shown that many human diseases, such as cancer and cardiovascular disease, are associated with the lack or underexpression of corresponding circular RNAs. Therefore, research on circular RNA expression technologies and improving the efficiency of circular RNA expression in cells is of great medical significance.

[0004] Eukaryotic circular RNAs are primarily formed by a non-canonical backsplicing mechanism, where a downstream splice donor site attacks an upstream splice acceptor site. Backsplicing is driven by paired reverse complementary sequences within the introns flanking the circular exon and by certain RNA-binding proteins. This mechanism is currently being exploited in the design of circular RNA expression plasmids and is a standard method for overexpressing circular RNAs. Adenoviruses, lentiviruses, nanoparticles, and exosomes can all be used as delivery vehicles for circular RNA expression plasmids to achieve disease treatment. For example, circular RNA overexpression plasmids can be obtained by inserting the circular RNA looping sequence into a backbone vector. While this can improve circular RNA expression levels to a certain extent, the improvement is very limited. Therefore, it is essential to explore strategies to further improve the efficiency of circular RNA expression based on existing circular RNA overexpression plasmids, which is of great significance for the treatment of diseases that rely on circular RNAs. Summary of the Invention

[0005] In view of this, the primary purpose of the present invention is to provide a sequence that improves the expression efficiency of circular RNA, as well as a sequence combination containing the sequence. The present invention reports for the first time that the use of the ZC3H14 protein binding sequence or a sequence combination containing the sequence can significantly improve the expression efficiency of circular RNA in vitro, thereby providing new strategies and methods for the research and application of circular RNA-related drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a sequence for improving the expression efficiency of circular RNA, which is a ZC3H14 protein binding sequence.

[0008] Herein, the circular RNA refers to a non-coding circular RNA or a protein-coding circular RNA that relies on IRES to initiate translation. In some specific embodiments of the present invention, the circular RNA can be circLARP1B, circSDHB, circSMARCA5, circPPFIA1 or circFBXW4.

[0009] Herein, the ZC3H14 protein binding sequence is any sequence identified by iCLIP experiments that can bind to the ZC3H14 protein. In the present invention, any of the above sequences can achieve the effect of improving the expression efficiency of circular RNA.

[0010] In some specific embodiments of the present invention, the ZC3H14 protein binding sequence has a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, which are respectively designated as UTR1 and UTR2 herein.

[0011] The second aspect of the present invention provides a sequence combination for improving the expression efficiency of circular RNA, wherein the sequence combination sequentially comprises the ring-forming sequence of the target circular RNA and the ZC3H14 protein binding sequence described in the first aspect of the present invention.

[0012] Wherein, the cyclic sequence of the target circular RNA includes the upstream promoting cyclic sequence, the cyclic exon sequence and the downstream promoting cyclic sequence of the target circular RNA in sequence, wherein the ZC3H14 protein binding sequence is connected to the 3' end of the downstream promoting cyclic sequence of the target circular RNA. In some specific implementation cases, the upstream and downstream promoting cyclic sequences include the upstream and downstream flanking intron sequences of the target circular RNA. In other specific implementation cases, the upstream and downstream promoting cyclic sequences include the upstream and downstream exons and the upstream and downstream partial intron sequences. It is understandable that the cyclic sequence of the circular RNA can be obtained by consulting or calculating by methods known to those skilled in the art without particular limitation.

[0013] Preferably, in the present invention, the sequence combinations described above are linked sequentially and continuously without gaps.

[0014] The third aspect of the present invention provides a circular RNA expression plasmid, wherein the circular RNA expression plasmid contains the sequence combination described in the second aspect of the present invention.

[0015] It is understandable that the circular RNA expression plasmid also includes a backbone vector, and the circular RNA expression plasmid can be constructed by inserting the sequence combination described in the second aspect of the present invention into the multiple cloning site of the backbone vector.

[0016] In this article, the backbone vector is a self-replicating DNA molecule used to bring the target gene into the host cell. It can provide the transport of the target gene, provide replication or integration capabilities, and provide conditions for the amplification or expression of the target gene. In the present invention, the backbone vector can specifically be a eukaryotic expression vector, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes virus vector, etc., but is not limited thereto. In some specific implementation cases of the present invention, the backbone vector used is pCDNA3.0.

[0017] The circular RNA expression plasmid constructed in the present invention is expressed in cells and allows the circular sequence of the target circular RNA to form a ring, thereby improving the expression efficiency of the target circular RNA in the cells.

[0018] The fourth aspect of the present invention is a method for constructing a circular RNA expression plasmid as described in the third aspect of the present invention, wherein the upstream cyclization-promoting sequence, cyclization exon sequence, downstream cyclization-promoting sequence and ZC3H14 protein binding sequence of the target circular RNA are inserted into the multiple cloning site of the backbone vector in this order to construct a circular RNA expression plasmid.

[0019] The specific multiple cloning site and sequence insertion method can be obtained by those skilled in the art based on known methods and data, and will not be described in detail here.

[0020] The fifth aspect of the present invention provides a method for improving the expression efficiency of circular RNA for non-therapeutic or diagnostic purposes, comprising the step of expressing the circular RNA expression plasmid described in the third aspect of the present invention in a cell.

[0021] Specifically, the constructed circular RNA expression plasmid is delivered to the target cells for expression in the target cells. In the present invention, the cells are preferably in vitro cells. The delivery methods described above can be carried out using methods known to those skilled in the art, and specific examples include, but are not limited to, liposomes, adenovirus, lentivirus, nanoparticles, and exosomes.

[0022] A sixth aspect of the present invention provides a nucleic acid drug comprising the circular RNA expression plasmid described in the third aspect of the present invention. This nucleic acid drug has the effect of treating a disease, wherein the disease referred to herein is any disease caused by the absence or low expression of circular RNA, such as cancer, cardiovascular disease, neurological disease, diabetes, etc.

[0023] It is understood that the nucleic acid drug described herein also includes any pharmaceutically acceptable carrier and / or adjuvant. In some specific implementation cases of the present invention, the pharmaceutically acceptable adjuvant and / or carrier include but are not limited to at least one of a diluent, an adhesive, a surfactant, an adsorption carrier, a lubricant, a filler, and a disintegrant. The selection of specific adjuvants and / or carriers can be carried out according to the dosage form of the drug. When specifically selected, they can be adapted to the active substance, or can effectively improve the stability and solubility of the active ingredient contained in the drug, or can change the release rate and absorption rate of the active substance, thereby ensuring or enhancing the administration effect of the active ingredient. In the present invention, the dosage form of the drug is not particularly limited, and any dosage form known in the art that is conducive to administration can be used without specific limitations. Being conducive to administration described herein refers to being able to improve therapeutic effects or improve bioavailability or reduce toxic and side effects or improve patient adaptability, etc.

[0024] Furthermore, the circular RNA expression plasmid contained in the nucleic acid drug is an effective dose, where the effective dose refers to the minimum dose that can stably express the circular RNA and increase the expression level and is within a safe range (generally meaning that no adverse reactions will occur).

[0025] Furthermore, the nucleic acid drug can be administered in a manner commonly used in the art according to the specific disease, and the specific dosage and frequency of administration are determined according to the subject's condition, the type of disease, and the extent of the disease. These can be determined by those skilled in the art through methods known in the art or clinical trials, and therefore there are no special limitations.

[0026] The seventh aspect of the present invention discloses at least one of the following uses of the sequence described in the first aspect of the present invention or the sequence combination described in the second aspect of the present invention:

[0027] (1) Improve the expression efficiency of circular RNA;

[0028] (2) preparing circular RNA expression plasmids;

[0029] (3) Preparation of nucleic acid drugs for treating diseases.

[0030] The eighth aspect of the present invention discloses at least one of the following uses of the circular RNA expression plasmid described in the third aspect of the present invention:

[0031] (1) Improve the expression efficiency of circular RNA;

[0032] (2) Preparation of nucleic acid drugs for treating diseases.

[0033] Beneficial effects of the present invention:

[0034] The ZC3H14 protein binding sequence and sequence combinations containing it in the present invention can significantly improve the expression efficiency of circular RNA. The circular RNA expression plasmid constructed based on this can express efficiently and stably, and the expression efficiency of its circular RNA is significantly improved compared to known circular RNA expression plasmids. In addition, the circular RNA expression plasmid constructed in the present invention can be used in combination with existing circular RNA expression plasmids to further improve the expression efficiency of circular RNA. The present invention can provide new drug regimens and strategies for the treatment of diseases caused by low or absent circular RNA expression, which is of great significance for the treatment of diseases that rely on circular RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The experimental results of circLARP1B in Example 1; wherein, Figure 1 A is a schematic diagram of plasmid construction. Figure 1 B is the expression level of circLARP1B in cells transfected with different plasmids.

[0036] Figure 2 The experimental results of circSDHB in Example 2; wherein, Figure 2 A is a schematic diagram of plasmid construction. Figure 2 B is the expression level of circSDHB in cells transfected with different plasmids.

[0037] Figure 3 is the experimental result of circSMARCA5 in Example 3; wherein, Figure 3 A is a schematic diagram of plasmid construction. Figure 3B is the expression level of circSMARCA5 in cells transfected with different plasmids.

[0038] Figure 4 The experimental results of circPPFIA1 in Example 4; wherein, Figure 4 A is a schematic diagram of plasmid construction. Figure 4 B is the expression level of circPPFIA1 in cells transfected with different plasmids.

[0039] Figure 5 The experimental results of circFBXW4 in Example 5; wherein, Figure 5 A is a schematic diagram of plasmid construction. Figure 5 B is the expression level of circFBXW4 in cells transfected with different plasmids. DETAILED DESCRIPTION

[0040] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. In addition, the technology involved in the present invention is conventional technical means such as vector construction, cell culture, plasmid transfection, and RT-qPCR, wherein the enzymes, primers, reagents, cell culture reaction conditions, cell transfection conditions, etc. involved can be reasonably selected by known methods or experiments according to the experience of those skilled in the art without explanation, and the detection means involved are well known and mastered by those skilled in the art.

[0042] Some material information in the following examples:

[0043] pCDNA 3.0 plasmid: purchased from Newpro Biotechnology, catalog number V011312;

[0044] HEK293T cells: purchased from ATCC, catalog number CRL-3216.

[0045] Example 1

[0046] In this example, circLARP1B was used as an example to construct a corresponding circular RNA expression plasmid, and the expression efficiency of circLARP1B was detected by cell transfection and RT-qPCR.

[0047] 1. Construction of circLARP1B expression plasmid

[0048] (1) The upstream circLARP1B flanking intron sequence (containing an Alu element) (SEQ ID NO. 3), the circLARP1B circular exon sequence (SEQ ID NO. 4), and the downstream circLARP1B flanking intron sequence (containing an Alu element that is reverse complementary to the upstream Alu element) (SEQ ID NO. 5) were inserted between the HindⅢ and ApaⅠ multiple cloning sites of the pCDNA 3.0 plasmid to obtain the circLARP1B w / o UTR plasmid.

[0049] (2) The same as step (1), except that the ZC3H14 binding sequences UTR1 and UTR2 as shown in SEQ ID NO.1 and SEQ ID NO.2 were inserted after the downstream circLARP1B flanking intron sequence, respectively, to construct circLARP1B UTR1 and circLARP1B UTR2 plasmids, respectively.

[0050] See the schematic diagram of plasmids in steps (1) and (2) for details. Figure 1 A.

[0051] 2. Transfection of HEK293T cells

[0052] pcDNA3.0 empty vector (EV), circLARP1B w / o UTR, circLARP1B UTR1, and circLARP1B UTR2 plasmids were transfected into HEK293T cells using Lipofectamine 2000 reagent. The detailed transfection steps are as follows:

[0053] (1) Cells were passaged and plated one day before transfection so that the cells were in the logarithmic growth phase and the cell density was controlled at around 70-80%;

[0054] (2) Take 125 μl of serum-reduced OPTI-MEM medium, add 5 μl of lipofectamine 2000, mix gently and let stand for 5 minutes;

[0055] (3) Dilute 2 μg of plasmid in 125 μl of OPTI-MEM medium, mix gently, add the lipofectamine-OPTI-MEM mixture that has been mixed and allowed to stand, mix again, and incubate for 20 min;

[0056] (4) Replace the complete medium in the 6-well plate with medium without double antibody, add the prepared transfection reagent to a volume of 750 μl, shake gently to mix, and continue culturing in a 37°C, 5% CO2 incubator;

[0057] (5) Six hours after transfection, the culture medium in the 6-well plate can be replaced with complete culture medium and transfected for 48 hours.

[0058] 3. Total RNA Extraction

[0059] During RNA extraction, be careful to avoid RNA degradation by RNase in the environment. Wipe the workbench with 3% H2O2 solution and use RNase-free pipette tips and centrifuge tubes. The detailed extraction steps are as follows:

[0060] (1) Discard the culture medium in the 6-well plate, wash the cells twice with 1 ml of PBS, and discard the PBS;

[0061] (2) Add 1 ml of TRIzol reagent to the cells in the 6-well plate, pipette the cells to completely lyse them in the TRIzol reagent, transfer them to a 1.5 ml centrifuge tube, and place the centrifuge tube in a vertical mixer to lyse for about 10 minutes;

[0062] (3) Add 1 / 5 volume (200 μl) of chloroform to the centrifuge tube, mix vigorously for about 15 seconds, let stand and separate, and then centrifuge at 12,000 g at 4°C for 15 minutes;

[0063] (4) After centrifugation, carefully transfer the upper aqueous phase of the centrifuge tube to a new 1.5 ml centrifuge tube to avoid aspirating the middle protein layer. Add an equal volume of isopropanol to precipitate the RNA and precipitate at -20°C for about 30 minutes.

[0064] (5) Centrifuge at 12000g for 15 min at 4°C. After centrifugation, discard the supernatant. A white precipitate can be seen at the bottom of the centrifuge tube. Add 1 ml of 80% ethanol and wash the precipitate by inverting it. Centrifuge at 12000g for 5 min at 4°C. Discard the supernatant and dry the precipitate by turning the centrifuge tube upside down.

[0065] (6) After the precipitate is dried, DNA digestion is performed. Taking Thermo DNase I digestion reagent as an example, the DNA digestion system is: 39 μl H2O, 1 μl RNase Inhibitor, 5 μl reaction buffer, 5 μl DNase I, and the reaction conditions are 37°C for 30 min. After the reaction is completed, 5 μl 0.5 M EDTA is added and the reaction is terminated at 70°C for 5 min.

[0066] (7) Add 45 μl of H O to the digested solution to make up the volume to 100 μl. Add 2.5 volumes of anhydrous ethanol and 1 / 10 volume of 3 M sodium acetate to precipitate the RNA again at -80°C for two hours.

[0067] (8) Centrifuge at 12000g for 15 min at 4°C. After centrifugation, discard the supernatant and add 1 ml of 75% ethanol to wash the precipitate by inverting. Centrifuge at 12000g for 5 min at 4°C. Discard the supernatant and dry the precipitate by turning the tube upside down.

[0068] (9) Add DEPC water to the dried precipitate and let it stand on ice until the RNA is completely dissolved.

[0069] 4. First-strand cDNA synthesis

[0070] cDNA synthesis was performed according to the instructions of ABScript II cDNA First Strand Synthesis Kit provided on the ABclonal website.

[0071] (1) Add the following system to an RNase-free PCR tube and mix thoroughly by pipetting;

[0072]

[0073] (2) Denature the RNA template and primers at 65°C for 5 min, centrifuge briefly, and quickly place on ice;

[0074] (3) Add 10 μl of Reaction Mix and 2 μl of Enzyme Mix to the above PCR tube and mix well;

[0075] (4) Reverse transcription was performed using a PCR instrument, incubated at 25°C for 5 min, incubated at 42°C for 1 hr, and heated at 80°C for 5 min to inactivate the enzyme. The obtained cDNA was used for subsequent RT-qPCR experiments.

[0076] 5. Real-time quantitative PCR (RT-qPCR)

[0077] The specific steps are as follows:

[0078] (1) Before conducting the experiment, first design highly specific primers for fluorescent quantitative PCR for the target fragment to be amplified. The melting curve should be single and the Ct value should increase linearly after gradient dilution of the template.

[0079] (2) Prepare the q-PCR reaction system:

[0080]

[0081] The primer information involved is as follows:

[0082]

[0083] After the q-PCR system was prepared, the mixture was centrifuged and the sample was divided into triplicate wells, and 15 μl was added to each well of a q-PCR 96-well plate;

[0084] (3) Place the 96-well plate in a fluorescence quantitative PCR instrument, set the reaction program and run:

[0085]

[0086] After the program is finished, the Ct values of the target gene circLARP1B and the reference gene 18S in different samples can be obtained. -ΔΔCt The relative expression level of the target gene circLARP1B was calculated by the method of

[0087] The results are as attached Figure 1 As shown in Figure 2B, it can be seen that compared with circLARP1B w / o UTR, the insertion of the ZC3H14 protein binding sequence significantly enhanced the overexpression efficiency of circLARP1B.

[0088] Example 2

[0089] In this example, circSDHB was used as an example to construct a corresponding circular RNA expression plasmid, and the expression efficiency of circSDHB was detected by cell transfection and RT-qPCR.

[0090] 1. Construction of circSDHB expression plasmid

[0091] (1) The upstream circSDHB flanking intron sequence (containing an Alu element) (SEQ ID NO. 10), the circSDHB looping exon sequence (SEQ ID NO. 11), and the downstream circSDHB flanking intron sequence (containing an Alu element that is reverse complementary to the upstream Alu) (SEQ ID NO. 12) were inserted between the HindⅢ and ApaⅠ multiple cloning sites of the pCDNA3.0 plasmid to obtain the circSDHB w / o UTR plasmid.

[0092] (2) The same as step (1), except that the nucleotide sequences of ZC3H14 binding sequences UTR1 and UTR2 as shown in SEQ ID NO.1 and SEQ ID NO.2 were inserted after the downstream circSDHB flanking intron sequence, respectively, to construct circSDHB UTR1 and circSDHB UTR2 plasmids, respectively.

[0093] See the schematic diagram of plasmids in steps (1) and (2) for details. Figure 2 A.

[0094] 2. HEK293T cells were transfected with pcDNA3.0 empty vector (EV), circSDHB w / o UTR, circSDHB UTR1, and circSDHB UTR2 plasmids using Lipofectamine 2000. After 48 h of culture, total RNA was extracted using TRIZol reagent and reverse transcribed to obtain cDNA. The efficiency of circular RNA overexpression was detected by RT-qPCR. The cell transfection, total RNA extraction, reverse transcription, and RT-qPCR methods were the same as in Example 1. The primer information used in the qPCR reaction system in this example was as follows:

[0095]

[0096] The results are as attached Figure 2 As shown in Figure 2, compared with circSDHB w / o UTR, the insertion of the ZC3H14 protein binding sequence significantly enhanced the overexpression efficiency of circSDHB.

[0097] Example 3

[0098] In this example, circSMARCA5 was used as an example to construct a corresponding circular RNA expression plasmid, and the expression efficiency of circSMARCA5 was detected by cell transfection and RT-qPCR.

[0099] 1. Construction of circSMARCA5 expression plasmid

[0100] (1) The upstream circSMARCA5 flanking intron sequence (SEQ ID NO. 15), the circSMARCA5 circular exon sequence (SEQ ID NO. 16), and the downstream circSMARCA5 flanking intron sequence (containing a sequence that is reverse complementary to the upstream circSMARCA5 flanking intron sequence) (SEQ ID NO. 17) were inserted between the HindⅢ and ApaⅠ multiple cloning sites of the pCDNA 3.0 plasmid to obtain the circSMARCA5 w / o UTR plasmid.

[0101] (2) The same as step (1), except that the ZC3H14 binding sequences UTR1 and UTR2 as shown in SEQ ID NO.1 and SEQ ID NO.2 were inserted after the downstream circSMARCA5 flanking intron sequence, respectively, to construct circSMARCA5 UTR1 and circSMARCA5 UTR2 plasmids, respectively.

[0102] See the schematic diagram of plasmids in steps (1) and (2) for details. Figure 3 A.

[0103] 2. HEK293T cells were transfected with pcDNA3.0 empty vector (EV), circSMARCA5 w / o UTR, circSMARCA5 UTR1, and circSMARCA5 UTR2 plasmids using Lipofectamine 2000. After 48 h of culture, total RNA was extracted using TRIZol reagent and reverse transcribed to obtain cDNA. The efficiency of circular RNA overexpression was detected by RT-qPCR. The cell transfection, total RNA extraction, reverse transcription, and RT-qPCR methods were the same as in Example 1. The primer information used in the qPCR reaction system in this example is as follows:

[0104]

[0105] The results are as attached Figure 3 As shown in B, compared with circSMARCA5 w / o UTR, the insertion of the ZC3H14 protein binding sequence significantly enhanced the overexpression efficiency of circSMARCA5.

[0106] Example 4

[0107] In this example, circPPFIA1 was used as an example to construct a corresponding circular RNA expression plasmid, and the expression efficiency of circPPFIA1 was detected by cell transfection and RT-qPCR.

[0108] 1. Construction of circPPFIA1 expression plasmid

[0109] (1) The sequences of upstream circPPFIA1 upstream exon (SEQ ID NO.20), upstream partial intron sequence of circPPFIA1 (SEQ ID NO.21), circular exon sequence of circPPFIA1 (SEQ ID NO.22), downstream partial intron sequence of circPPFIA1 (SEQ ID NO.23), and downstream exon of circPPFIA1 (SEQ ID NO.24) were inserted between the HindⅢ and ApaⅠ multiple cloning sites of the pCDNA3.0 plasmid to obtain the circPPFIA1 w / o UTR plasmid.

[0110] (2) The same as step (1), except that the nucleotide sequences of ZC3H14 binding sequences UTR1 and UTR2 as shown in SEQ ID NO.1 and SEQ ID NO.2 were inserted after the downstream exon of circPPFIA1, respectively, to construct circPPFIA1 UTR1 and circPPFIA1 UTR2 plasmids, respectively.

[0111] See the schematic diagram of plasmids in steps (1) and (2) for details. Figure 4 A.

[0112] 2. HEK293T cells were transfected with pcDNA3.0 empty vector (EV), circPPFIA1 w / o UTR, circPPFIA1 UTR1, and circPPFIA1 UTR2 plasmids using Lipofectamine 2000. After 48 h of culture, total RNA was extracted using TRIZol reagent and reverse transcribed to obtain cDNA. The efficiency of circular RNA overexpression was detected by RT-qPCR. The cell transfection, total RNA extraction, reverse transcription, and RT-qPCR methods were the same as in Example 1. The primer information used in the qPCR reaction system in this example was as follows:

[0113]

[0114] The results are as attached Figure 4 As shown in B, compared with circPPFIA1 w / o UTR, the insertion of the ZC3H14 protein binding sequence significantly enhanced the overexpression efficiency of circPPFIA1.

[0115] Example 5

[0116] In this example, circFBXW4 was used as an example to construct a corresponding circular RNA expression plasmid, and the expression efficiency of circFBXW4 was detected by cell transfection and RT-qPCR.

[0117] 1. Construction of circFBXW4 expression plasmid

[0118] (1) The circFBXW4 upstream intron sequence (SEQ ID NO. 27), circFBXW4 looping exon sequence (SEQ ID NO. 28), and circFBXW4 downstream intron sequence (SEQ ID NO. 29) were inserted between the HindⅢ and ApaⅠ multiple cloning sites of the pCDNA3.0 plasmid to obtain the circFBXW4 w / o UTR plasmid.

[0119] (2) The same as step (1), except that the nucleotide sequences of ZC3H14 binding sequences UTR1 and UTR2 as shown in SEQ ID NO.1 and SEQ ID NO.2 were inserted after the downstream intron sequence of circFBXW4, respectively, to construct circFBXW4 UTR1 and circFBXW4 UTR2 plasmids, respectively.

[0120] See the schematic diagram of plasmids in steps (1) and (2) for details. Figure 5 A.

[0121] 2. HEK293T cells were transfected with pcDNA3.0 empty vector (EV), circFBXW4 w / o UTR, circFBXW4 UTR1, and circFBXW4 UTR2 plasmids using Lipofectamine 2000. After 48 h of culture, total RNA was extracted using TRIZol reagent and reverse transcribed to obtain cDNA. The efficiency of circular RNA overexpression was determined by RT-qPCR. The cell transfection, total RNA extraction, reverse transcription, and RT-qPCR methods were the same as in Example 1. The primers used in the qPCR reaction system in this example were as follows:

[0122]

[0123] The results are as attached Figure 5 As shown in B, compared with circFBXW4 w / o UTR, the insertion of the ZC3H14 protein binding sequence significantly enhanced the overexpression efficiency of circFBXW4.

[0124] The examples described in the present invention illustrate that the sequences and sequence combinations provided in the present invention can significantly improve the expression efficiency of circular RNA, and this strategy is universal, and corresponding nucleic acid drugs can be developed based on this, which is of great significance for the treatment of diseases caused by low or missing circular RNA expression.

[0125] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0126] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A circular RNA expression plasmid, characterized in that: The circular RNA expression plasmid contains a ZC3H14 protein binding sequence, or a sequence combination; the sequence combination sequentially includes the ring-forming sequence of the target circular RNA and the ZC3H14 protein binding sequence; the nucleotide sequence of the ZC3H14 protein binding sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The circular RNA expression plasmid according to claim 1, wherein The circular sequence of the target circular RNA includes, in sequence, an upstream circularization-promoting sequence, a circularization exon sequence, and a downstream circularization-promoting sequence of the target circular RNA.

3. The circular RNA expression plasmid according to claim 1, wherein The circular RNA expression plasmid further comprises a backbone vector.

4. The circular RNA expression plasmid according to claim 3, wherein The backbone vector is pCDNA3.

0.

5. A method for constructing a circular RNA expression plasmid according to any one of claims 1 to 4, characterized in that: According to the order of the upstream promoting circularization sequence, circularization exon sequence, downstream promoting circularization sequence and ZC3H14 protein binding sequence of the target circular RNA, the above sequence combination is inserted into the multiple cloning site of the backbone vector to construct a circular RNA expression plasmid.

6. A method for improving the expression efficiency of circular RNA for non-therapeutic and non-diagnostic purposes, characterized in that: The method comprises the step of expressing the circular RNA expression plasmid according to any one of claims 1 to 4 in a cell.

7. A nucleic acid drug, characterized in that Contains the circular RNA expression plasmid according to any one of claims 1 to 4.

8. The nucleic acid drug according to claim 7, wherein The nucleic acid drug has the efficacy of treating diseases caused by the absence or low expression of circular RNA.

9. Use of sequences or sequence combinations that improve circular RNA expression efficiency in preparing circular RNA expression plasmids: The sequence is a ZC3H14 protein binding sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; The sequence combination includes the ring-forming sequence of the target circular RNA and the ZC3H14 protein binding sequence in sequence, and the nucleotide sequence of the ZC3H14 protein binding sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

10. The use according to claim 9, characterized in that The circular sequence of the target circular RNA includes, in sequence, an upstream circularization-promoting sequence, a circularization exon sequence, and a downstream circularization-promoting sequence of the target circular RNA.

Images