A method for overexpressing a target RNA in a cell via extrachromosomal circular DNA

Abstract

The application provides a method for overexpressing target RNA in cells through extrachromosomal circular DNA, and belongs to the technical field of biotechnology.The application provides an expression vector for target double-stranded RNA, which is extrachromosomal circular DNA (eccDNA), and the length and sequence of the double-stranded RNA and the eccDNA are not specially limited, and the overexpression of the target RNA is completed only by relying on the circular structure, and in particular, the target double-stranded RNA can be transcribed from almost any site on the template.The eccDNA can be adapted to various eukaryotic cells, and different lengths of dsRNA can be expressed, so that different requirements can be met.In the field of disease treatment, the eccDNA can be used for gene therapy of tumors, viral infections, genetic diseases and the like;in the field of agriculture, the eccDNA can be used for crop trait improvement and pest control;and in the field of basic research, the eccDNA is a powerful tool for gene function research.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for overexpressing target RNA in cells using extrachromosomal circular DNA. Background Technology

[0002] Double-stranded RNA (dsRNA) plays a crucial role in biomedical research and applications, including RNA interference (RNAi)-mediated gene silencing, viral mimicry, activation of immune responses (such as through activation of RIG-I-like receptors or PKR pathways), and as a key intermediate in the life cycle of certain viruses. Therefore, the efficient and controllable production of dsRNA within cells is a core requirement for many research and technological developments.

[0003] Currently, conventional methods for intracellular overexpression of dsRNA mainly include plasmid vector expression, viral vector expression, and direct introduction of in vitro synthesized dsRNA, but all have limitations. For example, plasmid vector expression utilizes plasmid DNA carrying RNA polymerase III (Pol III) promoters (such as U6 and H1 promoters) to drive the expression of short hairpin RNA (shRNA) or synthesize siRNA precursors. However, plasmid DNA is easily degraded by exonucleases, resulting in poor expression persistence, and it requires antibiotic selection for maintenance, posing a risk of resistance gene diffusion. Viral vector expression uses lentiviruses, adeno-associated viruses (AAVs), or retroviral vectors to deliver shRNA expression cassettes. Although viral vector transduction efficiency is high, viral elements may trigger immune responses, or pose insertional mutation and oncogenic risks, and viral preparation is complex and costly. dsRNA is prepared through chemical synthesis or in vitro transcription and introduced into cells using liposomes or electroporation, but RNA has poor intracellular stability and is easily degraded. Some large dsRNA molecules (>30 bp) can activate innate immune responses (such as the TLR3 and RIG-I pathways), leading to cytotoxicity.

[0004] In summary, current methods for overexpressing dsRNA in cells mainly suffer from drawbacks such as poor stability, low efficiency, insufficient safety, and cumbersome preparation processes. Summary of the Invention

[0005] This invention provides a method for overexpressing target RNA in cells using extrachromosomal circular DNA, achieving efficient, stable, and safe preparation of dsRNA, and providing technical support for RNAi-related research and applications.

[0006] This invention provides a method for constructing an extrachromosomal circular DNA vector containing target RNA, comprising the following steps: synthesizing a first linear double-stranded DNA fragment and a second linear double-stranded DNA fragment, wherein the first linear double-stranded DNA fragment contains a target RNA sequence, and the first linear double-stranded DNA fragment and the second linear double-stranded DNA fragment are anti-cross complementary; Using the first and second linear double-stranded DNA fragments as templates, a circularization reaction was performed to construct an extrachromosomal circular DNA vector containing the target RNA.

[0007] In one specific embodiment of the present invention, the mass ratio of the first linear double-stranded DNA fragment to the second linear double-stranded DNA fragment is 1:1.

[0008] In one specific embodiment of the present invention, the cyclization reaction procedure includes: denaturation at 95°C for 20 seconds; annealing at 4°C for 1 minute; and linkage at 65°C for 20 minutes.

[0009] The present invention also provides an extrachromosomal circular DNA vector containing target RNA constructed using the above-described construction method.

[0010] The present invention also provides the application of the above-mentioned extrachromosomal circular DNA vector containing target RNA in the intracellular overexpression of double-stranded RNA.

[0011] The present invention also provides a method for overexpressing double-stranded RNA in cells, comprising the following steps: introducing the above-mentioned extrachromosomal circular DNA vector containing the target RNA into cells.

[0012] In one specific embodiment of the present invention, the extrachromosomal circular DNA vector containing the target RNA further includes the removal of linear DNA before use.

[0013] In one specific embodiment of the present invention, the method of introduction includes electroporation or transfection.

[0014] This invention also provides the application of the above-mentioned extrachromosomal circular DNA vector containing target RNA in silencing target gene expression.

[0015] The present invention also provides a method for expressing a target gene in silent cells, comprising the following steps: introducing the above-mentioned extrachromosomal circular DNA vector containing the target RNA into the cell; The target gene corresponds to the target RNA.

[0016] Beneficial effects: This invention provides an expression vector for target double-stranded RNA using extrachromosomal circular DNA (eccDNA), and there are no special limitations on the length and sequence of the double-stranded RNA and eccDNA. The overexpression of the target RNA is completed solely by its circular structure. In particular, the target double-stranded RNA can be transcribed from almost any site on the template.

[0017] The eccDNA described in this invention has a wide range of sources. After in vitro synthesis, eccDNA does not contain viral protein-coding sequences, bacterial origins of replication, or immunostimulatory elements such as antibiotic resistance genes. Compared with viral vectors, eccDNA does not activate pattern recognition receptors such as TLR9 and RIG-I. Compared with in vitro synthesized long-chain dsRNA, intracellularly generated dsRNA is processed into siRNA, avoiding innate immune responses and cytotoxicity caused by direct introduction of exogenous dsRNA, thus significantly reducing immunogenicity. It can be introduced into cells via non-viral transfection methods, without the need for viral vectors, and without the risks of insertional mutations or immunogenicity. Furthermore, eccDNA is randomly distributed during cell division and gradually diluted with cell passages, exhibiting self-limiting properties, reducing long-term safety risks, and significantly improving safety. Due to the covalently closed circular structure of eccDNA, its resistance to exonuclease degradation is significantly superior to that of linear DNA. Experiments show that eccDNA can stably exist in cells and continuously express dsRNA for more than 4 weeks, far exceeding the expression time of traditional plasmid vectors (which usually decline significantly within 1-2 weeks). This long-term expression characteristic reduces the need for repeated drug administration, making it particularly suitable for the treatment of diseases requiring long-term gene silencing. Experiments also show that eccDNA can generate tandem repeat RNA chains through rolling circle transcription, and compared with traditional plasmids, the formation efficiency of dsRNA is greatly improved, enabling high-abundance overexpression.

[0018] The eccDNA described in this invention is adaptable to various eukaryotic cells and expresses dsRNA of different lengths, thereby meeting diverse needs. In the field of disease treatment, it can be used for gene therapy of tumors, viral infections, and hereditary diseases; in the agricultural field, it can be used for crop trait improvement and pest and disease control; and in the field of basic research, it is a powerful tool for gene function research. Attached Figure Description

[0019] Figure 1This diagram shows the structure of RNA products transcribed from eccDNA, containing tandem positive and antisense strands. A: Synthesized eccDNA #1, #2, eccRandom, and their corresponding linear DNA were transfected into mouse CT26 cells and then sequenced using third-generation RNA sequencing after 48 hours. B: A 12bp sequence containing the junction site of the eccDNA transcript was used as a junction tag for RNA product structure analysis. C: The eccDNA transfected group showed many sequences containing junctions. The RNA products of the tag were rare or absent in the linear DNA group and the negative control group; D: Tandem transcripts derived from eccDNA may contain both sense and antisense RNA products; EG: Schematic diagrams of representative tandem RNA products from the three eccDNAs, green represents the sense strand RNA product aligned with the eccDNA template, and brick red represents the antisense strand product; H: Copy number statistics of the eccDNA template contained in each transcript; I: The ratio of sense to antisense strand copy numbers in each eccDNA transcript is close to 1:1; J: Analysis of the start site of each eccDNA transcript, the column height represents the relative depth of the start RNA in this region; K: From Schematic diagram of eccDNA from different locations of the FGFR2 gene; L: Read length of eccFGFR2 transcripts analyzed by third-generation RNA single-molecule sequencing, and its comparison with the length of endogenous mRNA; M: Graph showing the copy number of the template DNA positive and negative sequences in all eccFGFR2 transcripts; N: Pie chart showing the proportion of RNA products containing only the sense strand, antisense strand, or both in eccFGFR2 transcripts; O: Pie chart showing the copy number proportion of the sense and antisense strand transcripts; P: Specific structure of representative transcripts from different eccFGFR2 genes, with green representing products from the sense strand and orange representing products from the antisense strand. Figure 2 The diagram shows the results of efficient transcription of dsRNA from eccDNA. In the diagram, A: secondary structure (RNAfold) of RNA transcripts derived from endogenous eccFGFR2; B: tertiary structure prediction (3dRNA / DNA); C: extraction of HEK293T endogenous eccDNA and removal of mitochondrial DNA (mtDNA); lower right: atomic force imaging of purified HEK293T endogenous eccDNA; D: Cy5-labeled linear DNA and HEK293T endo-eccDNA both showed high transfection efficiency in cells; E: J2 immunofluorescence results showed that both synthesized eccFGFR2 and HEK293T endogenous eccDNA could be transcribed into dsRNA in cells, generating stress granular aggregates of bright spots. Detailed Implementation

[0020] This invention provides a method for constructing an extrachromosomal circular DNA vector containing target RNA, comprising the following steps: synthesizing a first linear double-stranded DNA fragment and a second linear double-stranded DNA fragment, wherein the first linear double-stranded DNA fragment contains a target RNA sequence, and the first linear double-stranded DNA fragment and the second linear double-stranded DNA fragment are anti-cross complementary; Using the first and second linear double-stranded DNA fragments as templates, a circularization reaction was performed to construct an extrachromosomal circular DNA vector containing the target RNA.

[0021] This invention uses eccDNA as the expression vector for target RNA. The eccDNA can be derived from known cellular sequences or constructed from random sequences. In one embodiment, eccDNA sequences detected in tumor samples and randomly constructed eccDNA sequences using artificial random DNA sequences, after being transformed into cells using the same method, exhibited the same transcriptional characteristics, demonstrating that the expression of the target RNA described in this invention is an inherent property of the circular structure of eccDNA and does not depend on a specific sequence. In another embodiment, 12 representative eccDNA sequences from different regions of the FGFR2 gene were screened from tumor samples and synthesized in vitro, demonstrating that regardless of whether the eccDNA originates from the promoter, intron, exon, or UTR region of the coding gene, it can be efficiently transcribed in cells, producing a large amount of specific RNA products. This proves that the method described in this invention is applicable to eccDNA from various sequence sources and has broad application prospects.

[0022] The present invention does not have any particular limitation on the sequence and length of the target RNA, and can express RNA of any sequence and length.

[0023] The eccDNA described in this invention can be obtained by in vitro synthesis of two linear double-stranded DNA fragments, and target RNA is inserted into one of the two linear DNA fragments, with the two linear DNA fragments being anti-cross complementary.

[0024] This invention does not specifically limit the preparation method of the two linear double-stranded DNA fragments. They can be obtained by chemical synthesis or by PCR amplification. The two reverse-crossing complementary double-stranded DNA fragments contain the target RNA sequence to be overexpressed and are referred to as linear A and B. To facilitate subsequent ligation and circularization, the 5' end of the linear fragments needs to be phosphorylated.

[0025] In this invention, linear A and B obtained from the construction are mixed in a 1:1 mass ratio and used as a template for a cyclization reaction. In one embodiment, the cyclization reaction is a LAMA cycle reaction. The LAMA cycle reaction system of this invention, in 100 μL, comprises: 500 ng of linear A, 500 ng of linear B, 1 μL of Taq DNA ligase (NEB), 10 μL of Taq DNA ligase buffer, and the balance being water; the procedure includes: Denaturation at 95℃ for 20 seconds causes the double-stranded DNA fragments to unwind; annealing at 4℃ for 1 minute allows the fragments to pair complementaryly and form an open-loop intermediate; ligation at 65℃ for 20 minutes allows Taq DNA ligase to seal the nick and form a closed loop.

[0026] The present invention also provides an extrachromosomal circular DNA vector containing target RNA constructed using the above-described construction method.

[0027] In this invention, in vitro synthesized eccDNA can exist stably and be efficiently transcribed within cells, while linear DNA with the same sequence cannot achieve equivalent function, demonstrating that the circular structure is a prerequisite for the transcriptional function of eccDNA. eccDNA can undergo continuous foldback transcription, producing ultra-long RNA molecules containing tandem repeats of the sense and antisense strands. The total output of both the sense and antisense strands accounts for approximately 50%, providing an ideal precursor for the efficient assembly of double-stranded RNA, demonstrating that the eccDNA transcript possesses a unique tandem sense-antisense structure. The transcription start sites of eccDNA are widely distributed on the template, without obvious sequence bias, indicating that its transcriptional activity is mainly driven by the circular topology, rather than specific internal promoter elements, proving that transcription initiation is independent of specific sequences. Both tumor-derived natural sequences and artificially designed random sequences exhibit the same transcriptional characteristics, demonstrating that this phenomenon is an inherent property of the eccDNA circular structure and does not depend on specific sequences.

[0028] The eccDNA synthesized in vitro in this invention can generally be transcribed into RNA products containing continuous positive and antisense strands in tandem, and has the characteristics of long length, high copy number, and balanced positive and antisense ratio, which can serve as a universal and efficient intracellular double-stranded RNA overexpression platform.

[0029] The present invention also provides the application of the above-mentioned extrachromosomal circular DNA vector containing target RNA in the intracellular overexpression of double-stranded RNA.

[0030] The embodiments of this invention demonstrate that by introducing eccDNA into cells, it can be effectively transcribed and formed into double-stranded RNA, laying a solid foundation for subsequent research and applications.

[0031] The present invention also provides a method for overexpressing double-stranded RNA in cells, comprising the following steps: introducing the above-mentioned extrachromosomal circular DNA vector containing the target RNA into cells.

[0032] Before use, the eccDNA vector of the present invention can also remove linear DNA by enzymatic digestion. In one embodiment, Plasmid-Safe ATP-Dependent DNase (specifically degrades linear DNA) is added to the eccDNA vector, incubated at 37°C for 16 hours, and purified by magnetic beads to obtain pure eccDNA.

[0033] This invention enables the introduction of purified eccDNA into cells. The method of introduction is not particularly limited; it can be electroporation or chemical transfection methods such as liposomes to introduce the synthesized eccDNA into target cells. The cell type introduced is not particularly limited; it can be eukaryotic cells, prokaryotic cells, or viruses, etc.

[0034] This invention uses eccDNA that has entered the cell nucleus as a stable circular template, which is continuously transcribed by RNA polymerase to produce a long primary transcript RNA. This transcript naturally forms tandem positive and negative RNA strands within the cell. The specific mechanism involves complex RNA folding or the action of RNA-dependent RNA polymerase, but the final result is the formation of a double-stranded RNA structure within the cell. After multiple generations of cell passages, sequencing analysis can confirm the RNA sequence derived from eccDNA and the double-stranded structure it forms.

[0035] This invention also provides the application of the above-mentioned extrachromosomal circular DNA vector containing target RNA in silencing target gene expression.

[0036] The eccDNA described in this invention is adaptable to various cell types. In one embodiment, eukaryotic cells (such as HEK293T, HeLa, HepG2, CT26) and primary cells are used, which can express dsRNA of different lengths to meet different needs. In the field of disease treatment, it can be used for gene therapy of tumors, viral infections, and hereditary diseases; in the agricultural field, it can be used for crop trait improvement and pest and disease control; in the field of basic research, it is a powerful tool for gene function research.

[0037] The present invention also provides a method for expressing a target gene in silent cells, comprising the following steps: introducing the above-mentioned extrachromosomal circular DNA vector containing the target RNA into the cell; The target gene corresponds to the target RNA.

[0038] In one embodiment of this invention, structural prediction and immunofluorescence experiments demonstrated that eccDNA transcripts containing tandem sequences of sense and antisense (especially co-transcribed strands of sense and antisense) have an intrinsic tendency to form long and stable double-stranded RNA structures. Secondary and tertiary structure predictions both support this conclusion. Both exogenously synthesized and endogenously extracted eccDNA effectively induced dsRNA production after transfection into cells, while linear DNA controls could not, demonstrating the core inventive point that eccDNA relies on its circular structure to transcribe dsRNA. The perinuclear dsRNA derived from eccDNA aggregates in the cell, a feature suggesting its potential association with stress granule formation, opening a new perspective for understanding the biological function of eccDNA. Endogenous eccDNA also possesses this function, indicating that the method described in this invention mimics the intrinsic cellular mechanism and has broad applicability and biological relevance.

[0039] This invention introduces double-stranded circular DNA into cells, which can be effectively transcribed and formed into double-stranded RNA, laying a solid foundation for subsequent research and applications.

[0040] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for overexpressing target RNA in cells using extrachromosomal circular DNA, should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1: Verification of the transcription of in vitro synthesized eccDNA into tandem positive and negative RNA products in cells. 1. Experimental Materials and Methods 1.1 In vitro synthesis and preparation of eccDNA Three types of double-stranded circular DNA molecules were prepared using a combination of chemical synthesis and enzymatic cyclization via the LAMA cycle reaction: (1) Tumor-derived eccDNA: Using MicroRNA6502 and MicroRNA6516 as target RNAs, two representative eccDNA sequences detected in tumor samples were selected for synthesis and named eccDNA#1 (SEQ ID No.1) and eccDNA#2 (SEQ ID No.2), respectively.

[0042] (2) Random sequence eccDNA: A 400 bp artificial random DNA sequence was synthesized and named eccRandom (SEQ ID No.15).

[0043] BLAST alignment analysis showed that the above sequences had only 2-4% homology with the mouse reference genome, ensuring that they do not encode known endogenous genes or regulatory elements and eliminating the influence of sequence specificity on experimental results.

[0044] 1.2 Cell Culture and Transfection Mouse-derived CT26 colon cancer cells (Sevier) were seeded into 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum until 70-80% confluence. The experiment was divided into three groups: (1) eccDNA transfection group: 1 μg of eccDNA#1, eccDNA#2 and eccRandom were transfected into CT26 cells using liposome transfection reagent Lipofectamine 3000, and 3 biological replicates were set up for each sample.

[0045] (2) Linear DNA transfection group: As a control, cells were transfected with the same mass of corresponding linear DNA molecules under the same transfection conditions as above.

[0046] (3) Blank control group: only transfection reagent was added, and no DNA was added.

[0047] Forty-eight hours after transfection, the culture medium was discarded, the cells were washed twice with pre-cooled PBS, and the cells were lysed with TRIzol reagent. The samples were then collected for total RNA extraction.

[0048] 1.3 Third-generation single-molecule real-time sequencing The extracted total RNA samples were sent to the BGI Cyclone single-molecule sequencing platform for third-generation sequencing. Sequencing library preparation was performed according to the Cyclone platform standard operating procedure, including RNA fragmentation, adapter ligation, and sequencing. Figure 1 (A) 1.4 Bioinformatics Analysis and Results The raw sequencing data underwent quality control filtering to remove low-quality reads and adapter sequences, yielding high-quality long RNA reads. Using Bowtie2 software (v2.4.1), the reads were aligned to the mouse reference genome (mm10), filtering out reads originating from endogenous genes. Subsequently, all long RNA reads from these eccDNA and linear DNA sequences were retrieved, and the 12bp sequence at the eccDNA tandem transcript junction was used as a junction tag for RNA read length alignment analysis. Figure 1 (B) The results showed that the eccDNA transfected group could match many RNA products containing junction tags, while the linear DNA group and the negative control group had few or no similar products. Figure 1 In addition, tandem transcription products derived from eccDNA may contain both positive and antisense RNA products (C). Figure 1After aligning these sense and antisense strand products to the reference template sequence of eccDNA, it was found that these eccDNAs can undergo continuous tandem foldback transcription. Figure 1 In the middle EG), the copy number of the transcription tandem template varies from one to more than ten. Figure 1 (H), and the total percentage of positive and negative copies is approximately 50% (H). Figure 1 Analysis of the origins of these RNA transcripts revealed that eccDNA can be transcribed from almost any site on the template, and no evidence of a clear preference for transcription initiation sites was found. Figure 1 (J), suggesting that the transcription of eccDNA depends on its circular structure rather than a specific sequence.

[0049] In summary, (1) the circular structure is a prerequisite for the transcriptional function of eccDNA: the eccDNA synthesized in vitro can exist stably in cells and be transcribed efficiently, while the linear DNA with the same sequence cannot achieve the equivalent function.

[0050] (2) The eccDNA transcript has a unique tandem sense-antisense structure: eccDNA can undergo continuous foldback transcription to produce ultra-long RNA molecules containing tandem repeats of sense and antisense strands. The total output of sense and antisense strands is about 50%, providing an ideal precursor for efficient assembly of double-stranded RNA.

[0051] (3) Transcription initiation is not dependent on specific sequences: The transcription start sites of eccDNA are widely distributed on the template and there is no obvious sequence preference, indicating that its transcriptional activity is mainly driven by the circular topology rather than specific internal promoter elements.

[0052] (4) This phenomenon is universal: whether it is a natural sequence (eccDNA1, eccDNA2) from tumors or an artificially designed random sequence (eccRandom), they all exhibit the same transcriptional characteristics, proving that this phenomenon is an inherent property of the circular structure of eccDNA and does not depend on a specific sequence.

[0053] Example 2: Verification of the widespread transcription of eccDNA from different sources into tandem positive and negative RNA products in cells 1. Experimental Materials and Methods 1.1 In vitro synthesis of FGFR2-derived eccDNA eccDNA sequence information was detected in tumor samples. Twelve representative eccDNA sequences from different regions of the FGFR2 gene were selected for in vitro synthesis and named eccFGFR2#1 to eccFGFR2#12, respectively. Figure 1(K). All eccDNA was synthesized in vitro using LAMA technology, followed by exonuclease digestion and magnetic bead purification to ensure the removal of residual linear DNA molecules.

[0054] 1.2 Cell Culture and Transfection Mouse CT26 cells were seeded in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum to 70-80% confluence. Using the liposome transfection reagent Lipofectamine 3000, 1 μg of each of the 12 eccFGFR2 strains (eccFGFR2#1-#12) was transfected into CT26 cells, with three biological replicates for each sample. Forty-eight hours post-transfection, the culture medium was discarded, cells were washed twice with pre-cooled PBS, and TRIzol reagent was added to lyse the cells. Samples were collected for total RNA extraction.

[0055] 1.3 Third-generation single-molecule real-time sequencing The extracted total RNA samples were sent to the BGI Cyclone single-molecule sequencing platform for third-generation sequencing.

[0056] 1.4 Bioinformatics Analysis 1.4.1 Data Preprocessing and Identification of ECCDNA-Source Transcripts Raw sequencing data underwent quality control filtering to remove low-quality reads and adapter sequences. Reads were aligned to the mouse reference genome (mm10) using Bowtie2 software, filtering out reads clearly derived from endogenous genes. Subsequently, unaligned reads were extracted and aligned back to the corresponding eccFGFR2 reference template sequence using BLAST to identify transcripts derived from exogenously transfected eccDNA.

[0057] 1.4.2 Transcript Length Analysis The length distribution of all transcripts derived from eccFGFR2 was statistically analyzed, and the median length, mean length, and length range were calculated. Simultaneously, endogenous mRNA transcripts from CT26 cells were extracted from the same sequencing data, and their length distribution was statistically analyzed as a control. The Mann-Whitney U test was used to compare the length differences between eccFGFR2-derived transcripts and endogenous mRNA.

[0058] 1.4.3 Expression level analysis The frequency of each eccFGFR2 transcript in the sequencing data was counted as an indicator of its relative expression level. The expression distribution range, median expression level, and highest expression level of all detected eccFGFR2 transcripts were calculated.

[0059] 4.4 Analysis of the Composition of Think and Antisense Chains The identified eccFGFR2-derived transcripts were compared with their corresponding eccDNA reference template sequences, and the transcripts were classified into three categories based on the comparison results: Product of the positive strand only: The transcript sequence is completely aligned with the template sequence; Antisense strand product only: The transcript sequence is completely opposite in orientation to the template sequence; Mixed products: Transcript sequences containing both sense and antisense sequences.

[0060] The proportion of the three types of products in each eccFGFR2 sample was statistically analyzed. Simultaneously, the ratio of the total number of bases in the sense strand to the total number of bases in the antisense strand was calculated for all transcripts.

[0061] 1.4.5 Structural analysis of representative transcripts Representative transcripts of different eccFGFR2 were selected, and their complete sequences were compared with the corresponding eccDNA templates. Structural diagrams of the transcripts were drawn, showing the arrangement of the sense and antisense strands and their tandem repeat characteristics. Figure 1 (China P).

[0062] 2. Experimental Results 2.1 Efficient transcription of eccFGFR2 in cells Third-generation sequencing data analysis showed that after transfection of CT26 cells, all 12 eccFGFR2 strains (eccFGFR2#1-#12) produced a large number of specific transcripts derived from exogenous eccDNA. These transcripts clearly aligned back to their corresponding eccFGFR2 reference template sequences, and no similar signals were detected in the blank control group. These results indicate that regardless of whether the eccDNA originates from the promoter, intron, exon, or UTR region of the FGFR2 gene, it can serve as a stable transcription template within the cell, efficiently producing RNA products, thus confirming the universality of eccDNA transcriptional function.

[0063] 2.2 Length characteristics of eccFGFR2 transcripts Statistical analysis of the lengths of eccFGFR2-derived transcripts yielded the following results: Figure 1 As shown in L: (1) eccFGFR2 transcripts: The median length of all 12 eccFGFR2-derived transcripts was 4,690 nucleotides (nt). Some transcripts were over 10,000 nt in length, showing extremely long transcriptional features.

[0064] (2) Endogenous mRNA: The median length of endogenous mRNA detected in CT26 cells in the same sequencing data was only 2,139 nt.

[0065] (3) Statistical comparison: The Mann-Whitney U test showed that the length of the eccFGFR2-derived transcripts was significantly longer than that of endogenous mRNA (p<0.001). This result indicates that the circular eccDNA template can support the generation of ultra-long transcripts, whose transcript length far exceeds that of typical linear mRNA molecules in cells. This ultra-long transcriptional feature provides a structural basis for the inclusion of multiple sense and antisense repeat sequences within the same RNA molecule.

[0066] 2.3 High copy number expression of the eccFGFR2 transcript The expression levels of eccFGFR2-derived transcripts were statistically analyzed, and the results are as follows: Figure 1 As shown in M: (1) Distribution range: The expression level of eccFGFR2 transcripts is widely distributed, ranging from low abundance transcripts to high abundance transcripts.

[0067] (2) Highest expression level: Among all the eccFGFR2 samples tested, the copy number of a single transcript reached as high as 135. This value is much higher than the average expression level of typical endogenous genes, indicating that the eccDNA template has extremely high transcriptional activity.

[0068] (3) Biological significance: High expression level means that a single eccDNA molecule can accumulate a large amount of RNA product in the cell, providing sufficient substrate for the subsequent formation of double-stranded RNA.

[0069] 2.4 The compositional rules of the sense and antisense strands of the eccFGFR2 transcript Analysis of the sense and antisense strand composition of eccFGFR2-derived transcripts showed a highly consistent pattern. Figure 1 (N and O) (1) Balanced distribution of the three types of products: Among all 12 eccFGFR2 samples, RNA products containing only the sense strand, RNA products containing only the antisense strand, and mixed products containing both sense and antisense strands each accounted for about 1 / 3. Figure 1(N). This balanced triadic distribution pattern was highly consistent across all detected eccFGFR2 samples, indicating that the transcriptional mechanism of eccDNA can produce pure sense, pure antisense, and mixed transcripts with similar probabilities.

[0070] (2) Balance of total positive and negative copy number: The total number of bases in the sense strand and the total number of bases in the antisense strand were statistically analyzed. The results showed that in all eccFGFR2 samples, the overall copy number ratio of the sense strand to the antisense strand was strictly close to 1:1. Figure 1 (O). This balance ratio is completely consistent with the experimental results of eccDNA1, eccDNA2 and eccRandom mentioned above, further confirming that the positive and negative sense balance of eccDNA transcription is an inherent property of this type of molecule and does not depend on specific sequences.

[0071] (3) Structural characteristics of the mixed products: Further analysis of the mixed products showed that these transcripts also exhibited a continuous reverted transcription pattern, that is, the sense strand and the antisense strand were arranged alternately in a single RNA molecule to form a tandem repeat structure.

[0072] 2.5 Structural representation of representative transcripts Figure 1 The diagrams in the middle list the specific structures of representative transcripts derived from different eccFGFR2 sources. These structural diagrams visually demonstrate the alternating arrangement of the sense strand sequence (indicated by forward arrows) and the antisense strand sequence (indicated by reverse arrows) within a single RNA molecule; the number of tandem repeats varies from a few to more than ten times; transcripts from different eccFGFR2 sources differ in their revert sites and repeat patterns, but all exhibit common tandem features of sense and antisense.

[0073] This embodiment systematically analyzed 12 types of eccDNA derived from different regions of the FGFR2 gene and drew the following conclusions: (1) Verification of universality: Regardless of whether the eccDNA originates from the promoter, intron, exon, or UTR region of the coding gene, it can be efficiently transcribed in the cell, producing a large amount of specific RNA products. This proves that the method described in this invention is applicable to eccDNA from various sequence sources and has broad application prospects.

[0074] (2) Ultra-long transcription characteristics: The median length of transcripts from eccFGFR2 (4,690 nt) was significantly longer than that of endogenous mRNA (2,139 nt), indicating that the circular template can support the generation of ultra-long transcripts and provide a structural basis for containing multiple positive and negative repeat sequences.

[0075] (3) High expression level: The copy number of the eccFGFR2 transcript can reach up to 135, showing extremely high transcriptional activity, which can provide sufficient RNA substrate for the formation of double-stranded RNA.

[0076] (4) The balance between sense and antisense: Of all the eccFGFR2 transcripts, the sense strand alone, the antisense strand alone, and the mixed product each account for about 1 / 3, and the total copy number ratio of the sense strand to the antisense strand is strictly maintained at 1:1. This balance characteristic is an inherent property of eccDNA, which does not depend on the specific sequence and provides an ideal stoichiometry for the efficient formation of double-stranded RNA.

[0077] (5) Structural diversity: Transcripts from different eccFGFR2 sources differ in specific revert sites and tandem patterns, but all exhibit the common feature of continuous positive and antisense tandem, reflecting the combination of structural diversity and functional unity.

[0078] In summary, this embodiment, through systematic analysis of FGFR2-eccDNA from 12 different sources, fully demonstrates that in vitro synthesized eccDNA can generally be transcribed into RNA products containing tandemly linked positive and antisense strands, exhibiting characteristics of long length, high copy number, and balanced positive and antisense ratios. These results further solidify the technical basis of the method described in this invention, proving its feasibility and superiority as a universal and efficient intracellular double-stranded RNA overexpression platform.

[0079] Example 3: Structural prediction and immunofluorescence verification of double-stranded RNA formed from eccDNA-derived transcripts 1. Experimental Materials and Methods 1.1 Transcript sequence selection and structure prediction Three representative read sequences were randomly selected from the endogenous eccDNA transcripts obtained from previous sequencing: Using online tools for structural prediction: (1) RNA secondary structure prediction: Submit the above three sequences to RNAfold WebServer (http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi), calculate the minimum free energy structure using the default parameters (folding temperature 37℃), and output the structure diagram ( Figure 2 (A)

[0080] (2) RNA tertiary structure prediction: The sequence is submitted to the 3dRNA / DNA online server (http: / / biophy.hust.edu.cn / new / 3dRNA), and the tertiary structure is predicted based on homology modeling and fragment assembly methods, outputting a three-dimensional structure model. Figure 2 (B)

[0081] 1.2 Extraction and characterization of endogenous eccDNA 1.2.1 Extraction of endogenous eccDNA Endogenous eccDNA was enriched and extracted from HEK293T cells. The specific steps included: total cellular DNA extraction, removal of linear DNA using Exonuclease V, and removal of mitochondrial DNA (mtDNA) using PacI restriction endonuclease to avoid interference from mt-dsRNA transcribed from mtDNA in subsequent experiments. The processed samples were then purified and recovered.

[0082] 1.2.2 Atomic Force Microscopy (AFM) Characterization The purified endogenous eccDNA sample was diluted and dropped onto freshly peeled mica sheets. After adsorption at room temperature, it was dried under nitrogen. The DNA molecules were then scanned using an atomic force microscope (Bruker Dimension Icon) in tapping mode, with a scanning range of 2μm × 2μm, to observe the morphology and confirm circular structures, ensuring no wireless DNA contamination. Figure 2 (C)

[0083] 1.3 Cell transfection and dsRNA immunofluorescence detection 1.3.1 DNA Marking and Transfection Efficiency Verification To track DNA entry into cells, extracted endogenous eccDNA and control linear DNA (linearized fragments of the same sequence) were labeled with Cy5 fluorescent dye (Label IT Tracker kit, Mirus Bio). The labeled DNA was transfected into HEK293T cells. Twelve hours post-transfection, nuclei were stained with Hoechst 33342, and the distribution of Cy5 signal in the cytoplasm and nucleus was observed using a laser confocal microscope. Figure 2 (D).

[0084] 1.3.2 Experimental Grouping and Transfection HEK293T cells were seeded into 24-well plates (including slides) and cultured to 70% confluence. The experiment was divided into four groups: (1) eccFGFR2 mixture group: 12 kinds of eccFGFR2 (eccFGFR2#1-#12) synthesized in Example 2 were mixed in equal mass, with a total transfection amount of 100ng per well.

[0085] (2) Endogenous eccDNA group: Endogenous eccDNA extracted and purified from HEK293T cells, 100 ng per well.

[0086] (3) Linear DNA control group: The linear DNA fragments corresponding to the eccFGFR2 mixture were mixed in equal mass, and 100 ng were transfected into each well.

[0087] (4) Negative control group: only transfection reagent was added.

[0088] Each group had 3 replicates. Transfection was performed using Lipofectamine 3000, and immunofluorescence was detected 24 hours post-transfection.

[0089] 1.3.3 J2 antibody immunofluorescence staining Twenty-four hours after transfection, the culture medium was discarded, the samples were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour. Mouse anti-dsRNA monoclonal antibody (J2 antibody, Scocons) (1:200 dilution) was added and incubated overnight at 4°C. After washing with PBS, Alexa Fluor 488-labeled goat anti-mouse secondary antibody (1:500) was added and incubated at room temperature in the dark for 1 hour. After washing with PBS, the nuclei were stained with DAPI for 5 minutes and mounted with anti-quenching mounting medium. Images were observed and acquired using a laser confocal microscope (Zeiss LSM 880), and the intensity and distribution characteristics of dsRNA fluorescence signals in each group were compared. Figure 2 (E).

[0090] 2. Experimental Results 2.1 Transcripts derived from eccDNA can form stable dsRNA structures. The predictions of the secondary and tertiary structures of three representative transcripts are as follows: Figure 2 As shown in A and B, these transcripts can all form long and stable dsRNA structures, especially since the co-transcriptional strands of sense and antisense are almost all complementary double-stranded RNA structures.

[0091] 2.2 Confirmation of the circular structure of endogenous eccDNA Endogenous eccDNA extracted from HEK293T cells was treated with PacI and Exonuclease V, and atomic force microscopy imaging showed that... Figure 2 (C) Numerous circular DNA molecules were visible in the field of view, and no linear DNA fragments were observed, indicating that the enzymatic digestion treatment effectively removed mitochondrial DNA and other linear DNA contaminants.

[0092] 2.3 eccDNA can be efficiently transfected into cells. Confocal imaging 4 hours after Cy5-labeled DNA transfection showed ( Figure 2(D) Regardless of whether linear DNA or eccDNA is transfected, Cy5 signal is distributed in both the cytoplasm and the nucleus, and there is no significant difference in the signal intensity in the nucleus between the two treatment groups. This indicates that both linear and circular DNA can be efficiently delivered to the cytoplasm and partially enter the nucleus, providing a basis for subsequent transcription.

[0093] 2.4 eccDNA transfection induces intracellular dsRNA formation J2 antibody immunofluorescence detection results ( Figure 2 (China E) shows: (1) eccFGFR2 mixture group: 24 hours after transfection, significant dsRNA fluorescence signal appeared in the cells, and the signal intensity was much higher than that of the negative control group. The dsRNA signal was a bright punctate cluster, mainly distributed in the area around the cell nucleus, and some punctate structures extended into the cytoplasm. This punctate distribution pattern is similar to the typical characteristics of stress granules (SGs).

[0094] (2) Endogenous eccDNA group: Similar to the eccFGFR2 mixture group, obvious dsRNA signals were detected in the cells, also showing perinuclear punctate aggregation. This indicates that naturally occurring endogenous eccDNA also has the ability to transcribe and form dsRNA.

[0095] (3) Linear DNA control group: In cells transfected with the linear DNA mixture, only a weak background fluorescence signal was detected, similar to the negative control group, with no obvious punctate aggregation. This confirms that linear DNA cannot effectively produce detectable dsRNA.

[0096] (4) Negative control group: almost no specific fluorescence signal.

[0097] This embodiment draws the following conclusions based on structural prediction and immunofluorescence experiments: (1) Structural basis: eccDNA transcripts containing tandem sequences of sense and antisense (especially co-transcribed strands of sense and antisense) have an inherent tendency to form long and stable double-stranded RNA structures, and both secondary and tertiary structure predictions support this conclusion.

[0098] (2) Direct evidence of dsRNA formation: J2 antibody immunofluorescence assay confirmed that both exogenously synthesized eccFGFR2 mixture and intracellularly extracted eccDNA could effectively induce dsRNA production after transfection into cells, while linear DNA control could not. This directly proves the core invention of eccDNA relying on its circular structure to transcribe and produce dsRNA.

[0099] (3) Subcellular localization characteristics: dsRNA derived from eccDNA is aggregated in the perinuclear punctate pattern within the cell. This characteristic suggests that it may be related to the formation of stress granules, opening up a new perspective for understanding the biological function of eccDNA.

[0100] (4) Method universality: Endogenous eccDNA also has this function, indicating that the method described in this invention simulates the intrinsic cellular mechanism and has broad applicability and biological relevance.

[0101] In summary, this embodiment fully demonstrates the technical effects of the method of the present invention from both structural and functional perspectives: by introducing double-stranded circular DNA into cells, it can be effectively transcribed and formed into double-stranded RNA, laying a solid foundation for subsequent research and application.

[0102] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing an extrachromosomal circular DNA vector containing target RNA, characterized in that, Includes the following steps: A first linear double-stranded DNA fragment and a second linear double-stranded DNA fragment are synthesized, wherein the first linear double-stranded DNA fragment contains a target RNA sequence, and the first linear double-stranded DNA fragment and the second linear double-stranded DNA fragment are anti-cross complementary. Using the first and second linear double-stranded DNA fragments as templates, a circularization reaction was performed to construct an extrachromosomal circular DNA vector containing the target RNA.

2. The construction method according to claim 1, characterized in that, The mass ratio of the first linear double-stranded DNA fragment to the second linear double-stranded DNA fragment is 1:

1.

3. The construction method according to claim 1, characterized in that, The procedure for the cyclization reaction includes: denaturation at 95°C for 20 seconds; annealing at 4°C for 1 minute; and ligation at 65°C for 20 minutes.

4. An extrachromosomal circular DNA vector containing target RNA constructed using the construction method described in any one of claims 1 to 3.

5. The use of the extrachromosomal circular DNA vector containing the target RNA as described in claim 4 for intracellular overexpression of double-stranded RNA.

6. A method for overexpressing double-stranded RNA in cells, characterized in that, The method includes the following steps: introducing the extrachromosomal circular DNA vector containing the target RNA as described in claim 4 into a cell.

7. The method according to claim 6, characterized in that, The extrachromosomal circular DNA vector containing the target RNA also includes the removal of linear DNA before use.

8. The method according to claim 6 or 7, characterized in that, The methods for introducing the substance include electroporation or transfection.

9. The use of the extrachromosomal circular DNA vector containing target RNA as described in claim 4 in silencing target gene expression.

10. A method for silencing target gene expression in cells, characterized in that, The procedure includes the following steps: introducing the extrachromosomal circular DNA vector containing the target RNA as described in claim 4 into cells; The target gene corresponds to the target RNA.

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