Improved miR-OE backbones, methods of construction and uses thereof

Abstract

The present application relates to a kind of based on miR-30 skeleton and its construction method and its application in RNAi based on miR-30 skeleton.The skeleton construction method includes the following steps, with existing shRNAmiR30 vector as PCR template;Design two pairs of siRNA containing target gene complementary overlapping primers, respectively, primer F1 and R1, primer F2 and R2, respectively, to obtain P1, P2 as template, with primer F1 and R2 as upper and lower stream primers to carry out OE-PCR, obtain miR-OE.Further, the construction method of the present application also carries out the reply mutation of stem-loop structure and EcoRI enzyme cutting site in miR-OE skeleton.The miR-OE skeleton of the present application and the primer required to be synthesized in the construction method of miR-OE-EL are all short chain with length less than 60nt, and the difficulty and cost of synthesis are effectively reduced, and primer design is more flexible;And the interference efficiency of miR-OE-EL skeleton to target gene is significantly improved, and can be well applied to the preparation of shRNA drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an improved miR-OE backbone and its construction method and application. BACKGROUND

[0002] RNA interference (RNAi) refers to a gene silencing phenomenon in eukaryotes induced by double-stranded RNA, targeting the degradation of homologous complementary mRNA. There are three commonly used gene knockdown tools at present. Small interfering RNA (siRNA) is chemically synthesized in vitro, which is transiently expressed in cells and can only produce a short-term gene inhibition effect. Short hairpin RNA (shRNA) is stably expressed by using a plasmid vector, which generates siRNA through Dicer enzyme processing in cells. Although it can maintain long-term stable gene silencing, most shRNA vectors rely on the RNA polymerase III promoter (Pol III, such as U6, H1, etc.) for driving. This type of promoter is a constitutive high expression promoter, which cannot regulate gene expression in specific tissues and its overexpression will non-specifically compete with the processing elements of endogenous microRNA (miRNA), resulting in serious side effects. The shRNA system based on endogenous pri-miRNA (shRNAmiR) uses pri-miRNA as a backbone, replaces the miRNA mature sequence with the interference sequence of the target gene, which can be driven by various types of promoters and produce siRNA for gene silencing through the endogenous miRNA processing pathway.

[0003] Compared with the other two types of knockdown tools, shRNAmiR knockdown system has many advantages; it can be transcribed by various RNA polymerase II or III (Pol II, III) promoters, including constitutive promoters, tissue-specific promoters and inducible promoters; specific promoters such as muscle tissue-specific promoters MHCK7 and ACTA1, liver-specific promoters TBG and ALB, etc. can achieve target gene silencing in specific tissues or cells in vivo; and inducible promoters such as tetracycline-induced TRE promoters can be reversibly timed-regulated by small molecule drug binding or unbinding. In addition, the Pol II promoter can effectively transcribe longer RNA, enabling the expression of two or more shRNAmiRs in a multi-cistronic tandem, while knocking down multiple target genes; and the target gene can be co-expressed with multi-cistronic shRNAmiRs to achieve single vector, multi-functional use. Based on the above spatiotemporal specificity and multi-cistronic expression, shRNAmiR can effectively improve the efficiency of target gene silencing; in addition, compared with Pol III promoter, the transcriptional activity of Pol II promoter is lower, which can avoid the production of oversaturated shRNA and reduce cell toxicity.

[0004] Although the shRNAmiR knockdown system is more flexible and efficient, its complex construction method seriously restricts its use in vivo and in vitro. The most commonly used shRNAmiR vector is constructed using the miR-30 backbone, and the miR-30 backbone is introduced with XhoI and EcoRI enzyme digestion sites at the 5' and 3' flanking conserved sequences of the human endogenous miR-30 (Endo-miR-30) backbone to clone the target sequence, and in order to construct a 3' end 2 base overhang structure, the two conserved base pairs before and after the loop structure are changed from CU / GG to UA / UA Figure 1 A, B); Johanners Zuber team further repositioned the EcoRI enzyme digestion site of miR-30 to the 3' distal non-conserved region, and restored the 3' proximal conserved region to the natural sequence, i.e. "miR-E" backbone Figure 1C), which has a relatively higher efficiency than miR-30, but also requires a longer target sequence. The shRNA sequence of miR-30 and miR-E backbone both contain a flanking region, a specific target siRNA sequence and a top loop sequence. The commonly used construction method is to chemically synthesize a complementary long-chain oligonucleotide chain of more than 110 nt, then use the oligonucleotide annealing method to form a small fragment, and then use XhoI and EcoRI enzyme cutting sites to access the vector. Some studies use PCR amplification method, which needs to synthesize a complementary long-chain oligonucleotide chain of about 100 nt as a PCR template, and also needs to synthesize upstream and downstream primers containing the corresponding enzyme cutting sites. After amplification, the enzyme cutting, purification and ligation steps are required for cloning, which is more complex. It is worth noting that the synthesis of primers greater than 60 nt is more difficult, has a higher error probability, and is more expensive. Dapeng Wang et al. developed a miR-AB backbone based on the improvement of miR-E ( Figure 1 B), which introduces BamHI and Apal enzyme cutting sites in the stem, so that the shRNA sequence only contains a specific siRNA sequence and a loop structure. Two complementary oligonucleotides of 75 nt and 67 nt need to be synthesized for cloning, and there is no significant difference in RNAi interference effect compared with the miR-E backbone. Although miR-AB shortens the length of the vector fragment to be accessed, the oligonucleotide chain still needs to be synthesized more than 60 nt. In addition, the modification of the enzyme cutting site in the conserved sequence will have a certain impact on the RNAi structure. The introduction of BamHI and Apal enzyme cutting sites in addition to XhoI and EcoRI enzyme cutting sites makes the selection of multiple cloning sites for subsequent tandem access to other genes less. Moreover, there is the same problem as miR-30 and miR-E. The enzyme cutting site of their access fragment is fixed, so the shRNAmiR cloning vector cannot be randomly selected, which increases the step of replacing the vector later. Therefore, based on the miR-30 backbone, how to control the oligonucleotide chain synthesized for shRNA target sequence cloning to be a short chain of less than 60 nt, and to randomly replace the cloning site while maximizing the preservation of the shRNA conserved sequence, so as to reduce the synthesis difficulty and cost, make the cloning process more flexible and efficiently silence the target gene, is the key problem that needs to be solved for shRNAmiR system. SUMMARY

[0005] Therefore, based on the miR-30 backbone, how to control the oligonucleotide chain synthesized for shRNA target sequence cloning to be a short chain of less than 60 nt, and to randomly replace the cloning site while maximizing the preservation of the shRNA conserved sequence, so as to reduce the synthesis difficulty and cost, make the cloning process more flexible and efficiently silence the target gene, is the key problem that needs to be solved for shRNAmiR system.

[0006] The technical scheme for achieving the above-mentioned purpose comprises the following.

[0007] The first aspect of the present application is to provide a construction method of miR-OE backbone, comprising the following steps,

[0008] The shRNAmiR vector with miR-30 as its backbone was used as a PCR template;

[0009] Two pairs of complementary overlapping primers containing the target gene siRNA were designed, namely primers F1 and R1, and primers F2 and R2. F1 and R2 are respectively equipped with restriction enzyme sites, capable of amplifying the 5′UTR and 3′UTR sequences of the miR-30 backbone in the template. R1 consists of a 19-nucleotide fixed circular sequence of the miR-30 backbone, the target gene siRNA, and the corresponding 5′UTR sequence. F2 also consists of a 19-nucleotide fixed circular sequence of the miR-30 backbone, the target gene siRNA, and the corresponding 3′UTR sequence. Primers F1 and R1 amplify the full-length 5′UTR, while primers F2 and R2 amplify the full-length 3′UTR.

[0010] Amplification was performed separately using primers F1 and R1 to obtain amplification product P1, and primers F2 and R2 to obtain amplification product P2.

[0011] Using amplification products P1 and P2 as templates, and primers F1 and R2 as upstream and downstream primers, OE-PCR was performed to obtain the full-length pri-miRNA fragment miR-OE.

[0012] In some of the embodiments, the two-base overhangs on the left and right sides of the 19 nucleotides of the fixed circular sequence in R1 are ta and ta, respectively, and the two-base overhangs on the left and right sides of the 19 nucleotides of the fixed circular sequence in F2 are ta and ta, respectively.

[0013] In some of the embodiments, the two dangling bases on the left and right sides of the 19 nucleotides of the fixed circular sequence in R1 are cc and ag, respectively, and the two dangling bases on the left and right sides of the 19 nucleotides of the fixed circular sequence in F2 are ct and gg, respectively.

[0014] In some embodiments, the 19-nucleotide sequence of the fixed circular sequence in R1 is as shown in SEQ ID NO:39, and the 19-nucleotide sequence of the fixed circular sequence of the miR-30 backbone in F2 is as shown in SEQ ID NO:40; or

[0015] The 19-nucleotide sequence of the fixed circular sequence in R1 is shown in SEQ ID NO:37, and the 19-nucleotide sequence of the fixed circular sequence of the miR-30 backbone in F2 is shown in SEQ ID NO:38.

[0016] In some of these embodiments, a portion of the 3′UTR sequence in F2 is shown as in SEQ ID NO:41.

[0017] In some of these embodiments, a portion of the 3′UTR sequence in F2 is shown in SEQ ID NO:42.

[0018] In some embodiments, the restriction enzyme sites for F1 and R2 are XbaI and BamHI, respectively. In the miR-30 backbone construction method of this invention, other restriction enzyme sites can also be used as needed without affecting the successful construction of the miR-30 backbone. For example, they can be replaced with: BglII, BstBI, ClaI, EcoRI, EcoRV, HindIII, HpaI, KpnI, MluI, NdeI, NheI, NotI, PacI, PmeI, SalI, SmaI, SpeI, XhoI, etc.

[0019] In some of these embodiments, the sequence of F1 is as shown in SEQ ID NO:1.

[0020] In some of these embodiments, the sequence of R2 is as shown in SEQ ID NO:4.

[0021] The restriction enzyme sites in the F1 and R2 sequences can be replaced with other corresponding restriction enzyme sites as needed.

[0022] R1 is as shown in SEQ ID NO:25, and F2 is as shown in SEQ ID NO:26; or

[0023] R1 is as shown in SEQ ID NO:27, and F2 is as shown in SEQ ID NO:28; or

[0024] R1 is as shown in SEQ ID NO:25, and F2 is as shown in SEQ ID NO:33; or

[0025] R1 is shown as SEQ ID NO:27, and F2 is shown as SEQ ID NO:33.

[0026] A second aspect of the present invention is to provide the miR-OE skeleton obtained according to any of the above-described construction methods.

[0027] In some of these embodiments, the miR-OE skeleton is the miR-OE-E skeleton shown in SEQ ID NO:34.

[0028] In some of these embodiments, the miR-OE skeleton is the miR-OE-L skeleton as shown in SEQ ID NO:35.

[0029] In some of these embodiments, the miR-OE skeleton is the miR-OE-EL skeleton as shown in SEQ ID NO:36.

[0030] A third aspect of the present invention is to provide the application of the miR-OE backbone in siRNA.

[0031] A fourth aspect of the present invention is to provide the use of the miR-OE backbone in the preparation of shRNA drugs.

[0032] This invention provides a novel method for constructing a miR-30 backbone. This method utilizes OE-PCR, designs corresponding primers, and synthesizes short-chain primers (less than 60 nt in length), effectively reducing synthesis difficulty and cost, and enabling efficient and low-cost acquisition of miR-OE backbones. Furthermore, the construction method of this invention, particularly after reversing mutations at specific sites, significantly improves the interference efficiency of the obtained shRNA against target genes. Primer design is also more flexible, with the restriction sites in F1 and R2 arbitrarily replaceable, making the cloning process more flexible and simple. The construction method of this invention can flexibly obtain various miR-OE backbones, including miR-OE-E, miR-OE-L, and miR-OE-EL backbones. The miR-OE backbone obtained by this invention, through reversing mutations at EcoRI and loop structures in the existing miR-30 backbone, effectively improves the interference efficiency of RNA against target genes, making it well-suited for preparing corresponding shRNA drugs, achieving RNAi, and obtaining the desired therapeutic effect. Attached Figure Description

[0033] Figure 1 Schematic diagram of the skeleton structure; where A: Endo-miR30 skeleton and miR-30 skeleton;

[0034] B: miR-E skeleton and miR-AB skeleton; C: miR-OE skeleton construction method and miR-OE skeleton.

[0035] Figure 2 Map and sequence location of pTRIPZ-RFP-shRNAmiR30_CD274; where A is the map of pTRIPZ-RFP-shRNAmiR30_CD274; B is the design of the two primer pairs F1 / R1 and F2 / R2 and their positions in the plasmid sequence.

[0036] Figure 3 This is a schematic diagram of the electrophoresis results of miR-OE amplification in Example 1.

[0037] Figure 4 The sequence alignment results are for the interference vector in Example 2.

[0038] Figure 5 This is the sequencing peak diagram of the interference vector in Example 2.

[0039] Figure 6 This is a schematic diagram illustrating the interference effect of the shRNAmiR carried by the miR-OE backbone on the target gene in Example 2.

[0040] Figure 7 A schematic diagram of the new skeleton structure, where A: new skeleton miR-OE-X; B: new skeleton miR-OE-L; C: new skeleton miR-OE-E.

[0041] Figure 8 The sequence alignment results are for the interference vector in Example 3.

[0042] Figure 9 This is a sequencing peak diagram of the interference vector shRNAmiR-SQSTM1-EL1 / EL2 / EL3 / EL4 in Example 3.

[0043] Figure 10 This is a sequencing peak diagram of the interference vector shRNAmiR-TAX1BP1-EL1 / EL2 / EL3 / EL4 in Example 3.

[0044] Figure 11 This is a schematic diagram illustrating the interference effect of shRNAmiR on the target gene after the site reversion mutations of XhoI, EcoRI, and the loop structure in Example 3. In the diagram, A: SQSTM1, B: TAX1BP1.

[0045] Figure 12 This is a schematic diagram illustrating the interference effect of shRNAmiR on the target gene after simultaneous reversion mutations at EcoRI and loop structures in Example 4, where A: SQSTM1, B: TAX1BP1.

[0046] Figure 13 This is a schematic diagram of the new framework miR-OE-EL.

[0047] Figure 14 This is the structure of the miR-OE-E framework.

[0048] Figure 15 This is the structure of the miR-OE-L framework.

[0049] Figure 16 This is the structure of the miR-OE-EL framework. Detailed Implementation

[0050] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0051] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or under conditions recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0052] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0053] To facilitate understanding of this technology, some terms and phrases are defined below.

[0054] Overlap extension polymerase chain reaction (OE-PCR), hereinafter referred to as overlap PCR.

[0055] RNAi: Introducing antisense RNA corresponding to mRNA into cells can cause specific degradation of mRNA, leading to the silencing of its corresponding gene. This post-transcriptional gene silencing mechanism (PTGS) is called RNA interference. In RNAi, the antisense strand of siRNA cleaves the target mRNA to achieve gene silencing. One siRNA strand that is perfectly complementary to the target RNA is called the guide strand, and the other siRNA strand is called the passenger strand. The siRNA on the RISC complex unwinds into single strands; the passenger strand is cleaved, while the guide strand is preserved.

[0056] This invention develops a novel method for constructing shRNA miR based on the miR-30 backbone using overlap extension polymerase chain reaction (OE-PCR) technology.

[0057] The shRNAmiR construction method includes using an existing shRNAmiR vector containing a miR-30 backbone as a PCR template, using the 19 nucleotides of the fixed circular sequence of the miR-30 backbone as the overlapping complementary sequence for OE-PCR, introducing the 21nt target sequence of the silenced gene in the middle of the primer, and the fixed matching sequence of the miRNA backbone at the tail of the primer. The miRNA fragment can be obtained by overlapping PCR of the front and rear primers, and then cloned into any plasmid vector by enzyme digestion.

[0058] The present invention will be further described in detail below with reference to specific embodiments.

[0059] Example 1: Method for constructing the miR-OE backbone

[0060] Taking shControl (derived from the target sequence of the copepod green fluorescent protein variant TurboGFP) as an example, as shown in Table 1 and... Figure 1 As shown, the 19nt ring sequence is the overlapping portion (shaded area in Table 1). Figure 1 (Orange portion in the middle) Extending towards both ends, TurboGFPsiRNA (boxed portion) and adjacent miR-30 backbone UTR matching sequences are added to one positive and one antisense sequence in each of the two primer pairs, respectively, to design two complementary overlapping primers, R1 and F2; Complementary matching sequences are designed in the 5′ UTR and extended outward to introduce the restriction enzyme site XbaI (wavy line portion) as the F1 forward primer, and complementary matching sequences are designed at the end of the 3′ UTR region and extended outward to introduce the restriction enzyme site BamHI (wavy line portion) as the R2 reverse primer; All primers required to be synthesized are short oligonucleotides (<60nt).

[0061] Following the reaction system and conditions in Table 2, pTRIPZ-RFP-shRNAmiR30_CD274 (plasmid map see...) was used. Figure 2 A. The design of the two primer pairs F1 / R1 and F2 / R2 and their positions in the plasmid sequence are shown in [reference needed]. Figure 2 A and Figure 2 Using B) as a template, primers F1 and R1 were used to amplify the full-length 5′ UTR, siRNA, and loop region to obtain P1 (180bp), and primers F2 and R2 were used to amplify the full-length 3′ UTR, siRNA, and loop region to obtain P2 (196bp). Using P1 and P2 as templates, OE-PCR was performed with outer primers F1 and R2 as upstream and downstream primers. P1 and P2 were spliced ​​together by overlapping strand extension to form the full-length pri-miRNA fragment, referred to as miR-OE (376bp). Figure 1 C), the amplified electrophoresis results are as follows Figure 3As shown, after digestion with XbaI and BamHI, the plasmid was cloned into the pCDH-EF1α-MCS-IRES-Puro vector (purchased from SBISystem Biosciences, catalog number CD532A-2) to obtain the shRNAmiR negative control plasmid pCDH-EF1-mir30_shControl-IRES-Puro (hereinafter abbreviated as shCtrl).

[0062] Table 1. Primer sequences for synthesizing TurboGFP shRNA (shControl)

[0063]

[0064] Table 2. Reaction system and conditions for OE-PCR

[0065]

[0066]

[0067] Note: Prime MaxDNA Polymerase was purchased from Takara (Takara R045A).

[0068] In the miR-OE backbone construction process described in this invention, only short-chain primers with a length of less than 60 nt need to be synthesized, which significantly reduces the difficulty and cost of synthesis. Moreover, both restriction enzyme sites are outside the full-length region of pri-miRNA, without changing its conserved sequence and natural structure. The restriction enzyme sites can be changed at will according to the needs of the cloning vector, making the cloning operation simpler, more convenient and flexible.

[0069] Example 2: Validation of the miR-OE framework

[0070] Furthermore, to detect the miR-OE backbone-mediated RNAi effect, shRNA sequences for five target genes (CALCOCO2, NBR1, OPTN, SQSTM1, and TAX1BP1) were designed using software. Four target sequences were selected for each gene (Table 3). As shown in Table 4, the target sequences and their reverse complementary sequences from Table 3 were filled into the overlapping primers R1 and F2 boxes of the corresponding control vectors (resulting in R1_P and F2_G, where the boxes represent the positive and antisense sequences of the siRNA, which can be sequences of 19-21 bases, i.e., NNNNNNNNNNNNNNNNNNNNNNN represents an siRNA sequence of 19-21 bases in length, and this is the same in the following tables). The OE-PCR method was used, and the amplification reaction system and conditions were the same as in Table 2: the first round of PCR used the pCDH-EF1-mir30_shContr described in Example 1. Using ol-IRES-Puro as a template (or other existing shRNAmiR plasmids containing the miR-30 backbone as a template), primers F1 and R1_P, and F2_G and R2 amplified two fragments respectively. In the second round of PCR, the two amplified fragments were used as templates, and F1 and R2 were used as upstream and downstream primers to amplify the corresponding target sequence miR-OE. After restriction enzyme digestion and cloning into pCDH-EF1α-MCS-IRES-Puro, interference vectors were obtained, named shRNAmiR-CALCOCO2-1 / 2 / 3 / 4, shRNAmiR-NBR1-1 / 2 / 3 / 4, shRNAmiR-OPTN-1 / 2 / 3 / 4, shRNAmiR-SQSTM1-1 / 2 / 3 / 4, and shRNAmiR-TAX1BP1-1 / 2 / 3 / 4. The accuracy of the sequences was then verified by sequencing (sequence alignment results are shown in [link to sequencing data]). Figure 4 See sequencing peak diagram. Figure 5 The above-mentioned interference vectors and their corresponding target gene expression plasmids (Table 5) were co-transfected into cells for the detection of interference effects. The specific steps are as follows (taking shRNAmiR-CALCOCO2-1 as an example): 1) HEK293T cells were counted and processed at 5×10⁻⁶ cells per cell line. 5 Cells / well were seeded into 12-well plates. After 12 hours, shRNA miR-CALCOCO2-1 (2 μg) and overexpression plasmid pCMV-CALCOCO2(human)-3×Flag-Neo (abbreviated as Flag-CALCOCO2) (0.5 μg) were added together to 60 μL of Opti-MEM (Gibco). TMDilute the plasmid (Cat. No. 31985070) and immediately add it to PEI 25000 (Polysciences, Inc. 23966) at a ratio of 1:3 (plasmid (μg): transfection reagent (μL). After mixing, incubate at room temperature for 30 min and then add to HEK293T cells. After culturing at 37℃ for 6 h, replace with fresh complete medium and continue culturing for 48 h. Then lyse the cells and extract total protein. 2) Detect the knockdown level of CALCOCO2 using Western blotting with Flag-tagged antibody. Figure 6 As shown, the shRNA miRs carried by the miR-OE backbone can all exert a certain interference effect on their target genes. Since miR-OE is amplified based on the miR-30 backbone, its right wing conserved region still retains the introduction of the EcoRI restriction site and the mutation of the conserved base pairs on both sides of the loop, and the XhoI restriction site introduced in the left wing region, although it does not affect the conserved sequence, has mutated the backbone sequence; the mutations at these three sites may affect the target gene interference effect of shRNA miR; while the miR-OE backbone construction method of this invention is not based on the restriction ligation of XhoI and EcoRI cloning sites, and the middle loop region, as the complementary pairing sequence for overlapping PCR, can also be easily changed through primer design. We speculate that restoring these three mutations to the original miR-30 backbone sequence may improve its gene silencing efficiency.

[0071] Table 3. shRNA target sequences of each target gene

[0072]

[0073] Table 4. Primer sequences for shRNA of each target gene

[0074]

[0075]

[0076] Table 5. Overexpression plasmids corresponding to each interference vector in the co-transfection system

[0077]

[0078] Note: All overexpression plasmids in the table were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0079] Example 3: Construction of miR-OE-X, miR-OE-L, and miR-OE-E backbones and verification of RNAi effects.

[0080] This embodiment uses the target genes SQSTM1 and TAX1BP1 as examples to restore the three mutation sites back to their natural sequences and examine their impact on RNAi performance. As shown in Table 6, the two mutant base pairs TA / TA in the loop structure of the overlapping primers R1_P and F2_G were changed back to the natural structure CT / GG (italicized), resulting in overlapping primers R1_L and F2_L. As shown in Table 7, the two base pairs CG at the XhoI mutation site were changed back to AA (italicized), and the 23 base pairs upstream and downstream of this site were used as complementary overlapping sequences (bolded parts) to extend outwards to obtain primers R1_X and F2_X. Using the constructed shRNAmiR30 vectors of each target gene as templates, and referring to the amplification reaction system and conditions in Table 2, the outer primers were used to generate the primers. F1 and R2 were subjected to OE-PCR and cloned into pCDH-EF1α-MCS-IRES-Puro to obtain interference vectors with circular sequence mutations and XhoI site reversion mutations (i.e., shRNAmiR-SQSTM1-L1 / L2 / L3 / L4, shRNAmiR-TAX1BP1-L1 / L2 / L3 / L4, shRNAmiR-SQSTM1-X1 / X2 / X3 / X4, shRNAmiR-TAX1BP1-X1 / X2 / X3 / X4), with the backbones named miR-OE-L and miR-OE-X, respectively. Figure 7 A, B).

[0081] Because the EcoRI site is relatively close to the stem-loop structure, pCDH-EF1-mir30_shControl-IRES-Puro was used as a template. As shown in Table 8, the base A at the EcoRI mutation site was mutated back to C (italicized). Using 21 bases upstream and downstream of this site as complementary overlapping sequences (bolded text), primers R1_CE and F2_CE were extended outwards. OE-PCR and cloning were performed using the outer primers F1 and R2 to obtain pCDH-EF1-miR30_shControl_E-IRES-Puro. Using this as a control template, the loop structure was... The sequences were designed as overlapping complementary sequences (shaded areas). Forward overlapping primers F2_E were designed for each target gene (Table 9, bold text indicates sites where mutations have been restored in the control template). The reverse overlapping primer was R1_P from Table 4. OE-PCR was then performed using outer primers F1 and R2, and the results were cloned into pCDH-EF1α-MCS-IRES-Puro to obtain interference vectors for each target gene undergoing EcoRI site mutation repair (i.e., shRNAmiR-SQSTM1-E1 / E2 / E3 / E4 and shRNAmiR-TAX1BP1-E1 / E2 / E3 / E4). The backbone was named miR-OE-E. Figure 7 C).

[0082] The sequence accuracy of all the above-mentioned interference vectors for reversion mutations has been confirmed by sequencing (sequence alignment results are available in [link to sequencing data]). Figure 8 See sequencing peak diagram. Figure 9 and Figure 10 Further analysis using Western blotting revealed that site reversion mutations in EcoRI and the loop structure significantly increased the silencing effect of each shRNA on SQSTM1 (…). Figure 11 A) and TAX1BP1 ( Figure 11 B) interference efficiency; while XhoI, because it does not affect conserved sequences, its site reversion mutations did not significantly affect the silencing effect of the target gene.

[0083] Table 6. Primer sequences for loop sequence reversion mutation

[0084]

[0085] Table 7. Primer sequences for XhoI site reversion mutations

[0086]

[0087] Table 8. Primer sequences for shControlEcoRI site reversion mutations

[0088]

[0089] Table 9. Primer sequences for EcoRI site reversion mutation

[0090]

[0091] The 19 nucleotide sequences of the fixed circular sequence are as follows:

[0092] tacatctgtggcttcactaSEQ ID NO:37, tagtgaagccacagatgtaSEQ ID NO:38,

[0093] cccatctgtggcttcacagSEQ ID NO:39,ctgtgaagccacagatgggSEQ ID NO:40.

[0094] Example 4: Construction of the miR-OE-EL backbone and verification of RNAi effect

[0095] To further verify the importance of the high conservation of pri-miRNA sequences in gene expression regulation, EcoRI and loop site reversion mutations were performed simultaneously. As shown in Table 10, using the loop sequence mutation repair primer R1_L, an overlapping primer F2_EL to increase EcoRI site repair was designed. Using the shRNAmiR30 vectors of the SQSTM1 and TAX1BP1 target genes as templates, combined with primers F1 and R2 on both sides, OE-PCR was performed according to the reaction system and conditions in Table 2. The cloned shRNAmiR-SQSTM1-EL1 / EL2 / EL3 / EL4 and shRNAmiR-TAX1BP1-EL1 / EL2 / EL3 / EL4 were cloned into pCDH-EF1α-MCS-IRES-Puro and then sequenced for verification (sequence alignment results are shown in Table 10). Figure 8 See sequencing peak diagram. Figure 9 and Figure 10 Western blot results showed that the co-repair of EcoRI and loop sites in shRNA significantly improved the interference efficiency of the two target genes SQSTM1 and TAX1BP1 compared to the individual repair of the two sites, further demonstrating that the conserved sequence of pri-miRNA plays an important role in miRNA generation. Figure 12 Using the two primer pairs in Table 10 for OE-PCR, the required oligonucleotide chains to be synthesized are all short chains with a length of less than 60 nt, effectively reducing the difficulty and cost of synthesis; the cloning sites in F1 and R2 are not in conserved sequences, and can be arbitrarily replaced, making the cloning process more flexible and simple; the EcoRI and loop structures in the existing miR-30 backbone can be reversed by R1_L and F2_EL, effectively improving the interference efficiency of RNA on the target gene; the new backbone obtained by OE-PCR is called miR-OE-EL. Figure 13 The right-hand sequence of the unreversed mutation F2_EL is ttgcctactgcctcggaatSEQ ID NO:41, and the right-hand sequence of the reversed mutation F2_EL is ttgcctactgcctcggactSEQ ID NO:42.

[0096] Table 10. Primer sequences for EcoRI site and loop sequence site reversion mutations

[0097]

[0098] The structure of the obtained miR-OE-E framework is shown in the figure. Figure 14 The structure of the miR-OE-L framework, as shown in SEQ ID NO:34, is described below. Figure 15 The composition of SEQ ID NO:35 and the miR-OE-EL framework is shown in [link to documentation]. Figure 15and SEQ ID NO:36. Figure 14 to Figure 16 In the diagram, blue represents XbaI restriction sites, magenta represents XhoI restriction sites, yellow represents BamHI restriction sites, bold represents EcoRI sites or their reversion mutations, and wavy subscripts indicate the subscripts below the wavy lines. Figure 1 C and Figure 7 In the three skeleton diagrams, the content marked with three dots on the left and right is shown. The red text represents the circular structure or its reversion mutation. The boxes contain the positive and antisense sequences of siRNA, which can be sequences of 19-21 bases. For example, NNNNNNNNNNNNNNNNNNNNN represents an siRNA sequence of 19-21 bases in length.

[0099] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for constructing a miR-OE backbone, characterized in that, The construction method includes the following steps. The shRNAmiR vector with miR-30 as its backbone was used as a PCR template; Two pairs of complementary overlapping primers containing the target gene siRNA were designed, namely primers F1 and R1, and primers F2 and R2. F1 and R2 are respectively primers with restriction enzyme sites, capable of amplifying the 5′UTR and 3′UTR sequences of the miR-30 backbone in the template. R1 consists of the 19-nucleotide fixed circular sequence of the miR-30 backbone, the target gene siRNA, and the corresponding 5′UTR. F2 consists of the 19-nucleotide fixed circular sequence of the miR-30 backbone, the target gene siRNA, and the corresponding 3′UTR. Primers F1 and R1 amplify the full-length 5′UTR, while primers F2 and R2 amplify the full-length 3′UTR. Amplification was performed separately using primers F1 and R1 to obtain amplification product P1, and primers F2 and R2 to obtain amplification product P2. Using amplification products P1 and P2 as templates, and primers F1 and R2 as upstream and downstream primers, OE-PCR was performed to obtain the PCR product, i.e., the miR-OE backbone. The sequence of F1 is as shown in SEQ ID NO:1, or the sequence of F1 is as shown in SEQ ID NO:1, but with different restriction enzyme sites; and the sequence of R2 is as shown in SEQ ID NO:4, or the sequence of R2 is as shown in SEQ ID NO:4, but with different restriction enzyme sites; and R1 is as shown in SEQ ID NO:25, and F2 is as shown in SEQ ID NO:26; or R1 is as shown in SEQ ID NO:27, and F2 is as shown in SEQ ID NO:28; or R1 is as shown in SEQ ID NO:25, and F2 is as shown in SEQ ID NO:33; or R1 is shown as SEQ ID NO:27, and F2 is shown as SEQ ID NO:

33.

2. The miR-OE skeleton obtained by the construction method according to claim 1.

3. The miR-OE framework according to claim 2, wherein, The miR-OE framework is the miR-OE-E framework shown in SEQ ID NO:34; or The miR-OE framework is the miR-OE-L framework shown in SEQ ID NO:35; or The miR-OE skeleton is the miR-OE-EL skeleton as shown in SEQ ID NO:

36.

4. The use of the miR-OE backbone according to claim 2 or 3 in the preparation of shRNA drugs.

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